Effect of primary phase feline immunodeficiency virus infection on cats with chronic toxoplasmosis

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J.J. C A L L A N A N ET AL.

pathogenic mechanisms remain unclear (Folks, 1991 ). The direct or indirect role of FIV infection in the development of feline haematological disorders is not yet known. In this experiment, neutropenia appeared to be consumptive. In Case 5B, the profound neutropenia and lymphopenia was the result of toxic bone marrow suppression and stress. A direct effect of FIV infection on granulopoiesis could not however be ruled out. To date, limited information is available on the histopathological changes in experimental FIV infections. Yamamoto et al. (1988) observed that lymphadenopathy during the initial stages of FIV infection was a result of lymphoid hyperplasia and follicular dysplasia. Dow et al. ( 1990 ) reported on the histopathology of the brain in natural and experimental infected cats and showed that FIV is neurotropic and causes lesions consisting predominantly of perivascular mononuclear cell infiltrates. In this study, lymphadenopathy was also the result of generalised B- and T-cell stimulation. In addition, there was involvement of the non-lymphoid organs with the presence of perivascular aggregates of lymphoid tissue which in many cases became fully formed mature lymphoid follicles. FIV is a T-lymphotropic virus and studies have centred around the alteration of T-cell subsets, notably the T-helper cells (CD4) (Ackley et al., 1990). There is, however, a major activation of B-cells, and histopathological changes in the primary phase of infection are predominantly B-cell associated. The mechanisms of B-cell stimulation and their antigen and epitope specificity to FIV, as well as the ultimate fate of B-cell aggregates in non-lymphoid organs, have yet to be determined. ACKNOWLEDGEMENTS

The authors acknowledge the technical assistance of J. Cole, J. Murphy, R. Irvine and M. McDonald. Photographs were prepared by A. May. This work was supported by the Wellcome Trust, the Leukaemia Research Fund and the AIDS Directed Programme of the Medical Research Council.

REFERENCES Ackley, C.D., Yamamoto, J.K., Levy, N., Pedersen, N.C. and Cooper, M.D., 1990. Immunologic abnormalities in pathogen-free cats experimentally infected with feline immunodeficiency virus. J. Virol., 64: 5652-5655. Callanan, J.J., Hosie, M.J. and Jarrett, O., 1991a. Transmission of feline immunodeficiency virus from mother to kitten. Vet. Rec., 128: 332-333. Callanan, J.J., McCandlish, I.A.P., O'Neil, B., Lawrence, C.E., Rigby, M., Pacitti, A.M. and Jarrett, O., 1992. Lymphosarcoma in experimentally induced feline immunodeficiency virus infection. Vet. Rec., 130: 293-295. Dow, S.W., Poss, M.L. and Hoover, E.A., 1990. Feline immunodeficiency virus: a neutropic lentivirus. J. AIDS, 3: 658-668.

CLINICAL AND PATHOLOGICALFINDINGS IN FIV INFECTED CATS

13

Folks, T.M., 1991. Human immunodeficiency virus in bone marrow: still more questions than answers. Blood, 77: 1625-1626. Hopper, C.D., Sparkes, A.H., Gruffydd-Jones, T.J., Crispin, S.M., Muir, P., Harbour, D.A. and Stokes, C.R., 1989. Clinical and laboratory findings in cats infected with feline immunodeficiency virus. Vet. Rec., 125: 341-346. Hosie, M.J., Robertson, C. and Jarrett, O., 1989. Prevalence of feline leukaemia virus and antibodies to feline immunodeficiency virus in cats in the United Kingdom. Vet. Rec., 128: 293-297. Hosie, M.J. and Jarrett, O., 1990. Serological responses of cats to feline immunodeficiency virus. AIDS, 4:215-220. Ishida, T., Washizu, T., Toriyabe, K., Motoyoshi, S., Tomoda, I. and Pedersen, N.C., 1989. Feline immunodeficiency virus infection in cats of Japan. J. Am. Vet. Med. Assoc., 194: 221-225. O'Hara, C.J., 1989. The lymphoid and hematopoietic systems. In: S.J. Harawi and C.L O'Hara (Editors), Pathology and Pathophysiology of AIDS and HIV-Related Diseases. Chapman and Hall Medical, London, pp. 135-199. Pedersen, N.C., Ho, E.W., Brown, M.L. and Yamamoto, J.K., 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science, 235: 790793. Shelton, G.H., Waltier, R.M., Connor, S. and Grant, C.K., 1989. Prevalence of feline immunodeficiency virus and feline leukaemia virus infections in pet cats. J. Am. Anim. Hosp. Assoc., 25: 7-12. Shelton, G.H, Linenberger, M.L., Grant, C.K. and Abkowitz, J.L., 1990a. Hematologic manifestations of feline immunodeficiency virus infection. Blood, 76:1104-1109. Shelton, G.H., Grant, C.K., Cotter, S.M., Gardner, M.B., Hardy, W.D. and DiGiacomo, R.F., 1990b. Feline immunodeficiency virus and feline leukemia virus infections and their relationships to lymphoid malignancies in cats: a retrospective study (1968-1988). J. AIDS, 3: 623-630. Yamamoto, J.K., Sparger, E., Ho, E.W., Andersen, P.R., O'Connor, T.P., MandeU, C.P., Lowenstine, L, Munns, R. and Pedersen, N.C., 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res., 49: 1246-1258.

Veterinary Immunology and Immunopathology, 35 ( 1992) 15-22

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Elsevier Science Publishers B.V., Amsterdam

Long-term clinical observations on feline immunodeficiency virus infected asymptomatic carriers Takuo Ishida, Akiko Taniguchi, Sei Matsumura, Tsukimi Washizu and Isamu Tomoda Department of Clinical Pathology, Nippon Veterinary and Animal Science University, 1-7-1 Musashino-shi, Tokyo 180, Japan

ABSTRACT Ishida, T., Taniguchi, A., Matsumura, S., Washizu, T. and Tomoda, I., 1992. Long-term clinical observations on feline immunodeficiency virus infected asymptomatic carders. Vet. Immunol. Immunopathol., 35:15-22. Eleven feline immunodeficiency virus (FIV) infected asymptomatic carder (AC) cats were observed for 2 years for development of acquired immunodeficiency syndrome (AIDS). Four of the 11 (36.4%) showed progression of the clinical stage. Persistent generalized lymphadenopathy was noted in three cats as the first sign of illness after the AC phase, while the other showed lymphadenopathy with signs of AIDS-related complex. In all four cats the AIDS-related complex stage lasted for 10 months or longer, and two showed progression of the disease into AIDS. The two cats showing AIDS illnesses died within approximately 1 year after they had developed persistent generalized lymphadenopathy. Pathology confirmed the diagnosis of AIDS characterized by the presence of depletion lesions in the lymphoid organs, and of severe infections of an opportunistic nature. The overall mortality of FIV infected AC cats during a 2 year period was two out of 11 (18.2%). These cats showed decreased concanavalin A mitogen response of peripheral blood mononuclear cells as the disease progressed. ABBREVIATIONS AC, asymptomatic carder; AIDS, acquired immunodeficiency syndrome; ARC, AIDS-related complex; Con A, concanavalin A; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; IL-2, interleukin-2; PBMC, peripheral blood mononuclear cells; PCV, packed cell volume; POL, persistent generalized lymphadenopathy.

INTRODUCTION

The clinical course of feline immunodeficiency virus (FIV) infection is divided into five clinical stages (Ishida and Tomoda, 1990) which are comCorrespondence to: T. Ishida, Department of Clinical Pathology, Nippon Veterinary and Animal Science University, 1-7-1 Musashino-shi, Tokyo 180, Japan.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

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parable to those seen with human immunodeficiency virus (HIV) infection (Centers for Disease Control, 1986 ). The stages include acute phase, asymptomatic carrier (AC), persistent generalized lymphadenopathy (PGL), acquired immunodeficiency syndrome (AIDS )-related complex (ARC), and AIDS. The approximate duration of each stage was estimated from a number of clinical observations on the naturally infected and experimentally infected cats (Yamamoto et al., 1988, 1989; Ishida et al., 1989). Furthermore, the disease progression from ARC to AIDS has been closely followed innaturally infected cats initially showing ARC signs (Ishida and Tomoda, 1990). We have previously shown that about 12% of healthy outdoor cats in this country are infected with FIV (Ishida et al., 1989). Although we have also experienced a number of clinical and postmortem cases diagnosed as AIDS, this observation on the healthy cat population strongly suggests that there are a greater number of healthy infected cats than those with clinical diseases. The experimental infection of cats with FIV isolates, on the other hand, has been so far successful only in producing acute phase infection followed by the AC phase (Yamamoto et al., 1988 ). Although the epidemiologic data regarding the age of infected cats suggest that the asymptomatic phase may precede the period with chronic illnesses and death (Ishida et al., 1988 ), it is not known whether AC cats eventually develop AIDS. Furthermore, what proportion of AC cats develop ARC or AIDS in a fixed period of time is another question to be answered. In order to answer these questions, and to confirm the order of the clinical stages suggested previously, a long-term clinical observation study on naturally infected AC cats was carried out. The cats were closely observed for development of clinical signs, were monitored for immunologic alterations with lymphocyte blastogenesis, and pathology was performed on selected cases. MATERIALS AND METHODS

Naturally infected cats Eleven asymptomatic cats were randomly selected from a group of FIV positive and feline leukemia virus (FeLV) negative cats. They were all previously household pet cats with known history and were donated for the research (see Table 1 ). The cats were positive for anti-FIV p24 and gpl20 by immunoblot and positive for virus isolation by cultivating peripheral blood mononuclear cells (PBMC) with concanavalin A (Con A) and human recombinant interleukin-2 (IL-2) (Pedersen et al., 1987; Ishida et al., 1988 ).

Long-term observation The cats were kept in a conventional environment with air conditioning, housed in individual stainless steel cages, and were fed commercial diet

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17

(KalKan; Master Foods, Tokyo, Japan) and water ad libitum. Clinical observation was carded out every day for 2 years. Any change in clinical conditions was recorded, and clinical staging was carded out on the basis of criteria reported previously. The observation also included complete blood count, FIV and FeLV serology, and lymphocyte blastogenesis at regular intervals. The cats received only supportive therapies when conditions deteriorated. These included short-term fluid and antibiotic administration; no antiviral, immunomodulator or glucocorticoid therapy was carded out.

Lymphocyte blastogenesis The blastogenic activities of PBMC with Con A stimulation were measured by the glucose consumption test as described previously (Taniguchi et al., 1990). The assay was performed bimonthly or as needed.

Pathology The fatal cases were necropsied. The tissue from major viscera were fixed in 10% buffered formalin, and paraffin sections were made. RESULTS

Progression of disease stages Out of 11 AC cats, four (36.4%) showed progression of the disease in 2 years. Three of the four cats developed prominent PGL for short periods before developing ARC signs. The other cat showed lymphadenopathy together with ARC signs. Of the four cats with ARC illnesses, two eventually developed severe wasting disease and died. They were diagnosed as AIDS both clinically and pathologically. Therefore, the overall incidence of AIDS in 11 AC cats within 2 years was 18.2%. The rest of the cats remained clinically healthy throughout the observation period, but were antibody positive.

The clinical course of the four cats Cat 1, male, 3.5 years old at the start of observation, died after 18 months at 5 years of age. He remained clinically normal for the first 2 months. Then he started to show PGL involving mandibular, superficial cervical and popliteal nodes. The lymphadenopathy persisted for approximately 3 months ( 13 weeks), then moderate gingivitis was noticed in addition to PGL, and clinically diagnosed as ARC. The weight loss at this time was not marked. The ARC phase lasted for 10 months. During this time, the cat showed mild icte-

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rus and occasional upper respiratory signs, and the body weight gradually decreased. Then he showed marked emaciation ( 1.9 kg compared with an initial body weight of 3.0 kg), non-regenerative anemia and chronic gastrointestinal signs. The severe chronic gingivitis persisted, but lymphadenopathy was not marked at this time. The cat died after 3 months of the wasting illness. Cat 5, female, 3.5 years old at the start of observation, also died after 18 months at the age of 5 years. This cat showed a similar clinical course to that of Cat 1. PGL was seen after 2 months, which persisted for a slightly shorter period ( 11 weeks) than that of Cat 1. The ARC period which followed was characterized by chronic gingivitis, conjunctivitis and upper respiratory disease, and lasted for 10 months. Then the AIDS phase of the disease was noted, when emaciation (2.5 kg compared with an initial body weight of 4.0 kg), anemia, icterus and chronic gastrointestinal signs were seen. The duration of this terminal stage was also 3 months. Cat 6, female, initially 7 years old, developed PGL, gingivitis and a slight weight loss at the same time after 23 months, and was diagnosed as ARC. Cat 7, female, initially 3.5 years of age, started to show PGL after 20 months which lasted for 4 months. At the end of the 2 year observation period, she developed severe gingivitis. Both cats were still surviving with the same degree and type of illness after 10 months.

The clinicopathologic changes in the symptomatic cats During the PGL and ARC phases, there were no marked hematologic changes in each cat except for hyperproteinemia (Cats 1, 6 and 7; total plasma protein 8.6-12.0 g - ~dl- ~) and transient neutrophilia ( > 12 500/tl- 1). Lymphopenia was not seen in these stages, but slight lymphocytosis ( > 7000 #l- ~) was seen in one case (Cat 3 ). When Cats 1 and 5 developed AIDS signs, marginal anemia with packed cell volume (PCV) of 25 to 26% gradually deteriorated. The PCV shortly before death was 17.6% in Cat 1 with no evidence of regenerative response. The complete blood count was not possible in Cat 5 at the terminal stage, but the cat showed clinical anemia with non-regenerative red cell morphology in the blood smear. The lymphocyte count was decreased, being in the low normal range (2442 #1-1 ) in Cat 1 and severely decreased (3% of approximate white cell count of 10 000 #1- ~) in Cat 5. Total plasma protein was elevated in Cat 1 ( 8-10 g dl- 1) but not in Cat 5.

Lymphocyte blastogenesis At the beginning of the study, all 11 cats showed decreased Con A proliferative responses characteristic of AC with a mean _+SD of 49.1 _+5.5% (Table 1 ). The seven cats which remained healthy throughout the study did not show

19

CLINICALCOURSEOF NATURALFIV INFECTION TABLE 1 FIV infected AC cats used for 2 year observations

Cat

Age t (years)

Sex

Con A blastogenesis (SI%) 2

1 2 3 4 5 6 7 8 9 10

3.5 3.5 5 5 3.5 7 3.5 4 4 3.5 4

11

M M M M F F F F F F F

43.7 43.0 53.8 45.8 47.2 50.6 49.1 41.7 54.3 51.4 59.5

rAge at the start of observation period. 2SI, stimulation index.

any significant decrease in lymphocyte responsiveness (data not shown). On the other hand, the four cats developing illnesses showed gradual decreases in Con A response as the disease progressed (Figs. 1 and 2). The stimulation index at each disease stage was similar to those reported previously (Taniguchi et al., 1990). Blastogenesis assay for Cat 5 in the terminal AIDS stage was not possible.

Pathology The two cats that died of AIDS diseases were necropsied for histopathologic examination. Cat 1 had lymph node follicular depletion and fibrosis in 60 55

No 7

5o No 6

35 25 ~

3 AC

0~

. . . . . . . . .

PGL Clinical stages

ARC

Fig. 1. Changes in Con A blastogenesis stimulation index (SI) in the course of developing ARC.

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T. ISHIDA

ET AL.

6O

40

~

0 AC

I

I

PGL

ARC

AIDS

Clinical stages

Fig. 2. Changesin Con A blastogenesisstimulation index (SI) in the course of developingARC and AIDS. multiple nodes, including mandibular, parotid, axillary, popliteal, and mesenteric nodes. Chronic interstitial nephritis of intermediate severity was seen. In the small intestine and colon, moderate lymphocytic enteritis and colitis were observed. There was severe tapeworm infestation in the gut. Cat 5 also showed follicular segmentation and lymphadenitis in the multiple nodes. The spleen showed follicular depletion and amyloid deposition. There was severe systemic bacterial infection, and bacterial pleuritis and peritonitis were noted. Severe septic pleuritis and edema were found in the lung. Lymphocytic plasmacytic enteritis with severe ascariasis was seen. The liver showed cholangiohepatitis, amyloidosis in Disse's space, and severe atrophy and necrosis of hepatocytes was noted. Mild chronic interstitial nephritis was also seen. DISCUSSION The present investigation has shown that about one-third of FIV infected AC cats develop advanced diseases in 2 years. During the same period, a mortality of 18.2% was recorded. Since our previous observation showed that the ARC stage is invariably followed by development of AIDS within 1 year (Ishida and Tomoda, 1990), the two ARC cases seen in this study should eventually develop AIDS. If this is the case, the mortality may increase up to 36.2% (four of 11 ) in 3 years. When estimating the incidence of AIDS and incubation period in a FIV infected population, it is important to take the age of the subjects into consideration. In the present study, all the cats showing illnesses except Cat 6 were young adults aged 3.5 years and 5.5 years at the start and end of the experiment, respectively. This means that the longest possible duration of the FIV infection in these cases was 5.5 years if one assumes the infection occurred in

CLINICAL COURSE OF NATURAL FIV INFECTION

21

the first year of life. If we assume the exposure took place later in life, possibly at around 1 year of age as suggested by epidemiologic observations (Grindem et al., 1989; Ishida et al., 1989; Shelton et al., 1989c; Yamamoto et al., 1989), the incubation period in these cases must be shorter by 1 year. This is in good agreement with the mean age of AC cases (4.2_ 3.3 years) based on epidemiologic data from a large number of naturally infected cats (Ishida and Tomoda, 1990). The incidence of ARC and AIDS in these young adult cats excluding the older Cat 6 is thus three in ten (30%). Since their age at the end of the study was between 5.5 and 7 years, this incidence can be interpreted as one during the approximate period of 4.5-6 years after exposure. The present study has also shown that PGL develops before ARC. In one cat, however, the occurrence of PGL was noticed concurrently with other ARC signs. The order of each clinical stage previously suggested (Ishida and Tomoda, 1990) was confirmed. The duration of each disease stage was also in good agreement with the previous observations (Ishida and Tomoda, 1990). The most prominent clinicopathologic change in the AIDS stage in the present cases was lymphopenia. Also, it should be noted that three of four symptomatic cats had hyperproteinemia. These alterations may be closely associated with immune dysfunction. The progression of immunologic disturbance was monitored in individual cases by Con A blastogenesis as the disease stage proceeded. The stimulation index decreased as the stage progressed, and the index at each stage was in the characteristic range reported previously (Taniguchi et al., 1990). This observation further suggests that the development of wasting diseases in FIV infected cats is closely related to immunologic disturbances. The necropsy of the two fatal cases established a diagnosis of AIDS. The prominent features in these cases include the presence of lymphoid depletion and of opportunistic infections. The lymph node lesions have been well characterized in both human AIDS and FIV infected cats (Diebold et al., 1985; Brown et al., 1991 ). Although it is difficult to define opportunistic infections in the cat, the present cases at least had unusually severe infections with bacterial and/or parasite pathogens. Although the two cats had chronic interstitial nephritis, these lesions were interpreted as mild to intermediate which may not cause terminal renal failure and immunosuppression. The occurrence of amyloidosis in FIV infected cats warrants further studies on its pathogenesis related to dysproteinemia. ACKNOWLEDGMENTS

This study was partially supported by a Scientific Research Grant from the Ministry of Welfare, Japan. The authors thank Master Foods Ltd. for supplying the cat food.

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REFERENCES Brown, P.J., Hopper, D. and Harbour, D.A., 1991. Pathological features of lymphoid tissues in cats with natural feline immunodeficiency virus infection. J. Comp. Pathol., 104: 257-260. Centers for Disease Control, 1986. MMWR, Morbid. Mortal. Weekly Rep., 35: 334. Diebold, J., Marche, C.I., Audoouin, J., Aubert, J.P., Tourneau, A., Bouton, C.I., Reynes, M., Wizniak, J., Capron, F. and Tricottet, V., 1985. Lymph node modifcation in patients with the acquired immunodeficiency syndrome (AIDS) or with AIDS related complex (ARC). Pathol. Res. Pract., 180:590-611. Grindem, C.B., Corbett, W.T., Ammermann, B.E. and Tomkins, M.T., 1989. Seroepidemiologic survey of feline immunodeficiency virus infection in cats of Wake County, North Carolina, J. Am. Vet. Med. Assoc., 194: 226-228. Ishida, T. and Tomoda, I., 1990. Clinical staging of feline immunodeficiency virus infection. Jpn. J. Vet. Sci., 52: 657-660. Ishida, T., Washizu, T., Toriyabe, K. and Motoyoshi, S., 1988. Detection of feline T-lymphotropic Lentivirus (FTLV) infection in Japanese domestic cats. Jpn. J. Vet. Sci., 50: 39-44. Ishida, T., Washizu, T., Toriyabe, K., Motoyoshi, S., Tomoda, I. and Pedersen, N.C., 1989. Feline immunodeficiency virus infection in cats of Japan. J. Am. Vet. Med. Assoc., 194: 221-225. Pedersen, N.C., Ho, E.W., Brown, M.L. and Yamamoto, J.K., 1987. Isolation of a T lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science, 235: 790793. Shelton, G.H., Waltier, R.M., Connor, S.C. and Grant, C.K., 1989. Prevalence of feline immunodeficiency virus infections in pet cats. J. Am. Anim. Hosp. Assoc., 25:7-12. Taniguchi, A., Ishida, T., Konno, A., Washizu, T. and Tomoda, I., 1990. Altered mitogen response of peripheral blood lymphocytes in different stages of feline immunodefciency virus infection. Jpn. J. Vet. Sci., 52: 525-530. Yamamoto, J.K., Sparger, E., Ho, E.W., Andersen, P.R., O'Connor, P., Mandell, C.P., Lowenstine, L., Munn, R. and Pedersen, N.C., 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res., 49: 1246-1258. Yamamoto, J.K., Hansen, H., Ho, E.W., Morishita, T.Y. Okuda, T., Sawa, T.R., Nakamura, R.M. and Pedersen, N.C., 1989. Epidemiologic and clinical aspects of feline immunodeficiency virus infection in cats from the continental United States and Canada and possible mode of transmission. J. Am. Vet. Med. Assoc., 194: 213-220.

Veterinary Immunology and lmmunopathology, 35 (1992) 23-35

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Feline immunodeficiency virus neurotropism: evidence that astrocytes and microglia are the primary target cells Steven W. Dow, Matthew J. Dreitz and Edward A. Hoover Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80503, USA

ABSTRACT Dow, S.W., Dreitz, M.J. and Hoover, E.A., 1992. Feline immunodeficiency virus neurotropism: evidence that astrocytes and microglia are the primary target cells. Vet. lmmunol. Immunopathol., 35: 23-35. To investigate the neuropathogenesis of feline immunodeficiency virus (FIV) infection in vitro, we have utilized three populations of cultured feline neural cells (astrocytes, microglia, brain endothelium) to assess the relative susceptibility to FIV infection, ability to produce viral antigens, and effects of infection on cell survival. Astrocytes appeared to be the most susceptible to infection, followed by microglia, whereas brain endothelial cells were relatively resistant to infection. Astrocyte infection resulted in syncytium formation and cell death, while microglial cells remained persistently and productively infected, without obvious cytopathic effects. These results suggest that FIV entry into the central nervous system probably does not occur via infected endothelium and that both astrocytes and microglia are more likely target cells for the virus. ABBREVIATIONS ConA, concanavalin A; CrFK, Crandell feline kidney cells; ECGF, endothelial cell growth factor; FFU,focus-forming unit ; FIV, feline immunodeficiency virus; FITC, fluorescein isothiocyanate; GFAP, gilial fibrillary acidic protein; HIV, human immunodeficiency virus; IL, interleukin; PBL, peripheral blood lymphocytes; PI, post inoculation; SIV, simian immunodeficiency virus; SPF, specific pathogen free.

INTRODUCTION

All lentiviruses are neurotropic and most are capable of inducing central nervous system (CNS) dysfunction in at least a subpopulation of infected Correspondence to: E.A. Hoover, Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80503, USA.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

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s.w. DOW ET AL.

individuals. Studies of lentiviral neuropathogenesis have assumed new relevance since it is now apparent that most human immunodeficiency virus (HIV) infected patients will suffer from some form ofneurologic impairment during the course of their disease (McArthur, 1987). In the CNS of HIV infected humans and simian immunodeficiency virus (SIV) infected primates, the primary target cell is the microglial cell (or brain macrophage) (Koenig et al., 1986; Lackner et al., 1991 ). At present, it is unclear whether HIV and SIV infect only resident brain macrophages (microglia) or blood-borne monocytes that enter the brain and mature into macrophages within the brain parenchyma. Visna virus of sheep appears to have a broader neural cell tropism and infects, in addition to microglia/brain macrophages, glial cells and brain endothelial cells (Georgsson et al., 1989 ). We have shown previously that feline immunodeficiency virus (FIV) infects the CNS of most naturally infected cats, based on evidence of intrathecal synthesis of FIV specific antibodies and the presence of the virus in cerebrospinal fluid (Dow et al., 1990, 1992). Clinically apparent neurologic abnormalities may be present in up to 30% of infected cats and subclinical abnormalities detectable by electrophysiological evaluation may be present in an even greater percentage of cats (Wheeler et al., 1991; Dow et al., 1992). Experimentally, FIV infects the CNS of cats after intravenous or intrathecal inoculation and in some cases induces inflammatory lesions (primarily perivascular mononuclear cell infiltrates and gliosis) in subcortical grey matter structures, brainstem and cervical spinal cord. Early studies also showed that cultured feline astrocytes and microglia could be infected after in vitro inoculation with FIV-Petaluma (Dow et al., 1990). In order to more fully assess potential FIV CNS target cells and the effects of FIV infection, we have expanded our feline neural culture system to increase the yield of microglia and to include primary cultures of brain capillary endothelial cells. Endothelial cells are a potential target for a lentivirus such as FIV that circulates both in infected lymphocytes and free in plasma, and infected endothelium may provide a route for virus entry into the brain. This culture system has allowed us to compare relative FIV susceptibility amongst three potential neural target cells and to more directly assess the effect of FIV infection on cell function. MATERIALS AND METHODS

Virus stocks

The Petaluma strain of FIV was provided by J. Yamamoto (University of California, Davis) and was propagated in feline Crandell kidney cells (CrFK). The FIV-2546 isolate was obtained from a cat with immunodeficiency (leukopenia and opportunistic infections) and neurologic disease (behavioral

NEUROTROPISM OF FELINE IMMUNODEFICIENCY VIRUS

25

changes, compulsive chewing). Plasma from the original donor cat was provided by Dr. Steve Gardner, Albany Veterinary Clinic, Albany, CA. After inoculation of this plasma into a naive specific pathogen free (SPF) cat (Cat 2546), FIV was recovered from lymphocyte co-culture with ConA-stimulated lymphoblasts from retrovirus-free cats. Virus isolation was confirmed by FIV p26 antigen enzyme-linked immunosorbent assay (ELISA) (Dreitz et al., 1992). Stocks of this virus, designated FIV-2546, were screened by indirect immunofluorescence assay (IFA) with reference serum to assure that they were free of feline spumivirus and then adapted to grow in CrFK. Virus titers were determined by focal immunoassay, as described previously (Sitban et al., 1985). Neural cell culture

Primary cultures of feline neural cells were established from brain tissues of newborn SPF cats. Briefly, cerebral cortical tissue was removed after the cats had been killed with halothane. Meningeal tissue was carefully removed, the tissue minced in balanced salt solution, digested in 0.2% trypsin (Sigma, St. Louis, MO) and 80 #g DNase m1-1 (Sigma) at 37°C for 20 min with periodic agitation, triturated through a glass pipette until a single cell suspension was obtained. The cells were resuspended in Dulbecco's modified Eagle medium (DME) with 4500 mg glucose ml-1 (Sigma), 1% penicillin-streptomycin solution (Gibco-BRL, Grand Island, NY), 5% heat inactivated fetal bovine serum (FBS) and 5% calf serum (HyClone Laboratories, Logan, UT ), and 5 × 10 -5 M 2-mercaptoethanol (Sigma). The cells were seeded in 75 cm flasks (Costar, Van Nuys, CA) that had been precoated with 10 #g poly-Llysine ml- ~ (Sigma). Medium was changed every 3-4 days. At 7-10 days post seeding, microglia were removed from the astroglial monolayer by mechanical shaking. The astroglial cell monolayer was then trypsinized and reseeded to yield cultures highly enriched (90-95%) in astrocytes. Detached microgiia in the supernatant were pelleted by centrifugation and reseeded for subsequent experiments. Brain endothelial culture

Primary cultures of feline brain microvessels were established using slight modifications of a technique originally developed for culture of primate lymph node microvessels (Masinovsky et al., 1990). Cerebral cortices were prepared and digested in 0.2% trypsin as described for astrocyte cultures. The tissue fragments were dissociated by brief vortexing and the larger fragments were allowed to settle out by unit gravity sedimentation for 1-2 min; the supernatant, containing single cells plus brain microvessels, was then centrifuged at approximately 50 × g for 5 min to pellet the microvessels while leav-

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S.W. DOW ET AL.

ing single cells in suspension. The supernatant was discarded (or used for astrocyte culture) and the microvessels in the pellet were digested with 0.2% collagenase (Sigma) in medium with 1% FCS at 37°C for an additional 2-3 h with periodic agitation. The microvessels were then washed, pelleted, and resuspended in a small volume of endothelial growth medium (CS-C-0.55; Cell Systems, Kirkland, WA) or DME plus 10% FCS, supplemented with 50 /tg endothelial all growth factor (ECGF) m1-1 (Collaborative Biomedical, Bedford, MA) and 50 #g heparin m l - 1 (Sigma). The dissociated endothelial cells (usually clumps of 10-30 cells ) were allowed to adhere for 1 h to culture vessels that had been coated with 10/tg bovine plasma fibronectin ml-1 (Sigma); non-adherent cells were removed. Colonies of endothelial cells grew out from these microvessel fragments and were used for FIV infection experiments. One set of cultures yielded an outgrowth of several pure colonies of brain endothelial cells that could be propagated and passaged for up to 15 passages in vitro (S.W. Dow, unpublished results). These endothelial cells were used for experiments that required replating of cells. They were maintained in DME supplemented with 50 #g ECGF m l - 1 and 50/tg heparin m l - 1 or CS-C-0.55 medium (Cell Systems). The cells were passaged by brieftrypsinization and reseeded into wells that had been precoated with 10 #g fibronectin m l - 1.

FIV infection of neural cells by inoculation with cellfree virus Primary cultures of astrocytes and endothelial cells were detached from their original culture flasks by brief exposure to 0.25% trypsin and 0.1% EDTA (Gibco-BRL), pelleted by centrifugation at 400 × g, then reseeded at subconfluent density (approximately 5 × 105 cells per well) into individual wells of 6 well plates, and inoculated with virus the following day. Microglia that had been harvested by mechanical detachment were seeded at a similar density. The virus inoculum was 1 ml per 35 m m well for 6 well plates, or i00/A per well for 96 well plates, of supernatant from stock cultures of FIV-infected CrFK (approximately 1.5 × 103 FFU m1-1 ). Cells were incubated with the inoculum for 4 h at 37°C, then the inoculum was removed and replaced with medium. Cells were fixed and processed for IFA 4-5 days post inoculation (PI).

Co-culture of FIV infected lymphocytes with endothelial cells To assess the role of cell-to-cell contact in FIV infection of endothelial cells, we investigated whether FIV infected peripheral blood lymphocytes (PBL) might transmit the virus to endothelial cells more effectively than exposure to cell free virus. PBL were obtained from Cat 2546 by Ficoll gradient separation, stimulated with 5.0 pg ConA m l - 1 and co-cultured with lymphoblasts from uninfected cats. The PBL culture was tested twice weekly for FIV p26

NEUROTROPISM OF FELINE IMMUNODEFICIENCY VIRUS

27

antigen; once the culture became positive, additional uninfected lymphoblasts were added and cultured an additional week, when approximately 30% of lymphocytes were FIV infected as assessed by IFA. At this time, 2 X 106 total PBL were added to monolayer cultures of brain endothelial cells (in 35 mm wells) and co-cultured for an additional 4-5 days. The cells were then fixed and examined for FIV antigens by IFA.

FIV recoveryfrom infected brain tissue by culture A newborn cat (Cat 2243) was inoculated (0.25 ml tissue culture supernatant administered intracerebrally) with FIV-Petaluma and killed 6 months later. Primary regional brain cultures were established, using approximately 1 g of tissue each from cerebral cortex, cerebellum, caudate nucleus, thalamus, and brainstem. Cultures were prepared as described for astroglial cultures and seeded in poly-L-lysine coated 75 cm flasks. Cultures were monitored weekly for infection by FIV p26 ELISA for 1 month. Cells in positive cultures were passaged by trypsinization and replated. Infected cells were identified by dual label immunofluorescence for FIV antigens and cell specific antigens (see below ).

Identification of FIV antigens in infected cells and of neural antigens by IFA FIV infected cells were identified by immunostaining with a reference cat antiserum that recognizes all major FIV proteins. Cells were fixed in a 50/50 mixture of acetone and methanol at - 20 ° C for 5 min. The cell type was identified by staining with cell-type specific antisera: astrocytes (GFAP); endothelial cells (Factor VIII related antigen and uptake of low-density lipoprotein); fibroblasts (fibronectin). Microglia were identified by morphology, positive non-specific esterase activity, zymosan phagocytosis, uptake of acetylated low-density lipoprotein (Dow et al., 1990). Primary and secondary antibodies were diluted in IFA buffer (phosphate buffered saline (PBS), 1% bovine serum albumin, 5% heat-inactivated normal goat serum) (Sigma). Primary antibody dilutions were as follows: anti-FIV, 1 : 100; monoclonal antifibronectin (Sigma), 1:200; rabbit anti-Factor VIII related antigen (Dako, Santa Barbara, CA), 1 : 50; rabbit anti-GFAP (Dako, Santa Barbara, CA), 1 : 100. Primary antibodies were diluted in IFA buffer and incubated with the fixed cells for 45 min at room temperature, followed by two PBS washes for 15 min. Secondary antibodies were: goat anticat IgG, FITC conjugated (Kirkegaard and Perry, Gaithersburg, MD); goat antirabbit IgG, RITC conjugated (Chemicon, Temecula, CA); goat anti-mouse IgG, RITC conjugated (Boehringer-Mannheim, Indianapolis, IN); all were diluted 1:100 in IFA buffer and incubated for 30 min, followed by a final PBS rinse for 10 min. For dual label experiments, both primary antibodies were incubated to-

28

S.W. DOW ET AL.

gether, as were both secondary antibodies. Affinity purified and absorbed secondary antibodies were used in dual label experiments to minimize crossreactivity with cat immunoglobulins. For acetylated low-density lipoprotein uptake, cells were incubated with 20/A acetylated low-density lipoprotein mllabeled with Dil (Biomedical Technology, Stoughton, MA) for 4 h at 37°C, then fixed in 2% paraformaldehyde for 30 min at room temperature. After the final wash in PBS, wells were washed briefly in distilled water and coverslipped with a 50/50 mixture of PBS and glycerol. Wells were examined with a Nikon epifluorescence microscope equipped with interchangeable filters for both fluorescein and rhodamine excitation. Photomicrographs were taken with a Nikon camera on Ektacolor film.

Focal immunoassay to determine relative susceptibility to FIV infection Cells (astrocytes, CrFK, or endothelial cells) were seeded at 1 × 104 in individual wells of a 24 well plate, allowed to adhere overnight, and inoculated with 500/zl FIV-Petaluma (diluted to yield a titer of approximately 1.5 × 102 FFU m l - ~) for 4 h; the inoculum replaced with fresh medium, and cultured for 3 additional days. Wells were then fixed and stained with FIV reference serum as described for IFA, with the following modifications for immunoperoxidase staining: the secondary antibody was peroxidase conjugated goat anticat (IgG) (Organon Teknika, Durham, NC) diluted 1:200; after rinsing in PBS, the peroxidase substrate was developed by addition of aminoethyl carbazole (Sigma) plus 0.1% H202 for 10 min. Wells were scored by counting ten random fields and the average number of FIV positive foci per field was determined for triplicate wells. Controls included infected wells reacted with non-immune cat serum and uninfected wells reacted with immune serum.

FIV p26 antigen production by astrocytes, microglia, and brain endothelial cells Plates (96 wells) were seeded in quadruplicate with 5× 104 freshly detached (microglia) or trypsinized (astrocytes, endothelium) cells, which were allowed to adhere for 4 h. The wells were then inoculated with 100 pl FIV2546 supernatant for 4 h, rinsed twice with PBS, then 150/tl medium were added to each well. At 3 day intervals for 12 days PI, 100 #1 supernatant were removed from each well and replaced with 100/tl of flesh medium. Supernatants were assayed for p26 antigen by FIV p26 antigen ELISA. Optical density readings were converted to ng p26 by comparison with a standard curve generated with immunoaffinity purified FIV p26 antigen (Dreitz et al., 1992 ).

NEUROTROPISM OF FELINE IMMUNODEFICIENCY VIRUS

29

RESULTS

Characterization offeline neural cells in primary culture Astrocytes Feline astrocytes proliferated after passage with trypsin and assumed a stellate morphology in subconfluent culture. They were strongly positive for GFAP reactivity (Fig. 1 (a)) and were negative for fibronectin (not shown ). These cells could be passaged five to ten times without apparent decrease in survival or GFAP staining, although contaminating fibroblasts (fibronectin positive) tended to overgrow cultures after repeated passage.

Microglia Microglia could be highly enriched ( > 99%) by using mechanical agitation to separate them from underlayer cells and then pelleting from the supernarant. When replated, these cells rapidly adhered to plastic (Fig. 1 (c)). Over several days in culture, the cells began to spread out and assume a more flattened and irregular outline, with some microglia becoming multinucleate. We have found that these cells have properties typical of macrophages, including non-specific esterase activity (Fig. 1 (c)), phagocytosis of acetylated lowdensity lipoprotein, phagocytosis of zymosan, generation of respiratory burst activity, and release of interleukin- 1 (IL- 1), IL-6 and tumor necrosis factor (Dow, unpublished data, 1991 ).

Endothelial cells Feline brain capillary endothelial cells assumed a cobblestone morphology when grown to confluence. These cells phagocytosed acetylated low-density lipoprotein (Fig. I (e)) and were positive for Factor VIII related activity (not shown). They were more slow growing than astrocytes and required a fibronectin substrate and ECGS for optimal proliferation; CS-C-0.55 medium provided optimal growth relative to other media evaluated. Proliferative capacity began to decrease after 15 passages.

Identification of infected cells by IFA Feline immunodeficiency virus infection of microglia was persistent and non-cytopathic (Fig. 1 (d) ). Although syncytia developed in infected microglia several days PI, uninfected cultures also developed syncytia at a similar rate. By contrast, FIV infected astrocytes developed large syncytia (Fig. 1 (b)) within 48 h of inoculation and began to die within 72 h. Whereas FIV generally infected 80-90% of astrocytes within 4-5 days of inoculation, the number of infected microglia after this same interval rarely exceeded 30%. FIV-Petaluma and FIV-2546 both infected astrocytes and microglia, as did

30

S.W.DOWET AL.

Fig. 1. Dual label immunofluorescence staining of syncytium formation in FIV infected astrocytes 4 days PI. Astrocytes in (a) are positive for GFAP (rhodamine optics) and the same cells are also positive for FIV antigens in (b) (fluorescein optics). Freshly reseeded feline microglia in (c) are positive for non-specific esterase activity. In (d), infected microglia are positive for FIV antigens 10 days PI. (e) Feline brain capillary endothelial cell line, uniformly positive for phagocytosis of Dil-labeled acetylated low-density lipoprotein. (f) Rare FIV-infected brain capillary endothelial cell, 5 days after FIV inoculation. ( ( a ) - ( e ) X 500. )

NEUROTROPISM

OF FELINE IMMUNODEFICIENCY

VIRUS

31

eight other FIV isolates that we have evaluated (Dow, unpublished data, 1991). In all cases, astrocyte infection was associated with syncytium formation. After inoculation with either FIV-Petaluma or FIV-2546, occasional ( < 5%) infected brain capillary endothelial cells could be identified (Fig. 1 (e)), along with small syncytia that did not progress to cell death.

Relative susceptibility of astrocytes and endothelial cells to FIV infection By focal immunoassay, astrocytes were found to be approximately seven times more susceptible to infection by FIV-Petaluma than endothelial cells (Fig. 2 ). Astrocytes were three times less susceptible to infection than CrFK, the cells in which FIV-Petaluma was propagated. To assess the role of cell-to-cell contact in the entry of FIV into the CNS, FIV infected lymphocytes were co-cultured with brain capillary endothelial cells. Although numerous FIV infected lymphocytes were observed adhering to brain endothelial cells by immunofluorescence examination, infected endothelial cells were not observed. This experiment has also been repeated with five other FIV isolates with similar results (data not shown). Thus, it appears that brain capillary endothelial ceils are relatively resistant to FIV infection, whether virus contact occurs via infected lymphocytes or cell-free virus.

5.0' m

4.0"

3.0" E -,

2.0"

C C 111 0

1.0"

0

. CrFK

0

~ Astrocyte

Brain endothelium

Cell type Fig. 2. Results of the focal immunoassay for determination of relative susceptibility to FIV infection. Astrocytes, endothelial ceils, and CrFK were inoculated with FIV-Petaluma, then immunostained 3 days later and the average number of FIV positive foci per well quantitated. Results represent the mean of triplicate wells for each cell type, with the standard deviation.

S.W. DOW ETAL.

32

Comparison of FIV antigen release by infected neural cells Production of FIV p26 gag antigen was measured to compare the relative ability of three neural cells to produce virus after inoculation with FIV-2546 (Fig. 3). (FIV p26 gag concentrations as measured with this ELISA have been shown to correlate directly with reverse transcriptase activity (Dreitz et al., 1992).) Astrocytes began releasing high levels of p26 within 6 days of inoculation, whereas p26 production by microglia was low until 12 days PI. Brain endothelial cells did not release detectable p26 throughout the 12 day PI period. Thus, it appeared that astrocytes were the most permissive CNS G

:

600 ' /

fi 400 /

u.

a

Astro

...... t .....

Micro

. . . . . •A. .....

Endoth

200

....

0

5

0 Days

post

_.

10 inoculation

Fig. 3. FIV p26 production by astrocytes, microglia, and endothelial cells. Quadruplicate wells of a 96 well plate of each cell type were inoculated with FIV-2546 for 4 h, rinsed with PBS, and 150 #1 fresh medium added to each well; 100/tl aliquots were sampled at 3 day intervals for 12 days (and replaced with 100/zl fresh medium) and the p26 concentration in each sample determined by ELISA. Each point is the mean of quadruplicate p26 determinations. TABLE1 Results of in vitro experiments CNS cell

FIV susceptibility

Virus effects

Astrocyte Microglia Endothelium Oligodendrocyte Neuron (hippocampus)

+++ + + - /+ -

Syncytia, cell death Persistent infection None/small syncytia None Cell death

NEUROTROPISM OF FELINE IMMUNODEFICIENCYVIRUS

33

cell to FIV infection, followed by microglia, whereas endothelial cells were resistant to productive infection. Table 1 summarizes the results of in vitro experiments on feline neural cell susceptibility to FIV infection and effects of FIV infection.

FIV recovery from cultured CNS tissue Of the brain tissues from Cat 2243 that were cultured, FIV-Petaluma was recovered only from cerebellum and caudate nucleus. Cultured cells from caudate nucleus consisted of approximately 50% astrocytes and 50% fibroblasts, as identified by positive immunofluorescence staining for GFAP and fibronectin, respectively. When cells were dual labeled to identify FIV infected cells, nearly all astrocytes were FIV positive, whereas fibroblasts were not infected (not shown). Interestingly, syncytium formation by the infected astrocytes was not observed and the cells could be passaged multiple times, suggesting that these cells had adapted to FIV infection such that infection no longer induced cytopathic effects. Microglia were only present in low numbers during the first week of culture and did not survive passage; thus, we were unable to determine the level of microglial infection. DISCUSSION

In vitro culture of feline neural cells has allowed us to address several important questions regarding FIV neuropathogenesis, including relative susceptibility to infection and the effects of infection on cell survival. Results from these studies indicate that the primary CNS target cells for FIV infection are astrocytes and microglia. Of the two, astrocytes are apparently the most permissive to FIV infection and replication. In addition, we were able to recover FIV from primary cultures of brain tissue from an experimentally infected cat and identified the infected cell type as astrocytes. Although astrocytes appeared more susceptible to FIV infection than did microglia, based on percentage of cells infected and level of p26 gag antigen production, microglia are likely to play a more important role in FIV infection in vivo. Because microglia are able to remain persistently and non.-cytopathically infected, they may serve as a reservoir for infection within the CNS. In addition to releasing potentially harmful cytokines, lentivirus infected macrophages may also elaborate neurotoxic peptides (Giulian et al., 1990). For these reasons, further studies of FIV-microglial interactions are currently in progress in our laboratory. Brain capillary endothelial cells were found to be relatively resistant to infection with both cell-free and lymphocyte-associated FIV. Therefore, direct infection of endothelium probably does not serve as an important route for FIV entry into the CNS. However, this does not preclude a role for migration

34

s.w. DOW ET AL.

of infected lymphocytes across brain endothelium with subsequent infection of neural cells. FIV infected mononuclear cells have been observed in perivascular infiltrates within the CNS of FIV infected cats (Dandekar, personal communication, 1991 ). Previous data indicate that two other important CNS cells, oligodendrocytes and neurons, are also relatively resistant to FIV infection (Dow et al., 1990). By dual label immunocytochemistry, we found earlier that oligodendrocytes and hippocampal neurons were not productively infected after FIV inoculation. Although cultured neurons were not productively infected after inoculation, recent preliminary data suggests that FIV may still exert a neurotoxic effect on cultured feline hippocampal neurons, leading to neuronal death within 72 h PI (Dow, unpublished observations, 1991 ). These observations are consistent with the reported neurotoxicity of HIV envelope glycoprotein (gp 120) for cultured rodent and chicken neurons (Dreyer et al., 1990). Thus, it remains plausible that soluble virus envelope glycoproteins may induce neurotoxicity and neuronal dysfunction although the target cell is incapable of supporting a productive viral infection. Since infection of neurons and oligodendrocytes is very rare in most lentiviral CNS infections, neurologic dysfunction is likely to be mediated indirectly, possibly as the result of aberrant cytokine synthesis and release or by circulating viral proteins, particularly envelope glycoproteins. Using the neural culture system described here, additional studies are underway to investigate the effects of FIV infection on microglial and astrocyte function. The results of these studies may provide important insight into the mechanisms of lentivirus-induced neurologic dysfunction. ACKNOWLEDGMENTS This work was supported by grants NO1 AI 85007 and K11 AI 00952 from the National Institutes of Health and by a grant from the College Research Council, Colorado State University. The authors wish to acknowledge the expert technical assistance of Dr. Lynne O'Neil and Matt Myles and Esta Moutoux for secretarial assistance.

REFERENCES Dow, S.W., Poss, M.L. and Hoover, E.A., 1990. Feline immunodeficiencyvirus: a neurotropic retrovirus. J. AIDS, 3: 658-668. Dow, S.W., Dreitz, M.J. and Hoover, E.A., 1992. In press. Dreyer, E.B., Kaiser, P.K., Offermann, J.T. and Lipton, S.A., 1990. HIV coat protein neurotoxicity prevented by calcium channel antagonists. Science,248: 364-367. Georgsson, G., Houwers, D.J., Palsson, P.A. and Petursson, G., 1989. Expressionof viral anti-

NEUROTROPISMOF FELINEIMMUNODEFICIENCYVIRUS

35

gens in the CNS of visna-infected sheep: an immunohistochemical study on experimental visna induced by virus strains of increased neurovirulence. Acta Neuropathol., 77: 299-306. Koenig, S., Gendleman, H.E. and Orenstein, J.M., 1986. Detection of AIDS virus in macrophages in brain tissue from AIDS patients. Science, 233: 1089-1093. Lackner, A.A., Smith, M.O., Munn, R.J., Martfeld, D.J., Gardener, M.B., Marx, P.A. and Dandekar, S., 1991. Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys. Am. J. Pathol., 139: 609-621. Masinovsky, B., Urdel, D. and Gallatin, M., 1990. IL-4 acts synergistically with IL-1B to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-l. J. Immunol., 145: 2886-2895. McArthur, J.C., 1987. Neurologic manifestations of AIDS. Medicine, 66: 407-423. Sitban, M., Nishio, J., Wehrly, K., LodmeU, D. and Cheesebro, B., 1985. Use of a focal immunofluorescence assay on live cells for quantitation of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual tropic murine leukemia viruses. Virology, 141: 110-118. Wheeler, D.W., Mitchell, T.W., Gasper, P.W., Barr, M.C. and Whalen, L.R., 1991. FIV infection associated with neurologic abnormalities. Proceedings of First International Conference of Feline Immunodeficiency Virus Researchers, Davis, CA, p. 31.

Veterinary Immunology and Immunopathology, 35 (1992) 37-49

37

Elsevier Science Publishers B.V., Amsterdam

Detection of feline immunodeficiency virus infection in bone marrow of cats A m y M. B e e b e a, T o b i e G. G l u c k s t e r n a, J e a n n e G e o r g e b, N i e l s C. P e d e r s e n b a n d Satya D a n d e k a r ~

"Department of Internal Medicine, School of Medicine, University of California, Davis, CA 95616, USA bDepartment of Medicine, School of VeterinaryMedicine, Universityof California, Davis, CA 95616, USA

ABSTRACT Beebe, A.M., Gluckstern, T.G., George, J., Pedersen, N.C. and Dandekar, S., 1992. Detection of feline immunodeficiencyvirus infection in bone marrow of cats. Vet. Imrnunol. Immunopathol., 35: 3749. Natural or experimental feline immunodeficiencyvirus (FIV) infection in cats is often associated with hematologic abnormalities which are similar to those observed in human immunodeficiency virus (HIV) infected patients. To determine if cells in bone marrow are infected with FIV and whether severity of hematopoietic disorder is correlated with the level of viral infection, bone marrow tissues from ten experimentally and two naturally FIV infected cats were examined by in situ hybridization for presence of FIV RNA. Seven of the 12 FIV infected cats were also naturally or experimentally coinfected with feline leukemia virus (FeLV). FIV RNA was detected mainly in megakaryocytes and unidentified mononuclear cells in the bone marrow of cats that were sick and had marrow hypercellularity and immaturity. These included all cats in the acute phase of FIV infection and two of seven long term FIV infected cats. One long term FIV infected cat with lymphosarcoma was also positive for FIV RNA in bone marrow cells. The other four long term FIV infected cats were relatively healthy, with normal bone marrow morphology, and were negative for FIV infected cells. Bone marrow from three non-infected and two cats infected with FeLV alone were also negative for FIV RNA by in situ hybridization. We concluded that megakaryocytes and mononuclear cells were targets of the viral infection and that the presence of FIV RNA in cells of the bone marrow correlated with marrow hypercellularity and immaturity, and severity of illness. ABBREVIATIONS BFU-E, erythroid burst forming units; CFU-E, erythroid colony forming units; CFU-GM, granu-

Correspondence to: Satya Dandekar, Division of Infectious and Immunologic Diseases, Department of Internal Medicine, School of Medicine, Bldg. MS-1A Rm 3456, University of California, Davis, CA 95616, USA. Phone: (916) 752-3542 or 752-6128.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

38

A.M. BEEBE ET AL.

locyte macrophage colony forming units; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; SPF, specific pathogen free.

INTRODUCTION

Feline immunodeficiency virus (FIV) is a lentivirus (Pedersen et al., 1987) that causes immunosuppressive disease in cats which is similar to the acquired immune deficiency syndrome (AIDS) seen in human immunodeficiency virus (HIV) infected patients (Ishida et al., 1992). Hematologic abnormalities are a prevalent feature of FIV infection in cats. Following experimental FIV infection, cats experience a primary acute phase of illness followed by a long asymptomatic stage (Yamamoto et al., 1988; Torten et al., 1991 ). Naturally infected cats progress to clinical illness (Ishida et al., 1992 ). Abnormalities in blood and bone marrow have been observed in both the primary and terminal phases of illness (Hopper et al., 1989; Shelton et al., 1990; Mandell et al., 1992). Common blood abnormalities include leukopenia, neutropenia, lymphopenia, and anemia. Abnormalities in the bone marrow of symptomatic cats with FIV infection include hyperplasia, dysplasia and excess plasma cells and eosinophils (Hopper et al., 1989; Shelton et al., 1990; Mandell et al., 1992). The pathogenic mechanisms of these blood and marrow abnormalities have not been elucidated. FIV can infect a variety of cells in vitro, including feline T-lymphocytes, fibroblasts, glial cells and macrophages (Brunner and Pedersen, 1989; Miyazawa et al., 1989; Phillips et al., 1990; Tochikura et al., 1990). However, it has not been determined whether bone marrow cells can also be infected. This information is crucial for determining the role of FIV infection in the pathogenesis of blood and bone marrow abnormalities in FIV infected cats. The objectives of the present study were to determine if bone marrow was infected with FIV in vivo and whether bone marrow abnormalities and disease severity correlated with viral expression. The cats selected for this study were both naturally and experimentally infected with FIV, and many of them were also coinfected with feline leukemia virus (FeLV). Pre-existent FeLV infection increases the severity of FIV induced disease and the level of FIV replication in an experimental setting (Pedersen et al., 1990). This same disease synergism between FIV and FeLV infection also occurs in nature (Cohen et al., 1990; Moraillon, 1990). Although pre-existent FeLV infection enhances the level of FIV infection in many tissues, the FIV infection has no enhancing effect on FeLV replication (Pedersen et al., 1990). Therefore, asymptomatic FeLV carrier cats can be useful in potentiating FIV induced disease. Bone marrow tissues were examined for the presence of FIV infected cells by in situ hybridization, using an FIV DNA probe to detect viral RNA in individual cells within the tissue. This technique is useful for identifying cells that have been activated to produce viral RNA, as opposed to latently

FIV INFECTION IN BONE MARROW

39

infected cells. Histopathological changes in the bone marrow were examined in relation to FIV infection and clinical manifestations of the disease. MATERIALS AND METHODS

Animals and viral infections Two cats (nos. 31 and 90R335 ) which were naturally FIV infected were acquired through the Veterinary Medical Teaching Hospital, University of California, Davis. One of these two cats (no. 90R335 ) was also positive for FeLV by p27 antigen ELISA. Specific pathogen free (SPF) domestic cats were obtained from the breeding colony of the Feline Retrovirus Research Laboratory, University of California, Davis, and were housed in facilities of the Animal Resource Services. Four SPF cats were infected with FIV, two with FeLV, and six with both FIV and FeLV as described below. TABLE 1

Retroviral infection of cats Cat no.

Sex

Retrovirus infection status

Age (months) at time of: FeLV

FIV

Death

3156 91003 91006

F M M

Non-infected Non-infected Non-infected

-

-

49 9 9

3400 3410

M M

FeLV FeLV

7 5

-

15 15

31

M

FIV natural

-

t

67 68 2841 3394

M M F M

FIV FIV FIV FIV

-

12 12 3 15

x+12 18 18 5 17

90R335 3378 3386 3389 3397 3403 3404

F F F F F M F

FeLV + FIV natural FeLV+FIV 3 FeLV+FIV FeLV+FIV FeLV + F I V FeLV + FIV 3 FeLV + F I V

221 8 10 10 7 7 7

52 12 14 14 11 11 11

24 29 16 16 24 36 13

-

~Age at which tested positive for FeLV by ELISA. 2Age at which tested positive for FIV by IFA; tested negative 18 months later.

3Subsequently infected with

Haemobartonellafelisand recovered

from acute illness.

F, female; M, male; t , age unknown; x, age at which tested positive for FIV by Western blot.

40

A.M. BEEBEET AL.

In a previously reported study (Pedersen et al., 1990), SPF cats were experimentally infected with the CT600 and Rickard strains of FeLV. Six of the cats that remained persistently antigenemic for 4 months were then infected with FIV by intravenous inoculation of 0.5 ml of whole heparinized blood from Cat 39, a chronic carrier of the Petaluma strain of FIV. Four SPF cats were infected with FIV alone (nos. 67, 68, 2841, and 3394). Cat 3394 was inoculated with FIV as described above. Cat 2841 was inoculated intraperitoneally with whole blood from Cat 2429, a cat infected experimentally with the Petaluma strain of FIV (Yamamoto et al., 1988). Cats 67 and 68 were inoculated intraperitoneally with pooled whole blood from naturally FIV infected cats. A summary of the retroviral infection of the cats is given in Table 1. Blood and bone marrow evaluation Complete blood counts were performed by standard methods on most of the cats. Results were compared with reference values for SPF cats from our laboratory, and classified as either normal, high or low. Cats were killed with an intravenous overdosage of barbiturate (Pedersen et al., 1990). Bone marrow was fixed in an isotonic solution of 10% formalin in neutral phosphate buffer (Carson et al., 1973 ), embedded in paraffin, cut into 3-5/zm sections and placed on siliconized glass slides. Marrows were evaluated for cellularity, degree of maturation in the granulocytic and erythroid series, presence of megakaryocytes, and abnormalities of other bone marrow elements. Four categories were formed for bone marrow cellular patterns: normal, granulocytic hyperplasia with shift to immaturity (granulocytic dysplasia), myelogenous leukemia, myelofibrosis. Detection of F I V RNA by in situ hybridization The in situ hybridization procedure was modified from the technique of Brahic and Haase ( 1978 ). Bone marrow slides were deparaffinized in xylene then hydrated through graded ethanol to phosphate buffered saline (PBS). Hydrated tissues were treated with 1/tg proteinase K m l - 1 in 100 mM TrisHC1/50 mM EDTA, pH 7.4, for 15 min at 40°C, rinsed in PBS, incubated 15 min in 0.2 M Tris/0.1 M glycine, then dehydrated through graded ethanol and air dried. A 9.2 kb FIV genomic DNA fragment containing the entire gag, pol, and env regions was radioactively labeled by nick translation (Amersham N. 5000) to generate an FIV DNA probe. The probe was used at 3 × 106 counts m i n - 1 100/zl in a hybridization solution containing 50% deionized formamide, 10% dextran sulfate, 50 mM NaH2PO4, 0.6 M NaC1, 0.5 mM EDTA, 1 ×Denhardt's solution, 75/zg Escherichia coli tRNA m1-1, 100 #g salmon sperm DNA ml -~, and 20 mM dithiothreitol. The probe solution was denatured for 10 min at 95 ° C, cooled on ice for 5 rain, and 100-120/tl were placed

FIV INFECTION IN BONE MARROW

41

on each slide. Slides were coverslipped and incubated at 40 °C overnight in a humidification chamber. Slides were washed in 50% formamide/2 × SSC at 40°C, followed by 2 X SSC at 37 °C, dehydrated through graded ethanol containing 300 mM ammonium acetate, then air dried. Dried slides were dipped in NTB2 emulsion (Eastman Kodak, Rochester, NY) that had been diluted 1 : 2 in 600 mM ammonium acetate. Slides were exposed for 6-18 clays at 4 °C then developed in Kodak D 19 developer for 5 min, stopped in 2% acetic acid for 1 rain, and fixed with Kodak Rapid Fix for 5 min. Overnight staining with Giemsa stain (National Aniline Certified Giemsa Powder) was followed by differentiation in 2% acetic acid (2 X 1 min) and absolute ethanol (2 × 30 s). Each in situ hybridization experiment included controls of FIV infected and non-infected Crandell feline kidney cells. RESULTS

Clinical signs Clinical and pathologic findings for most of the experimentally infected cats have been reported previously (Yamamoto et al., 1988; Pedersen et al., 1990). Clinical signs are summarized in Table 2. Five cats were killed in the acute phase of illness. All of these cats were very sick and considered terminal. Of the seven long term FIV infected cats, only two were killed because of impending death. One (no. 3378) had lymphosarcoma, and four others were considered healthy. The two cats experimentally infected with FeLV alone had clinical signs distinct from those of the FIV and FIV/FeLV infected cats. Cat 3400 had marked anemia while Cat 3410 had bilateral panopthalmitic glaucoma and fever. Blood and bone marrow evaluation Four of the five cats in the acute phase of FIV infection were neutropenic, while one had neutrophilia (Table 3 ). Of four long term FIV infected cats tested, three had neutrophilia, and one had normal neutrophil counts. Immature neutrophils (bands or earlier) were not increased in any of the hemograms. The results of bone marrow evaluations are summarized in Table 3. The two cats infected with FeLV alone had myelofibrosis, an abnormality associated with that virus. Cat 2841, evaluated as having acute myelogenous leukemia, was previously reported to have myeloproliferative disease (Yamamoto et al., 1988 ). Four FIV infected cats had normal marrows, while seven had granulocytic myelodysplasia characterized by marrow hypercellularity and

+

+

+

.

+

3397

3403

3404

.

.

-

.

-

+

+

-

.

+

-

.

-

.

.

.

.

.

+

+

+

+

+

+

.

+

+

.

.

.

.

.

.

. . .

3Lymphosarcoma

that did not involve peripheral lymph nodes.

-

-

-

.

.

.

-

+

+

+

_

-

+

.

.

.

.

.

dration

dehy-

.

Lymphad-

.

+

+

+

+

.

_3

+

+

+

.

+

.

.

.

.

.

.

.

.

.

.

-

-

-

+ .

+

+

.

-

.

.

.

signs

Resp.

.

.

.

.

GI

.

.

.

+

+

+

+

-

+

-

.

.

compl,

Neuro., neurologic.

.

enopathy

Derm., dermatologic;

+

+ +

-

-

.

.

.

.

loss

sion

infected.

.

.

.

.

.

Weight

Depres-

2Resp., respiratory; GI compl., gastrointestinal complications;

.

.

Anorexia

1nat., n a t u r a l l y i n f e c t e d ; e x p t . , e x p e r i m e n t a l l y

FeLV + FIV expt.

3389

+

3394

3386

+

2841

-

.

68

90R335 3378

.

-

31

67

FIV nat.

FIV expt.

FeLV + FIV nat.

+

.

91006

-

.

3410

.

3156

Fever

C l i n i c a l signs 2

91003

Cat no.

3400

FeLV expt.

Non-infected

Infection ~

Clinical observations

TABLE 2

.

.

.

.

.

.

.

.

-

-

-

-

+

-

.

+

.

.

.

.

.

.

.

.

Gingivitis

.

.

.

Eye

-

+

+

-

+

+

-

+

.

.

.

lesion

Derm.

-

-

-

+

+

+

-

D

lesion

+

B

B

R

disorder

Neuro.

t ' ~

526 511 59 66 378 1089 46

3400 3410

31 67 68 2841 3394

90R335 3378 3386 3389 3397 3403 3404

FeLV expt.

FIV nat. FIV expt.

+ + + + + + + + +

+ + + +

-

-

Mononuclear cells

FIV in situ 2

+ + + + +

+ + +

-

-

-

-

-

m

Megakaryocytes

Granulocytic dysplasia (mild) Normal Normal Myelogenous leukemia Granulocytic dysplasia

Elective Elective Elective Illness Illness

Granulocytic dysplasia Normal Granulocytic dysplasia Granulocytic dysplasia Granulocytic dysplasia Normal Granulocytic dysplasia (mild)

Myelofibrosis Myelofibrosis

Illness Elective

Illness Lymphosarcoma Illness Illness Illness Elective Illness

Normal Normal Normal

Bone marrow evaluation

Elective Elective Elective

Reason for necropsy

1nat., naturally infected; expt., experimental infected. 2 + , < 4 positive cells per 10 × field; + + , 4-20 positive cells per l 0 X field; + + + , > 400 positive cells per l0 X field. aNT, not tested. Neutrophil counts: normal = 1.94-11.26 X 103/zl- 1. Hb ( hemoglobin ): normal = 10.6 l - 16.42 g d l - 1.

FeLV + FIV nat. FeLV + FIV expt.

335 119 121 63 59

3156 91003 91006

Non-infected

240 299

Cat no.

Infection I

Days after infection

Bone marrow infection and hematologic data

TABLE 3

NT NT NT Low Normal NT Normal Normal LOw Normal Low Low Normal Normal Normal Normal Normal

Normal Normal NT High Normal LOw Low NT High Low High High Normal LOw

a

NT NT NT

counts

Hb

cell

Neutrophils

Blood

4~

K

o z

"11 I'M

,<

4~

O

m

M

46

A.M. BEEBEETAL.

shift to immaturity. This lesion was found in both neutropenic and neutrophilic cats.

Detection of FIV RNA in bone marrow by in situ hybridization FIV RNA was detected by in situ hybridization in bone marrow cells of eight of 12 FIV infected cats (Table 3, Figs. 1 ( a ) - 1 (d) ). FIV RNA was found in megakaryocytes and unidentified mononuclear cells (Table 3, Figs. 1 ( b ) 1 ( d ) ) . In one dually infected cat, no. 3397, lymphoid follicles were present. Although this cat had extremely high levels of FIV RNA throughout the bone marrow, cells within its lymphoid follicles showed no hybridization of probe (Fig. 1 ( d ) ) . FIV RNA-containing cells were more frequently found in FIV infected or FIV/FeLV coinfected cats that had granulocytic myelodysplasia and severe illness (Table 3, Figs. 1 ( b ) - I ( d ) ) . Viral RNA was usually undetectable in bone marrow cells of FIV infected cats that had normal bone marrow histology and were not sick at the time of euthanasia. Cat 3378 was an exception. This cat had lymphosarcoma without bone marrow involvement. Despite normal marrow histology, infected cells were seen in both megakaryocytes and unidentified mononuclear cells in this cat. FIV RNA was not detected in bone marrow cells from non-infected SPF or FeLV infected cats. FIV infected Crandell feline kidney cells were included as positive controls for in situ hybridization. A scattered background of 1-2 grains per cell was found on all tissues, contrasting with distinct, well-defined clusters of grains over infected cells in the positive controls. DISCUSSION

The objective of this study was to correlate hematologic abnormalities that were seen in FIV infected cats and virus infection of bone marrow cells. FIV infected megakaryocytes and unidentified mononuclear cells were observed in the marrow of FIV and F I V / F e L V infected cats. Although the megakaryocytes in the present study were easily recognized through the overlying silver grains, it was not possible to definitively identify the infected mononuclear cells. They appear to have the morphology of either monocytes/macrophages or lymphocytes, but this remains to be determined with double staining techniques. Presence of FIV RNA in bone marrow cells appeared to be related to severity of disease and granulocytic myelodysplasia in long term infected cats. Healthy long term FIV infected cats had no infected cells in their bone mar-

Fig. 1. Detection of FIV RNA in bone marrow cells. In situ hybridization was performed on bone marrow tissues from: (a) healthy FIV infected Cat 67; (b) sick FIV infected Cat 2841; (c) sick FIV infected Cat 3394; (d) sick FIV/FeLV coinfected Cat 3397. Bar= 200/~m.

FIV INFECTION IN BONE MARROW

47

row. FIV RNA was present in bone marrow cells of all acute phase cats in this study. All of these cats were critically ill and had marrow abnormalities at the time of sample collection. Even though there appeared to be a relationship between the presence of hematologic abnormalities and FIV RNA in the bone marrow, the vast majority of bone marrow cells were apparently non-infected even in the sickest animals. Leukopenia, usually owing to an absolute neutropenia with more variable declines in lymphocyte counts, was the most florid hematologic abnormality observed in FIV or FIV/FeLV infected cats. The leukopenia was usually associated with granulocytic myelodysplasia characterized by hypercellularity of the bone marrow and a shift towards immature progenitor cells. No accompanying increase of neutrophilic precursors in the peripheral blood was observed. The pathogenic mechanisms responsible for these abnormalities have not been elucidated. The infection of relatively few cells in the bone marrow without extensive infection of myelopoietic precursors is evidence that the hematologic abnormalities observed in both primary and terminal stages of FIV infection are both complex and indirect in origin. Ineffective granulopoiesis, resulting from virus induced changes in the hematopoietic microenvironment at times of high viral load is one possible mechanism. Alternatively, immune mediated destruction of neutrophils in the bone marrow may be responsible. Supporting the fact that bone marrow precursor cells are not affected in a direct manner by FIV infection, Linenberger et al. ( 1991 ) have shown that bone marrow from asymptomatic, FIV infected cats is normal in numbers of granulocyte macrophage colony forming unit (CFU-GM), erythroid burst forming unit (BFU-E), and erythroid colony forming unit (CFUE) and responsiveness to growth factors. Furthermore, sera from these asymptomatic cats supported the growth of bone marrow progenitor cells as well as sera from non-infected cats, negating the presence of any soluble inhibitors. However, based on our study, we would expect that the bone marrow of such cats would be largely free of virus. In another study (Shelton et al., 1989), an FIV infected neutropenic cat was shown to also have normal frequencies of CFU-E, BFU-E, and CFU-GM progenitors. However, CFUGM colonies were decreased in the presence of sera from this neutropenic FIV infected cat. This would be consistent with the presence of a soluble inhibitor factor in cats with advanced disease rather than extensive destruction by infection of progenitors with FIV. The finding of FIV mainly in a small proportion of megakaryocytes and mononuclear cells in the bone marrow is not unique to the feline disease. HIV also replicates in a small number of megakaryocytes and mononuclear cells in the marrow of infected people (Zucker-Franklin and Cao, 1989; Sun et al., 1989; Stutte et al., 1990). Investigation of the mechanism of hematologic dysfunction in the face of relatively low levels of HIV infection in the bone marrow has yielded conflicting results (Donahue et al., 1987; Leiderman et al.,

48

A.M. BEEBE ET AL.

1987; Stella et al., 1987; Folks et al., 1988; Ganser et al., 1990). This may be a result of bone marrow suppressive therapies used for combating the HIV infection itself or of AIDS-related opportunistic infections. Based on the results of this and other papers, FIV infection of cats may be a good model for studying pathogenesis of hematopoietic abnormalities in AIDS. ACKNOWLEDGMENTS

We thank Dale Martfeld, Kim Floyd-Hawkins, and Nancy Delemus for excellent technical assistance and Bruce Rideout for performing necropsies. This study was supported by grant number AI30377 from the National Institutes of Health and by the University-wide AIDS Task Force from the University of California. REFERENCES Brahic, M. and Haase, A.T., 1978. Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc. Natl. Acad. Sci. USA, 75:6125-6129. Brunner, D. and Pedersen, N.C., 1989. Infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus. J. Virol., 63 ( 12 ): 5483-5488. Carson, F.L., Martin, J.H. and Lynn, J.A., 1973. Formalin fixation for electron microscopy: a re-evaluation. Am. J. Clin. Pathol., 59: 365-373. Cohen, N.D., Carter, C.N., Thomas, M.A., Lester, T.L. and Eugster, A.K., 1990. Feline immunodeficiency virus infection and feline leukemia virus seropositivity. J. Am. Vet. Med. Assoc., 197: 220-225. Donahue, R.E., Johnson, M.M., Zon, L.I., Clark, S.C. and Groopman, J.E., 1987. Suppression of in vitro haematopoiesis following human immunodeficiency virus infection. Nature, 326(6109): 200-203. Folks, T.M., Kessler, S.W., Orenstein, J.M., Justement, J.S., Jaffe, E.S. and Fauci, A.S., 1988. Infection and replication of HIV-1 in purified progenitor cells of normal human bone marrow, Science, 242(4880): 919-922. Ganser, A., Ottmann, O.G., yon Briesen, H., Volkers, B., Rubsamen, W.H. and Hoelzer, D., 1990. Changes in the haematopoietic progenitor cell compartment in the acquired immunodeficiency syndrome. Res. Virol., 141 (2): 185-193. Hopper, C.D., Sparkes, A.H., Gruffydd-Jones, T.J., Crispin, S.M., Muir, P., Harbour, D.A. and Stokes, C.R., 1989. Clinical and laboratory findings in cats infected with feline immunodeficiency virus. Vet. Rec., 125: 341-346. Ishida, T., Taniguchi, A., Matsumura, S., Washizu, T. and Tomoda, I., 1992. Long-term clinical observations on feline immunodeficiency virus infected asymptomatic carriers. Vet. Immunol. Immunopathol., 35:15-22. Leiderman, I.Z., Greenberg, M.L., Adelsberg, B.R. and Siegal, F.P., 1987. A glycoprotein inhibitor of in vitro granulopoiesis associated with AIDS. Blood, 70 (5): 1267-1272. Linenberger, M., Shelton, G.H., Persik, M.T. and Abkowitz, J.L., 1991. Hematopoiesis in asymptomatic cats infected with feline immunodeficiency virus. Blood, 78 (8): 1963-1968. Mandell, C.P., Sparger, E.E., Pedersen, N.C. and Jain, N.C., 1992. Long term hematologic changes in cats experimentally infected with feline immunodeficiency virus (FIV). 1992 Comp. Hematol. Int., 2: 8-17. Miyazawa, T., Furuya, T., Itagaki, S., Tohya, Y., Takahashi, E. and Mikami, T., 1989. Estab-

FIV INFECTION IN BONE MARROW

49

lishment of a feline T-lymphoblastoid cell line highly sensitive for replication of feline immunodeficiency virus. Arch. Virol., 108:131-135. Moraillon, A., 1990. Feline immunodepressive retrovirus infections in France. Vet. Rec., 126: 68-69. Pedersen, N.C., Ho, E.W., Brown, M.L. and Yamamoto, J.K., 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science, 235(4790): 790-793. Pedersen, N.C., Torten, M., Rideout, B., Sparger, E., Tonachini, T., Luciw, P., Ackley, C., Levy, N. and Yamamoto, J., 1990. Feline leukemia virus infection as a potentiating cofactor for the primary and secondary stages of experimentally induced feline immunodeficiency virus infection. J. Virol., 64(2): 598-606. Phillips, T.R., Talbott, R.L., Muir, S., Lovelace, K. and Elder, J.H., 1990. Comparison of two host cell range variants of feline immunodeficiency virus. J. Virol., 64 ( 10): 4605-4613. Shelton, G.H., Abkowitz, J.L., Linenberger, M.L., Russell, R.G. and Grant, C.K., 1989. Chronic leukopenia associated with feline immunodeficiency virus infection in a cat. J. Am. Vet. Med. Assoc., 194(2): 253-255. Shelton, G.H., Linenberger, M.L., Grant, C.K. and Abkowitz, J.L., 1990. Hematologic manifestations of feline immunodeficiency virus infection. Blood, 76 (6): 1104-1109. Stella, C.C., Ganser, A. and Hoelzer, D., 1987. Defective in vitro growth of hemopoietic progenitor cells in the acquired immunodeficiency syndrome. J. Clin. Invest., 80: 286-293. Stutte, H.J., Muller, H., Falk, S. and Schmidts, H.L., 1990. Pathophysiological mechanisms of HIV-induced defects in haematopoiesis: pathology of the bone marrow. Res. Virol., 141 (2): 195-200. Sun, N.C., Shapshak, P., Lachant, N.A., Hsu, M.-Y., Sieger, L., Schmid, P., Beall, G. and Imagawa, D.T., 1989. Bone marrow examination in patients with AIDS and AIDS-related complex (ARC). Morphologic and in situ hybridization studies. Am. J. Clin. Pathol., 92 (5): 589-594. Tochikura, T.S., Hayes, K.A., Cheney, C.M., Tanabe-Tochikura, A., Rojko, J.L., Mathes, L.E. and Olsen, R.G., 1990. In vitro replication and cytopathogenicity of the feline immunodeficiency virus for feline T4 thymic lymphoma 3201 cells. Virology, 179: 492-497. Torten, M., Franchini, M., Barlough, J.E., George, J.W., Mozes, E., Lutz, H. and Pedersen, N.C., 1991. Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus. J. Virol., 65 (5): 2225-2230. Yamamoto, J.K., Sparger, E., Ho, E.W., Andersen, P.R., O'Connor, T.P., Mandell, C.P., Lowenstine, L., Munn, R. and Pedersen, N.C., 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res., 49 (8): 1246-1258. Zucker-Franklin, D. and Cao, Y.Z., 1989. Megakaryocytes of human immunodeficiency virusinfected individuals express viral RNA. Proc. Natl. Acad. Sci. USA, 86( 14): 5595-5599.

Veterinary Immunology and Immunopathology, 35 ( 1992 ) 51-59

51

Elsevier Science Publishers B.V., Amsterdam

Decreased mitogen responsiveness and elevated tumor necrosis factor production in cats shortly after feline immunodeficiency virus infection Catherine E. Lawrence, John J. Callanan and Oswald Jarrett Department of Veterinary Pathology, Universityof Glasgow, Bearsden Road, Glasgow, G61 I QH, UK

ABSTRACT Lawrence, C.E., Callanan, J.J. and Jarrett, O., 1992. Decreased mitogen responsiveness and elevated tumor necrosis factor production in cats shortly after feline immunodefieiency virus infection. Vet. Immunol. ImmunopathoL, 35: 51-59. We present the results of an investigation into the effects of feline immunodeficiency virus (FIV) infection on the response to mitogens and cytokine production in the first month of infection. We were able to demonstrate a depression of response of peripheral blood mononudear cells to the mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen, with the response to pokeweed mitogen being most severely affected. The response of the cells of the spleen were affected by 10 days post infection and these could not be augmented by the addition of exogenous interleukin-2 (IL-2). The response of mesenteric lymph node ceils was not affected until 20 days post infection and this could be partially restored by the addition of exogenous IL-2. IL-2 production was unaffected in peripheral blood mononuclear cells, slightly depressed in mesenteric lymph node cells and slightly elevated in spleen cells. Tumor necrosis factor levels were significantly elevated with respect to controls within 10 days of infection. These studies suggest that there are a number of changes in the immune response of FIV infected cats early in infection and this may determine the subsequent outcome of the infection. ABBREVIATIONS AIDS, acquired immunodeficiency syndrome; Con A, concanavalin A; FIV, feline immunodeficiency virus; IL-2, interleukin-2; MLN mesenteric lymph node; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PHA, phytohaemagglutinin; PWM, pokeweed mitogen; rhlL-2, recombinant human interleukin-2; SPF, specific pathogen free; TNF, tumor necrosis factor.

INTRODUCTION

The effects of feline immunodeficiency virus (FIV) on the i m m u n e system of cats have been studied in field cases as well as in longitudinal experimental Correspondence to: C.E. Lawrence, Department of Veterinary Pathology, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK.

© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05,00

52

C.E. LAWRENCE ET AL.

infections (Siebelink et al., 1990; Torten et al., 1991 ). We have shown previously that major changes occur in the response of cats to mitogens during the first few months after infection which may predispose the animals to subsequent pathological effects. The present study was initiated in order to investigate the effects of FIV on immune function in the period immediately following infection. We have also examined the changes in the production of the cytokine interleukin 2 (IL-2) and tumor necrosis factor (TNF). In this way it may also be possible to determine why defects occur, and how early responses may determine the later pathological outcome. MATERIALS AND METHODS

Animals and sample preparation Six 12-week-old specific pathogen free (SPF) cats were inoculated intraperitoneally with 2 × 103 infectious doses of the Glasgow-8 strain of FIV (FIV/ GLA-8 ). Three age-matched cats were kept as naive controls. Blood samples were collected from the cats to assess mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC). PBMC were isolated from preservative-free heparinised blood by density gradient centrifugation on Lymphoprep (Nycomed, Birmingham, U K ) . The cells were washed twice with phosphate buffered saline (PBS) and once with RPMI-1640 supplemented with 10% heat-inactivated foetal calf serum, penicillin ( 100 IU m l - ~), streptomycin (100 /tg ml -~), L-glutamine (2 m M ) , and 2-mercaptoethanol ( 2 × 10 -5 M). At 10, 20 and 30 days post infection (p.i.) two infected and one control cat were killed and blood, spleen and mesenteric lymph nodes were taken. PBMC were isolated from the blood and cells were isolated from the spleen and MLN. The tissue was placed in 15 ml of RPMI- 1640 on a wire gauze in a sterile petri dish, and cells were forced through the gauze. The resulting cell suspension was collected and washed three times.

Mitogen-induced proliferation Cells were cultured in triplicate at 1 × 105 cells per well in 96 well flat-bottomed microtitre plates at 37 °C, with concanavalin A (Con A) (7.5/tg m l - 1) (Sigma Chemical Co., St. Louis, MO), pokeweed mitogen (PWM) (1 /~g m l - ~) ( Sigma ) or phytohaemagglutinin (PHA) ( 5 #g m l - ~) ( Sigma ) in the presence or absence of 100 IU of recombinant human IL-2 (rhIL-2). After 96 h the cells were pulsed with 1 #Ci [ 3H ]thymidine per well and after 16 h were harvested and counted in a scintillation counter.

MITOGEN RESPONSIVENESS AND TNF PRODUCTION IN FIV INFECTED CATS

53

IL-2 production Production of IL-2 was measured using the IL-2 dependent murine T-cell line, CTLL (Gillis et al., 1978). CTLL cells in the logarithmic phase were washed three times in culture m e d i u m and 100/zl were added to the wells of a 96 well flat-bottomed microtitre plate at a concentration of 45< 104 ml-1. Two-fold serial dilutions of cell supernatants from 24 h cultures of Con A stimulated and unstimulated cells were added. After 24 h the cells were pulsed with [3H]thymidine and after 16 h were harvested and counted in a scintillation counter. The units of IL-2 were determined by Probit analysis at 50% of the rhlL-2 standard.

TNF production The concentration of T N F in the plasma of cats was determined using the L929 cell line in the presence of actinomycin D (Carswell et al., 1975 ). After washing, 3 × 10 4 cells were added to each well of a 96 well microtitre plate in culture medium. Twenty-four hours later when the cells were confluent the m e d i u m was aspirated gently and serial dilutions of rhTNF or the sample were added to the wells in 100/tl of culture m e d i u m containing 1/zg actinomycin D m l - 1. After 24 h incubation the n u m b e r of viable cells was measured by staining with 0.05% amido blue, 9% acetic acid in 0.1 M sodium acetate for 30 min. The plate was then gently washed two times with PBS and the colour eluted using 50 m M NaOH. The optical density (OD) of the wells was then read at 620 n m using an ELISA reader. The concentration of T N F present in the plasma was calculated from the OD of the standard. RESULTS

Although only a few animals were used in the experiment several changes were apparent. Figure 1 illustrates the results from mitogen stimulation of PBMC. Incubation with Con A shows that there is a profound depression of the response within the first 30 days of infection when compared with age matched controls, and this effect was most obvious 10 days p.i. The results from PHA stimulated cells show a similar pattern with the infected cats responding to a lesser extent than the controls and the depression being most profound in the first 10 days. The response to PWM seemed to be most severely decreased 15 days p.i. Other investigations have shown that one mechanism of low T-cell responsiveness is failure to produce or respond to IL-2 (Alcocer-Varela and AlarconSegovia, 1982; Gilman et al., 1982 ). Hence experiments were performed to assess the role of IL-2 in mitogenic stimulation by measuring the augmentation of responses in the presence of IL-2. When cells of the MLN and spleen

54

C.E. LAWRENCE ET AL.

Con A

A 60-

A

40

O

x 0,.. tO

20 O

o !

!

!

10

20

30

PHA B

60

A

40 ¸

O

n O

g

I

20 ¸

o

•

8

•

8

o

o

0 10 '

0

2 '0

30

PWM 60

A

C

40

O o O-

20

o

o

1

oo

0

10

20

3'0

DAY POST INFECTION

Fig. 1. Response of feline PBMC to mitogens in the presence of IL-2. Cells were treated with (A) Con A, (B) PHA or (C) PWM. Infected cats are shown by open squares and control cats are shown by dosed squares. Proliferative response is expressed as counts m i n - ~ [ 3H ] thymidine incorporation.

MITOGENRESPONSIVENESSAND TNF PRODUCTIONIN FIV INFECTEDCATS

55

TABLE 1 The response of spleen and mesenteric lymph node cells to the mitogens Con A, PHA and PWM in the presence and absence of IL-2

[ 3H ] thymidine incorporation (counts min ~ × 10 -3) SPLEEN

MLN

-I~2

+IL-2

-I~2

+ID2

19.8 + 3.8

35.6 + 1

25.3 + 8.2

31.3 + 5.8

0.9; _1 25; 6 8; 19

14; 9 28; 7 7; 21

6:36 0.5; 7 17; 25

16; 37 2; 7 17; 34

4.1 +0.6

13+3

3.8+0.8

14.5+0.6

0.2; 0.2 3; 4 3;4

0.4; _1 15; 7 7; 18

0.4; 0.6 5; 4 2; 6

3; 2 14; 6 10; 31

22.2 + 5.2

28 +_5.2

26.3 + 5.6

43.1 _ 6.8

0.7; 1 27; 13 9; 11

2; 5 38; 17 10; 35

16; 54 28; 14 29; 28

15; 54 34; 18 36; 46

Con A

Controls FIV 10 d.p.i. 20 d.p.i. 30 d.p.i. PHA

Controls FIV 10 d.p.i. 20 d.p.i. 30 d.p.i. PWM

Controls FIV 10 d.p.i. 20 d.p.i. 30 d.p.i

Controls are the mean of three animals + SE. The figures for each animal at postmortem are shown. Underlined figures are those in which the response of both animals at that time point was significantly different from the control value. Proliferation is expressed as counts m i n - ~ [ 3H ] thymidine incorporation, d.p.i., days post infection.

were stimulated with Con A (Table 1 ), the proliferation of the spleen cells was depressed 10 days p.i. and that of the MLN by 20 days p.i. The addition of IL-2 partially restored the activity of the cells of the spleen at 10 days p.i. to normal levels, whereas the response at 20 days could not be restored in the MLN. The reactivity to PHA showed a broadly similar pattern with a depression of the spleen cells 10 days p.i. However, this could not be elevated with the addition of exogenous IL-2. The proliferation of the cells of the MLN did not appear to be severely affected. The response of spleen cells to PWM was severely affected at 10 days p.i. and the addition of IL-2 did not restore the response. The response of the cells of the MLN did not seem to be affected. When the IL-2 levels produced by the cells of the blood, spleen and MLN following exposure to Con A were measured (Fig. 2) it was seen that, first, the overall response of the MLN was greater than that of the spleen which was

N=

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oo

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INTERNATIONAL UNITS IL-2/ml

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O i

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0

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INTERNATIONAL UNITS IL-2/ml

130

O I

co O'

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o

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MITOGEN RESPONSIVENESS AND TNF PRODUCTION IN FIV INFECTED CATS

57

30

20

Z

n-

10

m

m

0

10

20

30

DAY POST INFECTION

Fig, 3. TNF levels in the plasma of infected and control cats. Controls are shown by closed columns and infected animals by open columns. Results are expressedas IU TNF ml- m. in turn greater than that of the PBMC. IL-2 production by any of the cells did not appear to alter significantly in the first 30 days p.i., although there was a suggestion that production by MLN cells was depressed with respect to controls while that of the spleen cells was elevated. Figure 3 illustrates that the levels of T N F measured in the plasma were slightly elevated by 10 days p.i. in comparison with controls and by 20 days p.i. were more than ten times those of the control. DISCUSSION

We have shown that the i m m u n e responsiveness of cats infected with FIV is affected as early as 10 days p.i., manifested by an inability to respond to mitogens, a defect in IL-2 production in the secondary lymphoid organs and an elevated T N F level. While FIV infected cats have previously been shown to exhibit various degrees of i m m u n e impairment, none have demonstrated such an early dysfunction. It was seen in this study that the PWM response was more severely affected

58

C.E. LAWRENCE ET AL,

than those to PHA or Con A and the addition of exogenous IL-2 did not appear to restore the response to control levels. This result implies that the defect in the response may lie primarily in the induction of the target cell from the resting to the activated state rather than the transition from activation to proliferation, since PWM stimulates activated cells via the C D 2 / C D 3 pathway whereas PHA and Con A stimulate both resting and activated cells (Hofm a n n et al., 1989). This result also suggests that the CD2 pathway is more severely affected than the CD3 pathway as PHA only stimulates cells through the CD3 pathway. The responses of the cells of the secondary lymphoid organs showed that initially the spleen cells were affected in the first 10 days and this response could not be elevated to control levels with the addition of exogenous IL-2. The cells of the MLN did not appear to be affected until Day 20 and their response could be elevated with the addition of IL-2. The ability of IL-2 supplementation to augment proliferation in the MLN cells but not the spleen cells indicates that lack of IL-2 responsiveness probably does not account for the defects seen in the MLN, but does in the spleen. There may be a decrease in the IL-2 receptor expression of the responding cells making them less able to become activated in response to this signal. Alternatively, there may be a decreased IL-2 production by the activated cells which makes them proliferate to a lesser extent; or there may be a decreased activation of the responding cells or a decrease in the number of the responding cells. The defect in the response of the MLN cells may represent an inability to produce IL-2 whereas that in the spleen may be the result of a defect in the ability to respond to native or exogenous IL-2, possibly as a result of decreased IL-2 receptor expression or an inability of the cells to be activated. Alternatively, it may be a combination of all three. It was not clear why there should be a different response of the cells in these two organs but it is possible that there are different cell populations present, some of which are more susceptible to the effects of both the virus and cytokines than others. The increase in T N F levels in the plasma of infected cats could be the result of infection a n d / o r subsequent activation of macrophages, leading to a dysregulated production of this cytokine. This may then be responsible for the decreased IL-2 production a n d / o r IL-2 receptor expression. In a previous study we were only able to demonstrate such levels in cats that had clinical signs of feline AIDS. However, in that study cats were infected as adults while in the present study kittens were infected. The difference in T N F production may be a reflection of a higher level of virus production in the younger animals. In conclusion, defects in the i m m u n e response occur very early after infection and these events may determine the subsequent i m m u n e dysfunction a n d / o r pathology. Further monitoring of animals will allow us to determine if there is an association between early i m m u n e dysfunction and rapid develo p m e n t of the clinical signs of feline AIDS.

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REFERENCES Alcocer-Varela, J. and Alarcon-Segovia, D., 1982. Decreased production of and response to intedeukin-2 by cultured lymphocytes from patients with systemic lupus erythematosus. J. Clin. Invest., 69: 1388-1392. Carswell, E.A., Old, L.S., Kassel, R.L., Green, S., Fiore, N. and Williamson, B., 1975. An endotoxin-induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. USA, 72: 3666-3670. Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A., 1978. T cell growth factor: parameters of production and a quantitative microassay. J. Immunol., 120: 2027-2032. Gilman, S.C., Rosenberg, J.S. and Feldman, J.D., 1982. T lymphocytes of young and aged rats. II. Functional defects and the role of interleukin-2. J. Immunol., 15:521-524. Hofmann, B., Jakobsen, K.D., Odum, N., Dickmeiss, E., Platz, P., Ryder, L.P., Pedersen, C., Mathiesen, L., Bygbjerg, I., Faber, V. and Svejgaard, A., 1989. HIV-induced immunodeficiency. Relatively preserved phytohemagglutinin as opposed to decreased pokeweed mitogen responses may be due to possibly preserved responses via CD2/phytohemagglutinin pathway. J. Immunol., 142:1874-1880. Siebelink, K.H.J., Chu, I.-H., Rimmelzwaan, G.F., Weijer, K., van Herwijnen, R., Knell, P., Egberink, H.F., Bosch, M.L. and Osterhaus, A.D.M.E., 1990. Feline immunodeficiency virus (FIV) infection in the cat as a model for HIV infection in man: FIV-induced impairment of immune function, AIDS Res. Hum. Retroviruses, 6:1373-1378. Torten, M., Franchini, M., Barlough, J.E., George, J.W., Mozes, E., Lutz, H. and Pedersen, N.C., 1991. Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus. J. Virol., 65: 2225-2230.

Veterinary Immunology and Immunopathology, 35 (1992) 61-69

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Tumor necrosis factor levels in cats experimentally infected with feline immunodeficiency virus: effects of immunization and feline leukemia virus infection R. Lehmann a, H. Joller b, B.L. H a a g m a n s c a n d H. L u t z a aDepartment of Veterinary Medicine, Universityof Zurich, Zurich, Switzerland bLaboratory of Clinical Immunology, Department of lnternal Medicine, Universityof Zurich, Zurich, Switzerland Clnstitute of Virology, Department of Infectious Diseases and Immunology, University of Utrecht, Utrecht, Netherlands

ABSTRACT Lehmann, R., Joller, H., Haagmans, B.L. and Lutz, H., 1992. Tumor necrosis factor c~ levels in cats experimentally infected with feline immunodeficiency virus: effects of immunization and feline leukemia virus infection. Vet. Immunol. ImmunopathoL, 35: 61-69. Tumor necrosis factor a (TNFct) levels were determined by enzyme-linked immunosorbent assay (ELISA) and by cell culture bioassay in supernatants of lipopolysaccharide-stimulated feline monocyte cultures and in cat serum samples. There was a good correlation between the results obtained by the two methods. From the fact that TNFct was neutralized quantitatively by antibodies to human T N F a in feline monocyte supernatants and in feline sera, it was concluded that feline T N F a immunologically cross-reacts with human TNFct and that the human TNFc~ ELISA can be used to quantitare feline TNFct. During the first 6 months after experimental feline immunodeficiency virus (FIV) infection no differences in serum TNFct values were observed between infected and non-infected cats. T N F a levels increased significantly after primary vaccination with a feline leukemia virus (FeLV) vaccine in FIV infected cats over those in the non-infected controls. During secondary immune response TNFt~ levels rose transiently for a period of a few days in both the FIV positive and the FIV negative cats. After FeLV challenge, TNFc~ levels increased in all animals challenged with virulent FeLV for a period of 3 weeks. This period corresponded to the time necessary to develop persistent FeLV viremia in the control cats. It was concluded from these experiments that in the asymptomatic phase of FIV infection no increased levels of TNFt~ are present, similar to the situation in asymptomatic HIV infected humans. Activation of monocytes/macrophages in FIV infected cats by stimuli such as vaccination or FeLV challenge readily leads to increased levels of TNFa.

Correspondence to: H. Lutz, Department of Veterinary Medicine, University of Zurich, Zurich, Switzerland.

© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

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ABBREVIATIONS FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; TNFa, tumor necrosis factor ce.

INTRODUCTION

Tumor necrosis factor a ( T N F a ) , also known as cachectin, was originally discovered by William B. Coley in 1893 who demonstrated necrosis of tumors after injection of bacterial products at the tumor site (Coley, 1893 ). Today T N F a is known to be a cytokine involved in the regulation of inflammation, immune reaction and erythropoiesis (for review see Rosenblum and Donato (1989) and Kunkel et al. (1989) ). T N F a is synthesized mainly by activated macrophages, monocytes and by a number of other cells of the immune system (Goh, 1990; Odeh, 1990). Although its exact function is not fully understood, it is known to be the principal mediator of septic shock, to be responsible for cachexia in chronic disease, and to act as the primary mediator in the pathogenesis of infection, tissue injury and inflammation (Gob, 1990; Odeh, 1990 ). Human TNFot is a non-glycosylated protein with a molecular weight of 17 500 (Goh, 1990). In contrast to most other cytokines T N F a shows a high cross-species reactivity (Rosenblum and Donato, 1989 ). TNFol was found to be elevated in the serum of human patients with AIDS, in about 50% of patients with AIDS-related complex and in some patients with lymphadenopathy in the early phase of human immunodeficiency virus (HIV) infection (Lahdevirta et al., 1988 ). Monocytes from HIV-positive donors produced higher levels of TNFce than those from non-infected controls (Wright et al., 1988 ). Furthermore, TNFot was shown to induce HIV expression in vitro (Folks et al., 1989) and to inhibit human B-cell differentiation (Kashiwa et al., 1987). From these and other observations it was proposed that treatment of AIDS patients should include T N F a antagonists (Odeh, 1990). Feline immunodeficiency virus (FIV)mfirst described in 1987 (Pedersen et al., 1987)--is widespread in the cat population and causes immune suppression. FIV infection is considered an important model of AIDS because FIV is a lentivirus closely related to HIV and because the infection leads to a loss of CD4 + cells and ultimately to immune suppression (Pedersen et al., 1987; Torten et al., 1991 ). It was the goal of the present report to study TNFot levels in cats experimentally infected with FIV during immunization with a feline leukemia virus (FeLV) vaccine and subsequent challenge with virulent FeLV.

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MATERIAL AND METHODS

Experimental design The serum and plasma samples used in this study were collected in a previous experiment described elsewhere (Lehmann et al., 1991 ). Briefly, 30 specified pathogen free cats were divided into two age- and sex-matched groups. Group I, 15 cats served as FIV negative control; Group II, 15 cats were infected intraperitoneally with a Swiss isolate of FIV (FIV Z Ga). Nine cats in each group were vaccinated twice with a commercial FeLV vaccine (Laboratoires Virbac, Carros, France), six cats in each group with a placebo vaccine. Three months later all cats were challenged intraperitoneally with FeLV (subtype A, Glasgow). All but one vaccinated cat were protected ( 17 of 18 ), while in each of the two non-vaccinated control groups five of six cats became persistently infected with FeLV.

Cats Specified pathogen free cats ( 16 female, 14 male) were obtained from CibaGeigy, Basel, Switzerland. This cattery is free of any known feline pathogen. All animals were housed in two identical climatized rooms at the animal experimental station of the Veterinary Faculty of the University of Zurich. They received canned and dry food (Whiskas and Brekkies; Effems, Zug, Switzerland). All animals were clinically examined weekly.

Quantitation of TNFa by ELISA T N F a was measured in cat serum samples by an enzyme-linked immunosorbent assay (ELISA) developed for the quantitation of biologically active natural and recombinant human T N F a in plasma, serum, and tissue culture supernatants (Endogen, Boston, MA, USA). The assay was a 'sandwich type' system, based on anti-TNFa antibodies derived from two species. The test samples were incubated on immunoplates coated with a mouse monoclonal anti-TNFa antibody. While T N F a present in the samples was captured and bound to the solid phase, unbound material was removed by washing. Polyclonal rabbit anti-TNFa serum was added. A second wash step removed unbound antibodies. Enzyme-labelled goat anti-rabbit IgG, conjugated with alkaline phosphatase was incubated and the optical density was measured by an ELISA reader. When displayed on a semilog scale, the measured absorbance was proportional to the concentration of T N F a in the test sample up to 7500 pg m l - k According to the manufacturer's specification, the lower detection limit was 10 pg m l - 1.

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Quantitation of TNFa by cell culture bioassay T N F a levels in selected cat serum samples and cell culture supernatants collected from stimulated feline monocyte cultures were determined according to the cell culture method described by Espevik and Nissen-Meyer ( 1986 ) utilizing the TNFc~-sensitive WEHI-164 sarcoma cell line as target cells. To obtain natural feline TNFtx, feline monocytes derived from peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS) ( 1/lg m1-1 ) for 24 h. TNFot in the supernatant of feline monocyte cultures and in cat serum samples was neutralized by incubation with rabbit antihuman TNFtx antibodies for 1 h at 37°C before adding to the WEHI-164 cells.

Data analysis and statistics All results were compiled in a computer data base. The null hypothesis postulated that the test parameters in FIV infected cats would not differ significantly from non-infected animals. Mean values of TNFc~ levels were analyzed for significant differences by the Student's t-test and a corresponding nonparametric method, the Mann-Whitney U-test (Sachs, 1984). Frequencies were compared using Fisher's exact test for small numbers (Sachs, 1984). Differences were considered significant if P < 0.05. RESULTS

Comparison of two methods of TNFa detection: ELISA vs. cell culture bioassay Six serum samples found to contain low, intermediate and high levels of TNFo< by ELISA were also tested by the WEHI-164 cell assay. There was a good agreement between the two methods (Fig. 1 ).

Neutralization of feline TNFa by antihuman TNFa antibodies Feline monocyte cultures were stimulated in vitro by LPS to synthesize TNFa. In different assays TNFot activity could be neutralized completely by polyclonal rabbit antihuman T N F a antibodies. T N F a in serum samples was also neutralized > 90% by the anti-TNFa antiserum.

TNFo~ levels during the early phase of FIV infection TNFce levels were measured by ELISA in the serum of all cats on six occasions between Day 0 and Day 180 after experimental FIV infection. No dif-

TNFot LEVELS IN CATS INFECTED WITH

65

FIV

100-

75-

<

50.

to H Ld

25 ¸

I

< 10

40-45

Cell culture

technique

80-105 (pg/ml)

Fig. 1. TNFot detection by two different methods: ELISA (y-axis) vs cell culture bioassay (xaxis).

ferences in T N F a levels were measured between FIV infected cats and the controls (data not shown).

TNFot levels during FeL V vaccination After the first vaccination with the recombinant FeLV and placebo vaccine the FIV positive cats showed an increase in TNFot levels over the FIV negative cats resulting in significantly higher concentrations by Day 14 (U-test, P<0.01; t-test, P<0.05; Fig. 2). The peak of T N F a levels on Day 14 after primary immunization coincided with a significantly higher frequency of cats with fever ( > 39°C) in the FIV positive group ( P < 0 . 0 5 ) . On Day 24, 4 days after the second vaccination, both the FIV positive and negative cats responded with a transient increase in TNFt~ levels.

TNFot levels during FeL V challenge For a 3 week period after the FeLV challenge the FIV positive and negative cats showed a transient increase in T N F a levels (Fig. 3 ). During the first 4 weeks of the FeLV challenge five of the FIV positive cats and six of the FIV negative cats showed a transient lymphadenopathy of 1-2 weeks' duration. With the exception of lymphadenopathy and occasional episodes of fever ( > 39 °C) the cats did not demonstrate any clinical signs of disease and food intake was not decreased. Elevated TNFt~ levels could not be correlated with fever during challenge in every cat. For the remainder of the observation period the mean T N F a levels in both the FIV positive and negative cats persisted at concentrations between 2 and 5 pg m l - 1.

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10-

•

FIV

neg

•

FIV

pos

6~ eO cQ4b-

r

42

0

Days afteP FeLV vaccination

Fig. 2. TNFct during FeLV vaccination. TNFc~ levels measured by ELISA, displayed as mean _+SD. Arrows indicate the first (Day 0) and second (Day 21 ) vaccinations with the recombinant FeLV or the placebo vaccine. * Significant difference between the FIV negative and FIV positive cats ( U-test, P < 0.0 l; t-test, P < 0.05 ). •

FIV

neg

•

FIV

pos

~15t~

~10-

¢Q. .-4 o~ Z

V-

\

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56

84

112

Days after FeLV challenge exposure

Fig. 3. TNFot during FeLV challenge exposure. TNFct levels measured by ELISA, displayed as mean + SD. All cats were challenged by intraperitoneal injection of 106 FFU of FeLV subtype A on Day 0. DISCUSSION

Feline T N F a could readily be measured in cat serum samples and cell culture supernatants from feline monocytes stimulated with LPS. The ELISA used in the present study was originally designed to quantitate human TNFot. It was concluded that the ELISA also could be employed to measure feline T N F a for the following reasons: ( 1 ) There was good agreement between the

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ELISA values and the WEHI- 164 cell assay. (2) Feline T N F a in serum samples and in the supernatant of LPS-stimulated feline monocyte cultures was completely neutralized by rabbit antihuman T N F a antibodies. (3) T N F a is not species-specific and human and murine T N F a are about 80% homologous (Marmenout et al., 1985; Rosenblum and Donato, 1989). The immunologic cross-reactivity between feline and human TNFa demonstrated in the present study therefore does not come as a surprise. Although many of the cat sera contained T N F a in concentrations below the approved detection limit of the assay, they could still be quantitated because the OD values were clearly measurable. However, in the low range of the standard curve the T N F a ELISA showed a relatively high variation which may explain why differences in T N F a levels between FIV positive and negative cats were not statistically significant (e.g. Fig. 3, Days 7 and 14). T-lymphoyctes and monocytes/macrophages are among the target cells of FIV (Pedersen et al., 1987; Brunner and Pedersen, 1989). Elevated T N F a levels in FIV infected cats therefore may be explained by infection of these cells by FIV and induction of T N F a synthesis. During the first 180 days of FIV infection no elevated T N F a levels were observed. This may be explained by either a small number of infected macrophages, by a low degree of stimulation of infected cells or by local production of T N F a which did not enter the plasma compartment (Rosenblum and Donato, 1989). In this respect FIV infection parallels the asymptomatic phase of HIV infection where the majority of infected people show no increased concentrations of plasma T N F a (Lahdevirta et al., 1988 ). By Day 14 after primary vaccination with an FeLV or a placebo vaccine, T N F a increased to a level significantly higher in FIV positive over FIV negative cats. This may reflect an especially strong stimulation of macrophages and/or T-cells in FIV infected animals; macrophages have an important role in antigen presentation. This observation also parallels the much stronger humoral immune response in the same FIV positive cats reported elsewhere (Lehmann et al., 1991 ). The mechanisms that lead to an increase of T N F a levels in FIV positive cats 14 days after primary vaccination are not clear; reactivation of latent FIV in monocytes/macrophages has to be considered. During secondary response, both FIV positive and FIV negative cats demonstrated a strong T N F a reaction, the response in the FIV negative cats this time being higher. This, too, parallels the much stronger secondary humoral immune response in the FIV negative cats (Lehmann et al., 1991 ). Interestingly, by Day 14 after primary immunization the FIV positive cats demonstrated a significantly higher frequency of fever. This may well be explained by the elevated concentration of T N F a which acts directly on the hypothalamus as a pyrogen and/or induces the synthesis of interleukin-1, also a powerful pyrogen (Dinarello et al., 1986). During the first 3 weeks of FeLV challenge, elevated T N F a values were

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found in FIV positive and negative cats. Although FeLV also infects monocytes and macrophages (Hoover et al., 1980) and therefore may give rise to T N F a synthesis, direct inflammatory response of the peritoneum also has to be taken into account. Challenge virus was injected intraperitoneally in a volu m e of 5 ml of tissue culture fluid. Although no clinical signs, no hematologic abnormalities and no decrease in food intake were observed (Lehmann et al., 1991 ), it has to be expected that this injection resulted in mild peritonitis. The observation that TNFo~ levels in our cats were relatively low in both the FIV positive and negative cats during the primary phase of FIV infection and a few weeks after FeLV challenge does not come as a surprise. Our cats were kept under ideal hygienic and ethologic conditions and were free of stress and infectious agents other than FIV and FeLV which could have contributed to additional stimulation of macrophages. They were most likely in an early phase of FIV infection as judged by still elevated CD4 + numbers ( > 400/A- 1; Lehm a n n et al., 1992). This situation is very similar to the asymptomatic phase of HIV infection where low TNFo~ levels also were reported (Lahdevirta et al., 1988 ). Further experiments will have to demonstrate if and to what degree cats in the AIDS phase of FIV infection show increased levels of T N F a . Should this be the case, this would offer an additional aspect to the use of FIV infection as a model to study AIDS therapy. It should be relatively easy to test T N F a antagonists as adjuvant therapy in conjunction with zidovudine or other etiologic drugs for i m p r o v e m e n t of the clinical situation in FIV infected cats with clinical signs. As TNFo~ is immunologically related between different species the antibodies to h u m a n or murine TNFc~ already available could be utilized for therapeutic studies in cats. ACKNOWLEDGMENTS This study was supported by a grant from Virbac Laboratories, Carros, France, and by donations from three Swiss cat clubs (FIFE, Katzenclub Ostschweiz, Katzenclub Ziirileu). The cat food was kindly provided by Effems, Zug, Switzerland. We thank Peter Fidler and Kim-Lan P h a m for their help with the animals. We thank Dr. P.F. Suter for reading the manuscript and helpful suggestions.

REFERENCES Brunner, D. and Pedersen,N.C., 1989. Infection of peritoneal macrophagesin vitro and in vivo with feline immunodeficiencyvirus. J. Virol., 63: 5483-5488. Coley, W.B., 1893. The treatment of malignant tumors by repeated inoculation of erysipelas: with a report often original cases. Am. J. Med. Sci., 105:487-490. Dinarello, C.A., Cannon, J.G., Wolff, S.M., Bernheim, H.A., Beutler, C., Cerami, A., Figari,

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I.S., Palladino, M.A. and O'Conner, J.V., 1986. Tumour necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin- 1. J. Exp. Med., 163:1433-1450. Espevik, T. and Nissen-Meyer, J., 1986. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods, 95: 99-105. Folks, T.M., Clouse, K.A., Justement, J., Rabson, A., Duh, E., Kehrli, J.H. and Fauci, A.S., 1989. Tumor necrosis factor a induces expression of human immunodeficiency virus in a chronically infected T-cell clone. Proc. Natl. Acad. Sci. USA, 86: 2365-2368. Goh, C.R., 1990. Tumour necrosis factors in clinical practice. Ann. Acad. Med. Singapore, 19: 235-239. Hoover, E.A., Rojko, J.L., Wilson, P.L. and Olsen, R.G., 1980. Macrophages and the susceptibility of cats to feline leukemia virus infection. Dev. Cancer Res., 4: 195-202. Kashiwa, H., Wright, S.C. and Bonavida, B., 1987. Regulation of B cell maturation and differentiation. I. Suppression of pokeweed-mitogen induced B cell differentiation by tumor necrosis factor (TNF). J. Immunol., 138: 1383-1390. Kunkel, S.L., Remick, D.G., Stricter, R.M. and Larrick, J.W., 1989. Mechanisms that regulate the production and effects of tumor necrosis factor-tx. Crit. Rev. Immunol., 9: 93-117. Lahdevirta, J., Maury, C.P.J., Teppo, A.-M. and Repo, H., 1988. Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immunodeficiency syndrome. Am. J. Med., 85: 289-291. Lehmann, R., Franchini, M., Aubert, A., Wolfensberger, C., Cronier, J. and Lutz, H., 1991. Vaccination of cats experimentally infected with feline immunodeficiency virus with a recombinant feline leukemia virus vaccine. J. Am. Med. Assoc., 199: 1446-1452. Lehmann, R., von Beust, B., Niederer, E., Condrau, M., Fierz, W., Aubert, A., Ackley, C.D., Cooper, M.D., Tompkins, M.B. and Lutz, H., 1992. Immunization induced decrease of the CD4+:CD8 + ratio in cats experimentally infected with FIV. Vet. Immunol. Immunopathol., 35: 199-214. Marmenout, A., Fransen, L., Tavernier, J., van der Heyden, J., Tizard, R., Kawashima, E., Shaw, A., Johnson, M.J., Semon, D., Muller, R., Ruysschaert, M.R., van Vliet, A. and Fiers, W., 1985. Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor. Eur. J. Biochem., 152:515-522. Odeh, M., 1990. Review article: The role oftumour necrosis factor-a in acquired immunodeficiency syndrome. J. Intern. Med., 228: 549-556. Pedersen, N.C., Ho, E.W., Brown, M.L. and Yamamoto, J.K., 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science, 235: 790793. Rosenblum, M.G. and Donato, N.J., 1989. Tumor necrosis multifaceted peptide hormone. Crit. Rev. Immunol., 9: 21-44. Sachs, L., 1984. Angewandte Statistik. Springer-Verlag, Berlin. Torten, M., Franchini, M., Barlough, J.E., George, J.W., Mozes, E., Lutz, H. and Pedersen, N.C., 1991. Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus. J. Virol., 5: 2225-2230. Wright, S.C., Jewett, A., Mitsuyasu, R. and Bonavid, B., 1988. Spontaneous cytotoxicity and tumor necrosis factor production by peripheral blood monocytes from AIDS patients. J. Immunol., 141: 99-104.

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Persistent upregulation of MHC Class II antigen expression on T-lymphocytes from cats experimentally infected with feline immunodeficiency virus B r u c e A. R i d e o u P ,b, P e t e r F. M o o r e b a n d N i e l s C. P e d e r s e n a

aDepartment of Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616, USA bDepartment of Pathology, School of VeterinaryMedicine, Universityof California, Davis, CA 95616, USA

ABSTRACT Rideout, B.A., Moore, P.F. and Pedersen, N.C., 1992. Persistent upregnlation of MHC Class II antigen expression on T-lymphocytes from cats experimentally infected with feline immunodeficiency virus. Vet. Immunol. Immunopathol., 35:71-81. A significant elevation in the percentage of CD4 + and CD8 + T-lymphocytes expressing major histocompatibility complex (MHC) Class II antigens was observed in the blood of cats shortly after they were experimentally infected with feline immunodeficiencyvirus (HV). In addition to an increase in the relative proportion of T-lymphocytes expressing Class II antigens, there was an increase in the density of Class II antigens on the cell surface. These elevations were still evident at the completion of the 5 month study. A second group of cats that had been infected with HV for almost 5 years, and with either normal or abnormally low levels of CD4 + T-lymphocytes, had similar elevations in MHC II expression, suggesting that such abnormalities are lifelong. Cats with chronic (2 year) feline leukemia virus (FeLV) infection or dual FIV/FeLV infections also showed similar alterations in MHC II expression on CD4 + and CD8 + T-lymphocytes, suggesting that these alterations were not FIV specific. Feline T-lymphocytes expressed more MHC II antigen and interleukin-2 (IL2 ) receptor following stimulation in vitro with conconavalinA and IL-2, demonstrating that feline Tlymphocytes respond to activation signals in a manner similar to T-lymphocytes of other species. However, changes in MHC II expression on T-cells of HV infected cats were not explainable by viral induced T-cell activation alone, because HV infected cats with elevated MHC II expression did not have coincident elevations in IL-2 receptor expression. ABBREVIATIONS APC, antigen presenting cells; con-A, conconavalinA; FeLV, feline leukemia virus; HV, feline ira-

Correspondence to: B.A. Rideout, Department of Pathology, Center for Reproduction of Endangered Species, Zoological Society of San Diego, P.O. Box 551, San Diego, CA 92112, USA.

© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

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munodeficiency virus; HIV, human immunodeficiency virus; IL-2, interleukin-2; Mab, monoclonal antibody; MHC, major histocompatibility complex; PBL, peripheral blood leukocytes; PBMC, peripheral blood mononuclear cells; PI, post inoculation; rHu-, recombinant human.

INTRODUCTION

The distribution of major histocompatibility complex (MHC) Class II molecules in the domestic cat has been characterized in several tissues and on peripheral blood mononuclear cells (PBMC) (Neefj es et al., 1986; Pollack et al., 1988; Rideout et al., 1990). Like the dog (Deeg et al., 1982; Doveren et al., 1985, 1986) and horse (Crepaldi et al., 1986), feline MHC II antigens are constitutively expressed not only by antigen presenting cells, but by Tlymphocytes as well. In contrast, human and murine T-lymphocytes express MHC II antigens only after activation (Ko et al., 1979; Robbins et al., 1988 ). The expression of MHC II molecules on antigen presenting cells (APC; e.g. macrophages and B-lymphocytes) is important in restriction of the immune response (Qvigstad et al., 1986). Less is known about the role of MHC II expression by T-lymphocytes. Recent studies suggest that expression of MHC II molecules on activated human T-lymphocytes is important in mediating signal transduction (Odum et al., 1991 ). MHC II molecules may also play some role in the pathogenesis of the acquired immunodeficiencysyndrome (AIDS). Homology between human immunodeficiency virus (HIV) envelope glycoproteins and human MHC II molecules has been well documented (Young, 1988; Golding et al., 1988; Habeshaw et al., 1990), and may be important in a number of proposed indirect mechanisms of immune dysfunction in AIDS (Fauci, 1988; Wigzell, 1988; Golding et al., 1988; Edelman and Zolla, 1989; Ascher and Sheppard, 1990; Rosenberg and Fauci, 1990; Weimer et al., 1991 ). Infection with HIV also results in altered MHC II expression on APC in vivo (Belsito et al., 1984; Heagy et al., 1984). Feline immunodeficiency virus (FIV) infection of domestic cats has become an important animal model for HIV infection of man, and FIV and HIV produce extremely similar diseases (Pedersen et al., 1989; Sparger et al., 1989; Yamamoto et al., 1988, 1989 ). Considering the present base of knowledge of FIV infection and the feline MHC II, and the documented abnormalities in MHC II expression in HIV infection, it seemed appropriate to study the effects of FIV infection on MHC II expression by T-lymphocytes. MATERIALS AND METHODS

Animals

All cats used in this study were specific pathogen free animals of mixed breed and sex belonging to the Feline Retrovirology Research Lab at the Uni-

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73

versity of California, Davis. The cats ranged from 6 months to 5 years of age at the time of this study. Cats with experimental FIV infection were inoculated with the Petaluma strain between 2 and 7 months of age as previously described (Pedersen et al., 1990). Cats with experimental FeLV infection were inoculated with the CT600 strain of FeLV as previously described (Pedersen et al., 1990). Although not previously reported because of an oversight, these cats were also inoculated with the Rickard strain of FeLV.

Sample preparation and cell staining Peripheral blood cells for in vivo studies were collected by a whole blood lysis technique as previously described (Rideout et al., 1990). Peripheral blood samples for in vitro studies were collected by jugular venipuncture into EDTA tubes followed by isolation of mononuclear cells over Histopaque 1077 according to the manufacturer's instructions. Harvested cells were depleted of platelets by three low speed (200 X g) centrifugation steps, using Dulbecco's phosphate buffered saline (pH 7.3, without divalent cations) as wash buffer. Staining of cells was accomplished by a two color direct method as previously described (Rideout et al., 1990), and by a two color indirect method. Briefly, the indirect method involved a 20 min blocking step using buffer (PBS with 5 mM EDTA and 0.1% sodium azide) containing 10% serum from the species in which the secondary antibody was prepared (horse or goat), followedby a 30 rain incubation with unlabeled primary antibody. The ceils were then washed and incubated another 30 min with fluorochrome (fluorescein isothiocyanate (FITC) or R-phycoerythrin ) conjugated secondary antibody. The cells were washed again and incubated 30 rain in buffer containing 1% normal mouse serum, followed by addition of directly conjugated second color antibody. After a final wash, cells were fixed in 1% paraformaldehyde and analyzed within 4 days. Feline MHC II molecules were identified with monoclonal antibody (Mab) 42.3H2 (Rideout et al., 1990). Lymphocyte subsets were identified with mouse monoclonal antibodies to feline CD4 (Fel 7 ) (Ackley et al., 1990) and CD8 (FT2) (Klotz and Cooper, 1986) Mabs (a gift of Dr. Max D. Cooper and Christopher D. Ackley, University of Alabama, Birmingham). Interleukin-2 (IL-2) receptor expression was detected with biotinylated recombinant mouse IL-2 (a generous gift of Drs. Gerard and Sandy Zurawski, DNAX Research Institute, Palo Alto, CA) with avidin-FITC as the second reagent (Vector Laboratories, Burlingame, CA). Cells incubated in vitro with conconavalin A (con-A) and IL-2 were washed twice, incubated in media without IL-2 or con-A for 1 h, and washed again prior to labeling with biotinylated IL-2. Negative (substitution) control antibodies were included for each cat during each staining procedure; antibodies for this purpose included unlabeled and fluorochrome or biotin conjugated isotype matched irrelevant murine monoclonal antibodies (CA4.1D3

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B.A. RIDEOUT ET AL.

and CA4. IC12), murine anticanine Mabs recognizing CD45A and Class II molecules, respectively (Moore et al., 1990) which have no binding affinity for cat cells, and MOPC 21 (Sigma Chemicals, St. Louis, MO ). Flow cytometric analysis was performed on a FACSTAR-PLUS (BectonDickenson). In most cases, 10 000 events were collected, with gating for lymphocytes based on light scatter properties. Cell counts in some samples from FIV infected cats were too low to collect 10 000 events; for these samples, 5000-7500 events were collected.

Effects of retrovirus infections on MHC H expression The effects of short term FIV infection on MHC II expression by T-lymphocytes were determined by two color flow cytometry in cats before and at monthly intervals after inoculation. The effects of long term FIV infection (4-5 years) were determined (at single time points) by two color flow cytometry in two groups of cats: one group with normal numbers of CD4 + cells, and one with abnormally low CD4 + cell counts. In order to determine the specificity of any FIV induced changes, the effects of long term asymptomatic FeLV infection on Class II expression by T cells was also evaluated. Potential synergistic or antagonistic interactions between FIV and FeLV were evaluated by measuring T-cell Class II expression in two cats experimentally infected with both FIV and FeLV (approximately 3 year duration of infections). To determine whether the retrovirus induced alterations were secondary to T-lymphocyte activation, IL-2 receptor and MHC Class II antigen expression were measured in peripheral blood leukocyte (PBL) samples from two cats with long term FIV infection. IL-2 receptor expression alone was measured in an additional seven cats with short (n = 5 ) or long (n = 2) term FIV infection, and in one cat with long term FeLV infection, all of which had previously elevated Class II expression.

In vitro activation studies In order to determine whether MHC Class II antigen expression on feline T-lymphocytes can be modulated by exogenous activation stimuli in vitro, PBMC from individual cats were divided into three fractions. One sample was stained immediately for MHC II and T-lymphocyte subsets using two color fluorescence as described below. One fraction from each cat was incubated in vitro for 48 h in RPMI (RPMI 1640 with 10% fetal bovine serum and 25/zg gentamicin ml-1 containing 5/zg con-A ml-1 and 100 units of recombinant human IL-2 (Cetus, Emoryville, CA). Another fraction was incubated similarly in medium without con-A or IL-2, to serve as a control for effects of processing on MHC II expression. Cell concentrations were 1 X 106 cells m l - 1. To confirm in vitro activation, IL-2 receptor expression was mea-

EFFECTS OF FIV ON MHC II EXPRESSION BY T-LYMPHOCYTES

75

sured on the feline PBMC cultures (using biotinylated mouse recombinant IL-2), or in parallel cultures of human PBMC (using anti-Tat Mab; a gift of Dr. Thomas Waldmann, NIH).

Statistical analysis Results were analyzed by repeated measures ANOVA for unbalanced designs using SuperAnova statistical software for the Macintosh (Abacus Concepts, Berkeley, CA). The Tukey-Kramer procedure was used for multiple comparisons. RESULTS

The effects of short term FIV infection on MHC II expression by T-lymphocytes are summarized in Table 1. The percentage of CD4 + lymphocytes expressing Class II molecules was elevated by the first sampling date 1 month post inoculation (PI) and remained elevated until the last sampling date 5 months PI (P< 0.05 ). Class II expression by CD8 + lymphocytes was measured for the first 3 months only, with similar results (Table 1 ). The effects of long term FIV infection on Class II antigen expression by Tlymphocytes are summarized in Table 2. Apparently healthy FIV infected cats with either normal or low CD4 + cell numbers had a markedly elevated percentage of CD4 + cells expressing Class II antigens compared to FIV negative cats (P< 0.05 ). Although a slightly higher percentage of Class II positive Tlymphocytes was seen in FIV infected cats with low CD4 + cell numbers compared with those with normal CD4+ cell numbers, this difference was not statistically significant. Expression of Class II antigens by CD8 + cells was nearly identical to that by CD4 + cells. In addition to an increase in the percentage of Class II positive T-lymphocytes in the cats with long term FIV infection, cats with low CD4 + cell numbers had an increase in mean fluorescence intensity for Class II antigens on CD4 + cells when compared with FIV TABLE1 Summary of results: short term FIV infection Month post inoculation

CD4 + M H C I I +

CD8 + MHC I I +

0 1 2 3 4 5

80.04+6.15 93.75 ± 3.03 89.00 + 3.09 94.37+2.19 ND 89.05 _+3.05

65.80+ 14.24 82.99 + 16.22 87. I 0 ± 4.74 97.04± 1.67 ND ND

IL-2-R+

2,90 ± 1.90

Results are expressed as mean percentage antigen positive PBL ± SD. IL-2-R, interleukin-2 receptor; ND, not done.

85.48 + 7.85 93.60+7.22 96.26 + 3.63

Preculture In vitro, not activated In vitro, activated

74.34 + 9.58 95.07+4.97 99.19 + 0.73

84.62+8.86 94.98 + 5.83 96.38 + 5.75 98.66 + 1.42 96.31 + 5.51

CD8 + MHC II + l

437.00 + 25.09 281.71+41.94 585.25 _+38.08

454.13+28.09 483.46 + 39.97 499.69 + 38.16 516.67 + 32.88 506.00 + 21.79

on CD4 + cells

MHC II density2

383.20 + 10.62 456.75+42.52 613.75 + 28.09

399.94+22.04 455.61 + 40.15 467.18 + 38.77 483.00 + 51.73 489.00 + 31.19

on CD8 + cells

~Results expressed as mean percentage antigen positive PBL_+ SD. 2Expressed as mean channel number (an increase of approximately 62 channels represents a doubling of fluorescence intensity). 3Values are for individual cats (not means). IL-2-R, interleukin-2 receptor; NRM, normal (i.e. NRM CD4 = normal numbers of CD4 + cells in peripheral blood ).

86.97+ 14.40 95.56 + 5.80 97.77 + 2.91 98.13 + 0.42 99.07 + 0.40

SPF ( F I V - F e L V - ) FIV + NRM CD4 FIV + low CD4 FeLV + FIV + FeLV +

C D 4 + MHC I I + 1

Summary of results: long term retrovirus infections and in vitro studies

TABLE 2

1.04 + 0.495 20.52+4.72 46.62 + 15.48

2.55, 12.723 8.862

IL-2-R+ l

r~

o c

O',

EFFECTS OF FIV ON MHC II EXPRESSION BY T-LYMPHOCYTES

77

negative cats. The Class II antigen fluorescence intensity was also increased on C D 8 + lymphocytes from FIV positive eats with either normal or low C D 4 + cell numbers. In order to determine whether the effect of FIV on Class II expression is virus specific, Class II expression on T-lymphocytes was measured in cats infected with FeLV, or FIV and FeLV. There was a marked elevation in the percentage of Class II positive CD4 + and CD8 + lymphocytes in both groups, with accompanying elevations in mean Class II antigen fluorescence intensity on both CD4 + and CD8 + lymphocytes (Table 2). In order to determine whether general T-cell activation was present, which might account for the elevated Class II expression in retrovirus infected cats, expression of IL-2 receptor and MHC II was measured in two cats with long term FIV infection. IL-2 receptor expression was measured alone in an additional seven cats with short or long term FIV infection, and in one cat with long term FeLV infection, all of which had previously elevated Class II expression. All of these cats had similar percentages of IL-2 receptor positive PBL (Tables 1 and 2). The mean for short term FIV infected cats was 2.90%_+ 1.90 (range 1.47-5.96%). The two cats with long term FIV infection expressed IL-2 receptor on 2.55% and 12.72% of PBL, respectively, while the one cat with long term FeLV infection expressed IL-2 receptor on 8.86% of its PBL. None of these values was significantly different from normal (less than 20%; Table 2). Because feline T-lymphocytes constitutively express MHC II antigens, but human and murine do not, we wanted to determine whether feline T-lymphocytes are in fact able to further increase their MHC II expression in response to activation signals as human and murine T-lymphocytes do. PBMC were obtained from healthy specific pathogen free cats and were divided into three fractions: one fraction from each eat was stained the same day for Class II expression by T-cells; the second fraction was cultured in vitro in medium without mitogens or recombinant human IL-2 (rHu-IL-2); the third was cultured in vitro in medium containing optimum concentrations of con-A and rHu-IL-2. The results of these studies are illustrated in Table 2. In vitro activation resulted in a statistically significant increase in the percentage of CD4 + and CD8 + cells expressing Class II molecules. In addition, there was an increase in the Class II antigen density on both CD4 + and CD8 + cells as evidenced by the increased mean Class II fluorescence intensity on cells activated in vitro. In vitro activation was documented by marked elevations in IL-2 receptor expression on the cultured feline PBMC. A statistically nonsignificant increase in IL-2 receptor expression was observed in feline PBMC cultivated without mitogens or rHu-IL-2. DISCUSSION

Infection with FIV is associated with a significant elevation in the proportions of MHC Class II positive C D 4 + and CD8 + T-lymphocytes and in-

78

B.A. RIDEOUT ET AL.

creased levels of MHC II antigen expression on the cell surface. These abnormalities were present as early as 1 month following experimental FIV infection and appeared to persist, as did FIV infection, for the remainder of the cats' lives. There was a strong trend for cats with the lowest CD4 + lymphocyte levels to have the highest proportion of MHC Class II positive T-lymphocytes. Identical abnormalities in MHC II expression by T-lymphocytes were seen in cats chronically infected with another retrovirus, FeLV. This suggested the possibility that chronic immune stimulation provided by persistant viral infection might be responsible for the sustained elevations in MHC II expression. Although such upregulation of MHC II expression accompanies T-cell activation in vitro in the cat, as in man (Heron et al., 1978; Ko et al., 1979; Giacomini et al., 1988 ), the enhanced MHC II expression seen in this study did not coincide with T-lymphocyte activation in vivo, as evidenced by the lack of concomitant elevations in IL-2 receptor expression. Expression of MHC Class II antigens by APC is important in regulating the intensity of the immune response (Lechler, 1988; Lechler et al., 1985 ); therefore alterations in the expression of Class II antigens can have a profound effect on the host immune response. Less is known about the role of Class II molecules expressed by T-cells. Some studies have shown that T-cells are capable of limited antigen presentation in vitro, but this is generally inefficient (Engelman et al., 1980; Triebel et al., 1984; Corredor et al., 1986; Odum et al., 1991 ). Recent studies suggest that MHC Class II molecules may be important in T-cell signal transduction (Odum et al., 1991 ). The importance of Class II mediated signaling has already been well documented for B-cells (Mooney et al., 1989a, b; Lane et al., 1990; Fuleihan et al., 1991 ). The high levels of constitutive Class II antigen expression by feline T-cells implies that these molecules have an important role to play in normal T-cell function. FIV induced alterations in T-cell Class II expression may therefore have a significant impact on normal T-cell function. It has recently been shown that cats experimentally infected with FIV experience a progressive decline in both Tcell mitogen responsiveness and ability to mount a humoral immune response to T dependent, but not T independent, antigens (Torten et al., 1991 ). The mechanism behind this observation is not known. Further studies will be required to determine the role of altered T-cell MHC Class II antigen expression in this and other immunologic abnormalities observed in feline retrovims infections. ACKNOWLEDGMENTS

The authors would like to thank Kim Floyd-Hawkins, Nancy Delemus, and Paul Rossitto for excellent technical assistance. This work was supported in part by a Training Grant in Laboratory Animal Medicine ( N I H / D R R

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79

5T32RR07038 ) and by grants RO 1-CAS0179-01 and RO 1-AI25802-04 from the National Institutes o f Health.

REFERENCES Ackley, C.D., Hoover, E.A. and Cooper, M.D., 1990. Identification of a CD4 homologue in the cat. Tissue Antigens, 35: 92-98. Ascher, M.S. and Sheppard, H.W., 1990. AIDS as immune system activation. II. The panergic imnesia hypothesis. J. Acquired Immune Def. Syndr., 3: 177-191. Belsito, D.V., Sanchez, M.R., Baer, R.L., Valentine, F. and Thorbecke, G.J., 1984. Reduced Langerhan's cell Ia antigen and ATPase activity in patients with the acquired immunodeficiency syndrome. N. Engl. J. Med., 310: 1279-1282. Corredor, V., Matsiu, Y. and Yunis, E.J., 1986. Class II antigens: T cells. In: B.G. Solheim, E. Moller and S. Ferrone (Editors), HLA Class II Antigens: A Comprehensive Review of Structure and Function. Springer-Veflag, New York, pp. 374-386. Crepaldi, T., Crump, A., Newman, M., Ferrone, S. and Antczak, D.F., 1986. Equine T lymphocytes express MHC II antigens. J. Immunogen., 13: 349-360. Deeg, J.H., Wulff, J.C., DeRose, S., Sale, G.E., Braun, M., Brown, M.A., Springmeyer, S.C., Martin, P.J. and Storb, R., 1982. Unusual distribution of Ia-like antigens on canine lymphocytes. Immunogenetics, 16: 445-457. Doveren, R.F.C., Buurman, W.A., Schutte, B., Groenewegen, G. and van der Linden, C.J., 1985. Class II antigens on canine lymphocytes. Tissue Antigens, 25: 255-265. Doveren, R.F.C., van der Linden, C.J., Spronken, E.E.M., Grocnewegen, G. and Buurman, W.A., 1986. Canine MHC-class II antigens on B and T lymphocytes. Tissue Antigens, 27: 87-98. Edelman, A.S. and Zolla, P.S., 1989. AIDS: a syndrome of immune dysregulation, dysfunction, and deficiency. FASEB J. 3: 22-30. Engelman, E.G., Benike, C.J. and Charron, D.J., 1980. Ia antigen on peripheral blood mononuclear leukocytes in man. II. Functional studies of HLA-DR positive T cells activated in mixed lymphocyte reactions. J. Exp. Med., 152:114s. Fauci, A.S., 1988. The human immunodeficiency virus: infectivity and mechanisms of pathogenesis. Science, 239:617-622. Fuleihan, R., Mourad, W., Geha, R.S. and Chatila, T., 1991. Engagement of MHC-class II molecules by staphylococcal exotoxins delivers a comitogenic signal to human B cells. J. Immunol., 146: 1661-1666. Giacomini, P., Fisher, P.B., Duigou, G.J., Gambari, R. and Natali, P.G., 1988. Regulation of class II MHC gene expression by interferons: insights into the mechanism of action of interferon (review). Anticancer Res., 8:1153-1162. Golding, H., Robey, F.A., Gates, F.T., Linder, W., Beining, P.R., Hoffman, T. and Golding, B., 1988. Identification of homologous regions in human immunodeficiency virus I gp41 and human class II beta 1 domain. J. Exp. Med., 167: 914-923. Habeshaw, J.A., Dalgleish, A.G., Bountiff, L., Newell, A.L., Wilks, D., Walker, L.C. and Manca, F., 1990. AIDS pathogenesis: HIV envelope and its interaction with cell proteins. Immunol. Today, 11: 418-425. Heagy, W., Kelley, V.E., Strom, T.B., Mayer, K., Shapiro, H.M., Mandel, R. and Finberg, R., 1984. Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome. J. Clin. Invest., 74: 2089-2096. Heron, I., Hokland, M. and Berg, K., 1978. Enhanced expression of beta-2 microglobulin and HLA antigens on human lymphoid cells by interferon. Proc. Natl. Acad. Sci. USA, 75:62156219.

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Klotz, F.W. and Cooper, M.D., 1986. A feline thymocyte antigen defined by a monoclonal antibody (FT2) identifies a subpopulation of non-helper cells capable of specific cytotoxicity. J. Immunol., 136: 2510-2514. Ko, H., Fu, S.M., Winchester, R.J., Yu, D.T.T. and Kunkel, H.G., 1979. Ia determinants on stimulated human T lymphocytes: occurence on mitogen- and antigen-activated T cells. J. Exp. Med., 150: 246. Lane, P.J., McConnell, F.M., Schieven, G.L., Clark, E.A. and Ledbetter, J.A., 1990. The role of class II molecules in human B cell activation. Association with phosphatidyl inositol turnover, protein tyrosine phosphorylation, and proliferation. J. Immunol., 144: 3684-3692. Lechler, R.I., 1988. Structure-function relationships of MHC class II molecules. Immunol. Suppl., l: 25-26. Lechler, R.I., Norcross, M.A. and Germain, R.N., 1985. Qualitative and quantitative studies of antigen-presenting cell function by using I-A expressing L cells. J. Immunol., 135:29142922. Mooney, N., Grillot, C.C., Hivroz, C. and Charron, D., 1989a. A role for MHC class II antigens in B-cell activation. J. Autoimmun., Suppl. 2:215-223. Mooney, N., Hivroz, C., Ziai, T.S., Grillot, C.C. and Charron, D., 1989b. Signal transduction via MHC class II antigens on B lymphocytes. J. Immunogenet., 16: 273-281. Moore, P.F., Rossitto, P.V. and Danilenko, D.M., 1990. Canine leukocyte integrins" characterization of a CD 18 homologue. Tissue Antigens, 36:211-220. NeeOes, J.J., Hensen, E.J., de Kroon, T.I.P. and Ploegh, H.L., 1986. A biochemical characterization of feline MHC products: unusually high expression of class II antigens on peripheral blood lymphocytes. Immunogenetics, 23:341-347. Odum, N., Martin, P.J., Schieven, G.L., Hansen, J.A. and Ledbetter, J.A., 1991. Signal transduction by HLA class II antigens expressed on activated T cells. Eur. J. Immunol., 21: 123129. Pedersen, N.C., Yamamoto, J.K., Ishida, T. and Hansen, H., 1989. Feline immunodeficiency virus infection. Vet. Immunol. Immunopathol., 2 l: 11 l-129. Pedersen, N.C., Torten, M., Rideout, B., Sparger, E., Tonachini, T., Luciw, P.A., Ackley, C., Levy, N. and Yamamoto, J., 1990. Feline leukemia virus infection as a potentiating cofactor for the primary and secondary stages of experimentally induced feline immunodeficiency virus infection. J. Virol., 64: 598-606. Pollack, M.S., Hayes, A., Mooney, S., Pedersen, N.C. and Cook, R.G., 1988. The detection of conventional class I and class II I-E homologue major histocompatibility complex molecules on feline cells. Vet. Immunol. Immunopathol., 19:79-91. Qvigstad, E., Bruserud, O. and Thorsby, E., 1986. The role of human class II molecules in activation of T4 lymphocytes. In: B.G. Solheim, E. Moiler and S. Ferrone (Editors), HLA Class II Antigens: A Comprehensive Review of Structure and Function. Springer-Verlag, New York, pp. 473-488. Rideout, B.A., Moore, P.F. and Pedersen, N.C., 1990. Distribution of MHC class II antigens in feline tissues and peripheral blood. Tissue Antigens, 36: 221-227. Robbins, P.A., Maino, N.L., Warner, N.L. and Brodsky, F.M., 1988. Activated T cells and monocytes have characteristic patterns of class II antigen expression. J. Immunol., 141: 1281. Rosenberg, Z.F. and Fauci, A.S., 1990. Immunopathogenic mechanisms of HIV infection: cytokine induction of HIV expression. Immunol. Today, 1l: 176-180. Sparger, E.E., Luciw, P.A., Elder, J.H., Yamamoto, J.K., Lowenstine, L.J. and Pedersen, N.C., 1989. Feline immunodeficiency virus is a lentivirus associated with an AIDS-like disease in cats. AIDS, Vol. 2: $43-9. Torten, M., Franchini, M., Barlough, J.E., George, J.W., Mozes, E., Lutz, H. and Pedersen, N.C., 1991. Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus. J. Virol., 65: 2225-2230.

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Triebel, F., Missenard-Leblond, V., Autran, B., Conty, M., Charron, D. and Dibre, P., 1984. Antigen-specific proliferative human T cell clones with specificity for diphtheria toxoid: genetic and molecular restriction by class II antigens. Eur. J. Immunol., 14: 697. Weimer, W., Daniel, V., Zimmermann, R., Schimpf, K. and Opelz, G., 1991. Autoantibodies against CD4 cells are associated with CD4 helper defects in human immunodeficiency virusinfected patients. Blood, 77: 133-140. Wigzell, H., 1988. Immunopathogenesis of HIV infection. J. AIDS, 1: 559-565. Yamamoto, J.K., Sparger, E., Ho, E.W., Andersen, P.R., O'Connor, T.P., Mandell, C.P., Lowenstine, L., Munn, R. and Pedersen, N.C., 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res., 49: 1246-1258. Yamamoto, J.K., Hansen, H., Ho, E.W., Morishita, T.Y., Okuda, T., Sawa, T.R., Nakamura, R.M. and Pedersen, N.C., 1989. Epidemiologic and clinical aspects of feline immunodeficiency virus infection in cats from the continental United States and Canada and possible mode of transmission. J. Am. Vet. Med. Assoc., 194:213-220. Young, J.A.T., 1988. HIV and HLA similarity. Nature, 333:215.

Veterinary Immunology and lmmunopathology, 35 (1992) 83-93

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Elsevier Science Publishers B.V., Amsterdam

Transmission of feline immunodeficiency virus from infected queens to kittens Terri Wasmoen, Stephanie Armiger-Luhman, Claire Egan, Vicki Hall, HsienJue Chu, Lloyd Chavez and William Acree Department of Biological Research and Development, Fort Dodge Laboratories, Fort Dodge, IA 50501, USA

ABSTRACT Wasmoen, T., Armiger-Luhman, S., Egan, C., Hall, V., Chu, H.-J., Chavez, L. and Acree, W., 1992. Transmissionof feline immunodeficiencyvirus from infected queens to kittens. Vet. Immunol. Immunopathol., 35: 83-93. This study demonstrates the transmission of feline immunodeficiencyvirus (FIV) from infected queens to kittens in two separate litters. Queen I was infected by intravenous administration of FIV at 22 days prior to parturition. Two out of three kittens from the litter were found to be viremic at 10 weeks of age as detected by culture isolation and polymerase chain reaction detection of FIV DNA in peripheral blood mononuclear leukocytes. The third kitten remained aviremic through 40 weeks of age. Queen 2 was infected by subcutaneous administration of FIV 2 days prior to parturition. This litter also had two out of three kittens infected with FIV; however, viremia was not detected in one of the kittens until 21 weeks of age. Culture isolation was found to be superior to polymerase chain reaction for the early detection of FIV, and viremia was found to precede seroconversion by up to 4 weeks. Although all infected kittens have remained healthy, depressed CD4:CD8 lymphocyte ratios suggest that clinical disease may develop. This study suggests that FIV infection in cats may be a useful model system for the study of HIV transmission from mothers to infants. ABBREVIATIONS FIV, feline immunodeficiencyvirus; HIV, human immunodeficiencyvirus; PCR, polymerase chain reaction; SPF, specific pathogen free.

INTRODUCTION

Transmission of feline leukemia virus from infected queens to kittens has been reported by numerous investigators (Hardy et al., 1976; Pederson et al., 1984). However, transmission of another retrovirus, feline immunodeficiency virus (FIV), from mothers to kittens has not been documented. The Correspondence to: Terri Wasmoen, Department of Biological Research and Development, Fort Dodge Laboratories, Fort Dodge, IA 50501, USA.

© 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

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lack of data on the vertical transmission of FIV is somewhat surprising in light of the evidence for vertical transfer of other lentiviruses such as human immunodeficiency virus (HIV) (Scott et al., 1985; Peckham et al., 1988; Tovo et al., 1988; Blanche et al., 1989). This study demonstrates that a novel isolate of FIV can be transmitted from infected queens to their offspring in two separate cases. MATERIALS AND METHODS

Animals

Two 1-year-old female specific pathogen free (SPF) cats were used (Liberty Laboratories, Liberty Corners, N J). The females were impregnated by commingling with adult male SPF cats in isolation facilities. Kittens were born naturally and housed in isolated cages with their mothers until 14 (Case 1 ) or 12 (Case 2 ) weeks of age. V/rus The NCSU-1 isolate of FIV was obtained from Drs. Wayne and Mary Tompkins (North Carolina State University, NC) in EDTA-whole blood derived from a naturally infected cat. Queen 1 was infected with FIV by intravenous administration of 1 ml of EDTA-whole blood from the North Carolina State University cat. Queen 2 was infected by subcutaneous administration of cell-free tissue culture supernatants derived from mononuclear cell cultures from Queen 1 (see below). Virus isolation

Mononuclear leukocytes were isolated from EDTA-whole blood using discontinuous Percoll gradients. Briefly, blood was diluted with an equal volume of phosphate buffered saline (PBS), and 6 ml were overlayed onto 63/42.5% Percoll (Sigma Chemical Company, St. Louis, MO ) step gradients. Gradients were centrifuged for 5 min at 4 0 0 × g and then 25 min at 800Xg (22°C). Mononuclear cells were removed from the interface and washed twice with PBS containing 5% fetal calf serum. Mononuclear cells, 5 × 105, from infected cats were co-cultivated with 1 × 106 mononuclear cells from normal cats in 24 well plates using RPMI medium (supplemented with 20% MLA 144 cells (ATCC TTB 201 ) supernatant (interleukin-2 source), 10% fetal calf serum, 10 mM Hepes, 25/zM 2-mercaptoethanol, 2.5 #g polybrene m1-1, and 5/zg concanavalin A ml-1 and incubated at 37 °C in 5% CO2. Cultures were maintained up to 4 weeks and supernatants were analyzed for FIV by commercial antigen ELISA (IDEXX, Portland, ME) or by reverse transcriptase assay (see below).

FIV TRANSMISSION FROM QUEENS TO KITTENS

85

Reverse transcriptase assay Culture supernatant ( 10/tl) was added to 30 #1 of reaction cocktail (50 mM Tris-HC1 pH 7.8, 5 mM MgC12, 7.5 mM KC1, 2 mM dithiothreitol, 5 #g Poly(A). (dT)15 m1-1, 0.05% NP-40, 0.5 gCi [a-32p]dTTP) and incubated for 1 h at 37°C. Then 10 gl of the reaction mixture were spotted onto triplicate DE81 filters and washed four times with 2 × SSC (20 × SSC= 3 M NaC1, 0.3 M sodium citrate 2H20, pH 7.0). Filters were placed in 3 ml of Cytoscint (ICN Biomedicals, Irvine, CA) scintillation fluid and counted.

Polymerase chain reaction (PCR) detection of FIV DNA Mononuclear cells purified by 5 × 105 Percoll gradient were placed in 100 /tl oflysis buffer ( 10 mM Tris-HC1 pH 8.5, 50 mM KC1, 2.5 mM MgC12, 0.5% Tween 20, 0.1 mg proteinase K ml- ~) and incubated for 1 h at 50°C. Samples were boiled and 10 gl used in a 100 gl standard kit PCR reaction (Perkin Elmer Cetus, Norwalk, CT ) using 0.2 gg of each 21-oligomer oligonucleotide primer (primers flank gag region nucleotides 1056 and 1638 of the FIV.CG sequence). PCR reactions were run for 35 cycles as follows: 1.5 min 94°C, 2 min 59°C, 3 rain 72°C. PCR products were detected by gel electrophoresis on 4% NuSieve gels followed by transfer to nylon membranes for Southern blot. FIV-specific sequences were detected using a random hexamer 32p-labeled probe specific to the gag region being amplified.

Lymphocyte subset analysis CD4 and CD8 lymphocyte subsets were measured by FACS analysis using previously described monoclonal antibodies (Tompkins et al., 1990). Briefly, mononuclear cells were isolated on Histopaque 1083 (Sigma). A total of 250 000 ceils was incubated with monoclonal antibodies for 20 min at 4°C, then washed and incubated with the appropriate fluorescein isothiocyanate conjugates for 15 min at 4 ° C, fixed with paraformaldehyde, and analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). Lymphocyte counts were determined by Microcell Counter analysis (Sysmex CC-150, TOA Medical Electronics, Kobe, Japan) and differential staining.

Serology FIV-specific antibodies were detected in plasma using a commercial ELISA (IDEXX, Portland, ME ).

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RESULTS

Case I

Queen 1 was infected with FIV by intravenous administration of whole blood from a cat carrying the NCSU- 1 isolate. Viremia was detected by tissue culture isolation 17 days later and confirmed by PCR detection of proviral DNA in mononuclear cells at 31 days after challenge. Figure 1 shows the rapid and persistent inversion of C D 4 : C D 8 ratios induced by the NCSU-1 FIV isolate in this cat. Three kittens were born at 22 days post challenge. The kittens were housed in isolation with their mother until 14 weeks of age and then gang-housed with an uninfected, age-matched litter of kittens. Two out of the three kittens were confirmed to be viremic at 10 weeks of age by tissue culture isolation of FIV. The presence of FIV in these cultures was determined by antigen ELISA and magnesium-dependent reverse transcriptase assay (Table 1 ). In addition, FIV infection was demonstrated by the detection of proviral DNA in mononuclear cells by PCR (Fig. 2). Table 2 shows the time course for the detection of FIV infection by culture isolation, PCR, and detection of antibodies. The data for Kitten 35 indicate that culture isolation (positive at 10 weeks of age) was more sensitive than PCR (positive at 14 weeks of age) for 2.0

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POST-CHALLENGE

Fig. 1. CD4:CD8 lymphocyte ratios for Queen 1. The percentage of CD4 + and CD8 + lympho-

cytes was determined by FACSanalysis at various time points followinginfection with FIV.

87

FIV TRANSMISSIONFROMQUEENSTO KITTENS TABLE 1 Reverse transcriptase assay detection of FIV in culture fluids Description

Identification

Age-matched controls

32 33 34

175 142 165

Infected kittens

35 36

8 413 7 114

Uninfected littermate

37

383

Queen I Positive control Negative control

XH1

XH1 35 36 37

Average counts min- t

12 446 20 804 259

32 33 34

564 bp -

Fig. 2. Southern blot detection of PCR-amplified FIV DNA from peripheral blood mononuclear cells. DNA from mononuclear leukocytes was amplified by PCR and the 582 bp FIV-specific DNA fragment detected by Southern blot. XH 1, Queen 1; 35 and 36, FIV-infected kittens from Queen 1; 37, uninfected kitten from Queen 1; 32, 33, and 34, age-matched kittens born to an uninfected queen.

TABLE 2 Detection of FIV infection in Queen 1 kittens Kitten

35 36 37

Age at time of positive test (weeks) Culture isolation

PCR

Antibody

l0 l0 Negative

14 10 Negative

14 14 Negative

88

T. WASMOENETAL.

(a) NORMALS

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WEEKS OF AGE

Fig. 3. CD4 and CD8 lymphocyte subsets in Queen 1 FIV-infected kittens vs age-matched controls. FACS analysis of CD4 and CD8 lymphocyte subsets for various weeks of age are shown for: (a) the CD4: CD8 lymphocyte ratios for Queen 1 FIV-infected kittens (35 and 36 ), Queen I uninfected littermate, and the average for three age-matched control kittens ( 32, 33 and 24); (b) the absolute number of CD4 positive cells for Queen 1 FIV-infected kittens (35 and 36) vs their littermate; (c) the absolute number of CD8 + cells for Queen 1 FIV-infected kittens (35 and 36) vs their littermate.

89

FIV TRANSMISSION FROM QUEENS TO KITTENS

(c)

2ooo LITTERMATE

35 36

_1 -,1 .,....

-I -I I.U 0

1000

¢0

0

0

i

I

I

I

20

30

40

50

WEEKS OF AGE Fig. 3. continued. TABLE3 Detection of FIV infection in Queen 2 kittens Kitten

B23 B24 B25

Age at time of positive test (weeks) Culture isolation

PCR

Antibody

21 8 Negative

21 12 Negative

21 12 Negative

the detection of viremia. For both positive kittens, viremia could be detected prior to seroconversion. Both kittens remained viremic through 40 weeks of age, whereas the littermate and age-matched controls remained negative. The FIV-infected kittens remained clinically normal throughout the observation period. However, Fig. 3 (a) shows that the infected kittens have significantly depressed CD4:CD8 lymphocyte ratios when compared to their littermate and age-matched controls. The CD4:CD8 inversion in kitten 35 appears to be related to a depression in the absolute number of CD4+ cells (Fig. 3 (b)). However, while CD4 + cell numbers are dropping over time for kitten 36, an elevation in CD8 + cells is the major factor in the CD4:CD8 inversion (Fig. 3 (c)).

90

T, WASMOEN ET AL,

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Fig. 4. CD4 and CD8 lymphocyte subsets in Queen 2 FIV-infected kittens vs age-matched controis. FACS analysis of CD4 and CD8 lymphocyte subsets for various weeks of age are shown for: (a) the CD4:CD8 lymphocyte ratios for Queen 2 FIV-infected kittens (B23 and B24), Queen 2 uninfected littermate, and the average for three age-matched control kittens (32, 33 and 24); (b) the absolute number of CD4 + cells for Queen 2 FIV-infected kittens (B23 and B24) vs their littermate; (c) the absolute number of C D 8 + cells for Queen 2 FIV-infected kittens (B23 and B24) vs their littermate.

91

FIV TRANSMISSION FROM QUEENS TO KITTENS

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16

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WEEKS OF AGE Fig. 4. c o n t i n u e d .

Case 2

Queen 2 was infected with FIV by subcutaneous administration of cell-free tissue culture fluids. Viremia was detected at 13 days post challenge by tissue culture isolation of virus. The cat was positive for FIV by PCR at 20 days post challenge and seroconverted by 27 days post challenge. This cat developed a CD4:CD8 lymphocyte ratio of less than 1.0 by 48 days post challenge (data not shown). Two days post challenge, three kittens were born. The kittens were isolated in a cage until 12 weeks of age and then gang-housed with the kittens from Case 1. Table 3 shows the tests used and timing of detection of viremia in these kittens. At 8 weeks of age viremia was detected in kitten B24 by detection of antigen (ELISA) and reverse transcriptase activity in mononuclear cell culture supernatants (data not shown). Viremia was confirmed by PCR at 12 weeks of age, at the same time seroconversion was evident. Kitten B23 had a questionable culture at 8 weeks of age, tested negative at 10 weeks of age, and was confirmed viremic at 21 weeks of age by PCR and culture isolation. Figure 4 (a) shows that CD4:CD8 lymphocyte ratios in all three of these kittens are depressed when compared with age-matched controls. However, the kitten with the earliest onset of viremia (Kitten B23 ) has the lowest ratio. The low CD4:CD8 ratios in infected kittens are the result of a significant decrease in the number of CD4 + cells when compared with the littermate

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T. WASMOEN ET AL.

(Fig. 4 (b)). The low CD4:CD8 ratio in the littermate appears to be related to increases in CD8 + cell numbers (Fig. 4 (c)). DISCUSSION

An important route of HIV infection has been shown to be perinatal transmission from mothers to children, where the incidence rate has been reported to be as high as 65% (Tovo et al. 1985; Scott et al., 1985; Peckham et al., 1988; Blanche et al., 1989). Feline immunodeficiency virus is very similar to HIV in genomic organization, virion morphology, and clinical disease syndrome. Therefore, perinatal transmission of FIV might be expected. The above two cases document the transmission of FIV from infected queens to kittens. The exact mode of transmission cannot be deduced from these studies. In Case l, where the queen was infected in the second trimester, transmission could have occurred in utero, during the birthing process, or by contact after birth. In Case 2, where the kittens were born before viremia was detectable in the queen, postnatal infection with FIV is most likely. Breast milk has been documented to carry the HIV virus (Thiry et al., 1985 ) and, thus, may be a likely mode of transmission for FIV. In addition, saliva is a possible source of FIV that could be transmitted to kittens during maternal grooming. Because Kitten B23 was gang-housed with infected kittens from Case 1 prior to the detection of viremia, we cannot rule out direct transmission from other kittens. However, Kittens 32, 33, and 34 have been housed with the Case l kittens for over 25 weeks without developing FIV infections. For both litters, culture isolation was superior to PCR and antibody detection for the early diagnosis of FIV infection. All of the FIV-infected kittens have altered CD4"CD8 lymphocyte ratios. In three of the kittens this alteration is attributable to decreases in CD4 + cell numbers. In the fourth kitten, increases in CD8 + cells are a major factor in the CD4: CD8 inversion. It is unknown whether these early CD4: CD8 ratio changes will be predictive of the development of immune deficiency disease in these kittens. As of 40 and 21 weeks of age (Case 1 and Case 2, respectively), all kittens appear to be normal and healthy. The NCSU-1 isolate of FIV is unusual in its ability to induce rapid and persistent alterations in CD4:CD8 Iymphocyte ratios in infected cats (Fig. 1 ) (Tompkins et al., 1991 ). This virus also induces a much higher virus burden in infected cats than seen with other isolates (unpublished observations ). Therefore, the ease of transmission of NCSU-1 from queens to kittens may be the consequence of some unusual virulence characteristics for this isolate. In addition, the introduction of a large virus burden during pregnancy may have enhanced the rate of transmission to offspring in these cases. Additional studies are needed to examine the peri- or postnatal transmission rate in natural settings, the possible routes of transmission, and the ability of various

FIV TRANSMISSION FROM QUEENS TO KITTENS

93

isolates to b e t r a n s m i t t e d to o f f s p r i n g in this m a n n e r . F I V i n f e c t i o n in cats using the N C S U - 1 isolate m a y p r o v i d e a n i m p o r t a n t m o d e l for s t u d y o f the t r a n s m i s s i o n o f H I V f r o m m o t h e r s to infants. ACKNOWLEDGMENTS W e w o u l d like to t h a n k A. Niles, K. J e n n e n a n d L. I h r k e for t e c h n i c a l assistance.

REFERENCES Blanche, S., Rouzioux, C., Moscato, M.G. et al., 1989. A prospective study of infants born to women seropositive for human immunodeficiency virus type I. N. Engl. J. Med., 320:16431648. Hardy, Jr., W.D., Mess, P.W., MacEwan, E.G. et al., 1976. Biology of feline leukemia virus in the natural environment. Cancer Res., 36: 582-588. Peckham, C.S., Senturia, Y.D., Ades, A.E. et al., 1988. Mother to child transmission of HIV infection. Lancet, ii: 1039-1042. Pederson, N.C., Meric, S.M., Ho, E. et al., 1984. The clinical significance of latent feline leukemia virus infection in cats. Feline Pract., 14: 32-48. Scott, G.B., Fischi, M.A., Klimas, N. et al., 1985. Mothers of infants with the acquired immunodeficiency syndrome. J. Am. Med. Assoc., 253: 363-366. Thiry, L., Sprecher-Goldberger, S., Jonkheer, T. et al., 1985. Isolation of the AIDS virus from cell-free breast-milk of three healthy virus carriers. Lancet, ii: 896-898. Tompkins, M.B., Gebhard, D.H., Bingham, H.R. et al., 1990. Characterization of monoclonal antibodies to feline T lymphocytes and their use in the analysis of lymphocyte tissue distribution in the cat. Vet. Immunol. Immunopathol., 26: 305-317. Tompkins, M.B., Nelson, P.D., English, R.V. et al., 1991. Early events in the immunopathogenesis of feline retroviral infections. J. Am. Vet. Med. Assoc., 199:1311-1315. Tovo, P.A., DeMartino, M., Italian Multicenter Study Group, 1988. Epidemiology, clinical features, and prognostic factors of paediatric HIV infection. Lancet, ii: 1043-1045.

Veterinary Immunology and lmmunopathology, 35 (1992) 95-119

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Elsevier Science Publishers B.V., Amsterdam

Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats Gerhard H. Reubel a, Jeanne W. George a, Jeffrey E. Badough a, Joanne Higgins ~, Chris K. G r a n t b and Niels C. Pedersen a

aDepartment of Medicine, School of Veterinary Medicine, Universityof California, Davis, CA 95616, USA bDepartrnent of Immunology and Retrovirus Research, PacificNorthwest Research Foundation, Seattle, WA 98122, USA

ABSTRACT Reubel, G.H., George, J.W., Barlough, J.E., Higgins, J., Grant, C.K. and Pedersen, N.C., 1992. Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats. Vet. lmmunol. Immunopathol., 35: 95-119. Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4 + and CD8 + Tqymphocytes. A brief decrease in the CD4 + / C D 8 + T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8 + T-lymphocytes. CD4 + and CD8 + T-lymphocyte numbers and CD4 + / C D 8 + T-lymphocyte ratios returned to baseline within 4-8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV- 1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenie responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection. ABBREVIATIONS CAT, chloramphenicol acetyltransferase; Con A, concanavalin A; CPE, cytopathic effect; CrFK,

Correspondence to: Gerhard H. Reubel, Department of Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. Tel: (916 ) 752 7470.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

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Crandell feline kidney (cells); FHV-1, feline herpesvirus type 1; FITC, fluorescein isothiocyanate; FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; LTR, long terminal repeat; PWM, pokeweed mitogen; SIV, simian immunodeficiency virus; TCIDso, tissue culture infectious dose, 50%.

INTRODUCTION

The role of infectious or non-infectious cofactors in the progression of immunodeficiency in human immunodeficiency virus (HIV)-infected people is unknown. Some researchers believe that the decline in immune function is solely time-dependent (Jason et al., 1989) while according to others a number of cofactors might serve either to delay or accelerate the development of immunodeficiency. These cofactors include age at time of infection (Eyster et al., 1987), genetic predisposition (Mann et al., 1988), the use of drugs (Schechter et al., 1985; Winkelstein et al., 1987) or other incidental diseases (Quinn et al., 1987; Watkins et al., 1988; Webster, 1991 ). Unfortunately, it is difficult to study the role of cofactors in HIV infection of man, especially of the more common incidental infectious diseases. This is thus one area wherein animal models could be of great value. A number of lentivirus infections of animals exist, but only two have been shown to cause acquired immunodeficiency syndrome (AIDS )-like disease: simian immunodeficiency virus (SIV) infection of macaque monkeys and feline immunodeficiency virus (FIV) infection of domestic cats (Baskin et al., 1988; Pedersen et al., 1989 ). FIV infection of cats may be an appropriate model to study the role of incidental infectious diseases in lentivirus disease; cats are available in a specific pathogen free (SPF) state and can easily be housed in pathogen free quarters in comparatively large numbers. The common infectious diseases of domestic cats have also been very well studied under natural and experimental conditions (Pedersen, 1988 ). The study reported herein is one part of a long-term multistage experiment dealing with the effect of common infectious diseases on the rate of progression of immunodeficiency in FIV infected cats. The cats were infected as adolescents with the Petaluma strain of FIV and were kept in strict pathogen free facilities. FIV infected cats underwent a primary and transient illness 68 weeks after inoculation (Pedersen et al., 1990; Yamamoto et al., 1989). About 1 year after infection, the FIV infected cats and a group of their noninfected cohorts were infected with Toxoplasma gondii (Lappin et al., 1991 ). The present study involves a second common incidental infectious disease agent of cats, feline herpesvirus, type 1 (FHV-1). FHV- 1 is an alphaherpesvirus that causes severe upper respiratory diseases (rhinitis, conjunctivitis, pharyngitis, tracheitis ), and at times pneumonia, in domestic cats (Povey, 1979 ). Clinical signs normally resolve in 7-10 days in older cats. In common with herpesvirus infections of other species of animals

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and of man, FHV-1 can persist indefinitely in the trigeminal ganglia and related nerves, creating a chronic carder state. The carder state is usually latent, although active virus shedding and occasional disease recurrence can follow periods of stress (Gaskell and Goddard, 1984). Many cats naturally infected with FIV suffer from chronic upper respiratory disease (Yamamoto et al., 1989) and FHV-1 has been associated with some of these cases. The authors have also observed one FIV infected cat with widely disseminated herpesvirus infection (L. Lowenstine and N.C. Pedersen, personal communication, 1989). Feline herpesvirus infection may also be an appropriate model to study the role of various herpesvirus interactions in people with AIDS. Human cytomegalovirus, herpes simplex virus, Epstein-Barr virus and human herpesvirus type 6, have all been associated either in vivo or in vitro with HIV infection of man and may act as cofactors in the progression of AIDS (Ostrove et al., 1987; Tremblay et al., 1989; Laurence, 1990; Okubo and Yasunaga, 1990; Cardgan et al., 1990; Webster, 1991 ). The objective of the present communication is to describe the effect of chronic FIV infection on acute FHV-1 disease in terms of disease severity, virus shedding, hematology and immunity. The effect of acute FHV- 1 infection on the antibody response to FIV will also be described. MATERIALS AND METHODS

Experimental animals SPF domestic cats were obtained from the breeding colony of the Feline Retrovirus Research Laboratory of the School of Veterinary Medicine, University of California, Davis, and were housed in small groups in infectious disease isolation facilities provided by the Animal Resources Service at the same institution.

Experimental design A total of 60 age- and sex-matched cats were selected for the study and each was assigned to one of four experimental groups. Ten eats were not exposed to any infectious agent and served as naive controls. Thirty cats were inoculated with the Petaluma isolate of FIV at an average age of 10 months, 18 months before the initiation of the present study (Pedersen et al., 1990; Yamamoto et al., 1989). Ten of these FIV infected eats were not exposed to any additional infection and served as FIV infected control. Twenty naive cats were infected with FHV-1, as were 20 previously FIV infected cats. FIV infection was confirmed by serial testing for serum antibody to FIV (O'Connor et al., 1989).

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G.H. REUBEL ET AL.

Feline herpesvirus-1 infection Cats in the FHV-1 and FIV/FHV-1 groups were inoculated with 10 6.6 TCIDso 0.4 ml -l of FHV-1, strain C27 (ATCC VR 636) by administering two drops of virus-containing cell culture medium into both eyes and nostrils (0.1 ml each). Cats in the naive and FIV alone groups received an equivalent amount of cell culture medium without virus.

Clinical scoring Clinical signs were monitored for 14 days after FHV- 1 infection and scored daily, using a modified scoring method described elsewhere (Povey et al., 1980) (Table 1 ). In addition, rectal temperature was recorded daily for 14 days after challenge. TABLE 1 Scoring system for clinical signs following infection with FHV- 1 Clinical signs

Score

Body temperature > 39.0°C >__39.5°C >_40.0°C

2 3 4

Constitutional signs Malaise Dehydration Gauntness

1 2 3

Respiratory signs Nasal discharge/serous Nasal discharge/mucopurulent Pneumonia (dyspnea, coughing, depression) Eye lesions Conjunctivitis/serous Conjunctivitis/mucopurulent Blepharospasm/acute keratitis Oral lesions Salivation Pharyngitis Ulcers/single Ulcers/multiple

FELINE HERPES AND IMMUNODEFICIENCY VIRUS INFECTIONS

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Clinical treatment In order to palliate clinical signs resulting from FHV-1 infection (secondary bacterial infections and dehydration), severely affected cats were given amoxiciUin (20 nag kg -1 daily) and lactated Ringer's solution (200 ml per cat per day, subcutaneously) for varying numbers of days.

Cells and media For virus isolation, titration and evaluation of neutralizing antibodies, Crandell feline kidney (CrFK) cells were used as substrate. Cells were grown in a 1 : 1 dilution of Leibovitz's L 15 medium and Earle's minimum essential medium containing 10% fetal calf serum, 100 IU penicillin m l - l, 10/lg streptomycin m l - 1 and 5/tg gentamicin m l - i.

Isolation and quantification of FHV-1 Samples of conjunctival and oral secretions were obtained at different time points after challenge using sterile cotton swabs. After transfer into sterile tubes containing 1.0 ml of cell culture medium with 300 IU penicillin m1-1, 30 #g streptomycin ml-1 and 15 #g gentamicin ml-1, swabs were stored at - 7 0 ° C for processing. CrFK cells were grown to confluence in 24-well cell culture plates and inoculated in duplicate with 200/d of each swab sample. Cytopathic effect (CPE) was monitored for 5 days; CPE typical for FHV-1 (distinct plaques, ballooning cells, cell lysis) in at least one of two wells was considered a positive result. To determine virus titers of positive samples, ten-fold dilutions were prepared in cell culture medium. Quadruplicate samples of each dilution (50/zl) were inoculated onto CrFK cells grown in 96well microtiter plates. Virus titers were evaluated according to the method of Reed and Muench, ( 1938 ).

Virus neutralization assay FHV- 1 neutralizing antibody titers in heat-inactivated serum samples (30 min, 56°C) of all cats in the FHV-1 and FIV/FHV-1 infected groups were determined. Twofold dilutions of the serum were mixed in a 96-well microtiter plate with equal volumes of FHV-1, strain C27 ( 10-100 TCIDso m l - l ) and incubated for 90 rain at 37°C. Then 100 gl of the virus-serum mixtures were transferred to corresponding wells of a 96-well microtiter plate containing confluent monolayers of CrFK cells. Plates were monitored for CPE for 5 days. Antibody titers were expressed as the reciprocal of the highest serum dilution completely neutralizing CPE.

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G.H. REUBEL ET AL.

Immunofluorescence assay Specific IgG and IgM responses to FHV-1 were evaluated in sera from five cats each in the FHV-1 and FIV/FHV-1 infected groups. Confluent monolayers of CrFK cells were cultured in eight-chamber slides and infected with 1000 TCID5o ml-~ of FHV-1. The cells were fixed in acetone for 30 min at room temperature when approximately 25 virus-induced plaques per well were present. The slides were covered with twofold dilutions of the sera and incubated at room temperature for 60 min. Thereafter, they were incubated with mouse monoclonal antibodies specific for feline IgG or feline IgM (Pacific Northwestern Research Foundation, Seattle, WA) and subsequently stained with FITC-conjugated anti-mouse-IgG (Kirkegaard and Perry, Gaithersburg, M D ) . Serum from the naive group cats served as a control. Antibody titers were expressed as the reciprocal of the highest serum dilution that resulted in specific fluorescence of FHV-1 induced plaques.

FIV antibody ELISA FIV specific IgG antibody titers were determined in sera from ten FIV infected and ten F I V / F H V - 1 infected cats at different time points after FHV- 1 infection. Microtiter plates were coated with 0.2/tg of sucrose purified FIV, strain Petaluma, per well, tenfold dilutions of the sera added and the plates incubated for 1 h at 37°C. Biotin-labeled rabbit anti-cat IgG was used as secondary antibody for 30 rain at 37°C. Thereafter, horseradish peroxidase labeled streptavidin was added and the plates incubated for 20 min at 37 °C. Tetramethyl benzidine served as substrate for the enzymatic reaction which was stopped after approximately 3 m i n with 1 N H2SO4. The optical density (OD) was measured with an automatic plate reader at a test wavelength of 495 n m against a reference wavelength of 590 nm. Serum from an SPF cat was used as blank. Antibody titers were expressed as the reciprocal of the serum dilution that would produce an absorbance reading equal to 50% of that of a 1 : 100 dilution of a FIV antibody positive control serum.

Complete blood counts Complete blood counts included hemoglobin, mean corpuscle volume, mean corpuscle hemoglobin, total leukocyte count and differentials. Electronic counts were determined by impedance using a System 9000 cell counter (Baker Instruments). Differential leukocyte counts were obtained from Wright-Giemsa stained blood smears using standard methods (Jain, 1986). In order to determine whether peripheral blood neutrophil counts increased in proportion to the degree of FHV-1 illness, the absolute blood neutrophil counts on Day 7 post infection were divided by the sum of the clinical

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101

score. This ratio was a measure of the cat's ability to mount a neutrophil response proportionate to the disease severity.

Enumeration of CD4 + and CD8 + feline T-lymphocytes Murine monoclonal antibodies to feline C D 4 + (Ackley et al., 1990) and feline CD8 + (Klotz and Cooper, 1986) T-lymphocyte surface markers were used for T-lymphocyte subset enumeration by flow cytometric analysis (Barlough et al., 1991 ).

Lymphocyte blastogenes& assay This whole blood microassay was a modification of standard procedures described elsewhere and was performed in 96-well flat-bottomed microtiter plates (Pauly et al., 1973; Gregory et al., 1987; Barlough et al., 1991 ). Heparinized blood ( 10 #1) was incubated in a total volume of 200 #1 per well at 37°C in 5% CO2 with either pokeweed mitogen (PWM) (3/tg m1-1 ), concanavalin A (Con A) (6/2g m l - 1), FHV- 1 antigen ( 1 : 10 dilution ) or complete RPMI (RPMI supplemented with 10% bovine fetal serum, 2 mM Lglutamine, 1% non-essential amino acids, 100 IU penicillin m l - 1, and 10 #g streptomycin m l - 1) as unstimulated controls reflecting spontaneous proliferation. FHV- 1 antigen was prepared by heat inactivation (30 rain, 56 ° C) of CrFK cell culture supernatant containing 107.0TCIDso of FHV-1 ml-1. Complete RPMI served as diluent for the assay. The cultures were labeled on Day 4 with 1 #Ci per well [3H]thymidine (New England Nuclear) and 24 h later were harvested on filtermats with a 6 well Skatron cell harvester. [ 3H ] thymidine incorporation into DNA was measured with a Tri-Carb 2000 liquid scintillation counter (United Technologies/Packard). Each of the four treatments (PWM, Con A, FHV-1 and cell culture medium) was replicated three times for each blood sample. Mean values were calculated for use in statistical analysis. Lymphocyte-adjusted values were obtained by dividing mean counts m i n - 1per culture by the total numbers oflymphocytes/11-1 blood of corresponding cats multiplied with the dilution factor (0.1), which resulted in mean counts m i n - 1 per cell.

Statistical analys& Differences in mean values between groups were evaluated for statistical significance (P_< 0.05) by Student's t test (two-tailed) and by ANOVA for repeated measures (Armitage and Berry, 1987). Data were assembled on a computerized spreadsheet program (Excel, Microsoft, Richmond, WA) and statistical calculations performed by a statistics package (Statview SE + Graphics, Abacus Concepts, Berkeley, CA).

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G.H. REUBEL ET AL.

RESULTS

Clinical observations

Clinical signs of FHV-1 infection appeared 3 days post exposure in a similar proportion of FIV infected and non-infected cats (Fig. l ( a ) ) . Fever, sneezing, and serous ocular and nasal discharges were the most prominent clinical abnormalities. The FIV infected cats were more ill than the non-FIV infected cats from Day 4 post infection onward. Most of the FIV infected cats were partially or completely anorectic, 13 of 20 had pharyngitis a n d / o r rhinitis, and six of 20 became dehydrated. In comparison, eight of 20 of the nonFIV infected cats had pharyngitis a n d / o r rhinitis and three of 20 became dehydrated. Nine of 20 cats in the FIV infected group manifested signs of pneumonia (dyspnea, coughing, high fever and marked depression ) compared with one of 20 cats in the non-FIV infected group. Gingivitis and oral ulceration were observed in two of 20 FIV infected cats and in one of 20 of the non-FIV infected animals. Acute keratitis manifested by blepharospasm occurred in 50% of the cats in both FIV infected and non-infected groups; three of 20 of the FIV infected cats and none of the non-FIV infected cats developed corneal ulcers that healed within 10 days. No clinical signs of illness were observed among the two non-FHV-1 exposed control groups (naive cats and cats infected solely with FIV). FHV-1 infection was associated with a biphasic elevation of the rectal temperature on Days 3 and 7 post inoculation. The mean rectal temperature was significantly greater in FIV infected cats on Days 2, 4-7 and 11 post infection than in non-FIV infected animals (Fig. 1 ( c ) ) . Eleven of 20 cats in the FIV infected group had rectal temperatures that exceeded 40 °C at some point in their disease compared with five of 20 non-FIV infected cats. The mean rectal temperatures returned to preinfection levels in both groups of cats by Days 12-14 post infection. Based on the scoring system (Table 1 ), cats with pre-existing FIV infection were significantly (P_< 0.01 ) more ill than n o n - H V infected cats between Days 4 and 10 post infection (Fig. 1 ( a ) ) . Cats with clinical severity scores greater than 10 usually had fevers over 40 ° C. The comparative severity of the FHV-1 induced illness among FIV infected and non-FIV infected groups of cats was mirrored by the types and frequency of supportive treatment that were administered to treat dehydration and secondary bacterial infections. Seventeen of 20 FIV infected cats were treated a total of 73 times over a 9 day period. This was compared with 11/20 nonFIV infected cats that were treated 34 times over a 4 day period (Fig. 1 ( b ) ) .

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TABLE 2 Isolation of FHV-1 from oral and ocular secretions of FHV-1 and FIV/FHV-I infected cats following experimental FHV- 1 infection Days post infection

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Virus shedding F H V - 1 w a s r e c o v e r e d m o r e f r e q u e n t l y f r o m o c u l a r t h a n oral s e c r e t i o n s in b o t h F I V i n f e c t e d a n d n o n - F I V i n f e c t e d cats; 3 4 . 1 % a n d 3 2 . 8 % o f t h e t o t a l

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oral swabs taken from the FIV infected and non-FIV infected groups, respectively, yielded FHV- 1, compared with 43.5% and 43.5% of the respective ocular swabs. The proportion of cats that shed virus was similar between the two groups, except on Days 11 and 14 post infection. Eleven of 20 FIV infected cats were shedding virus on Day 11 post infection compared with four of 20 of the non-FIV infected cats; 12/20 and eight of 20 of the cats in the two respective groups were shedding FHV- 1 on Day 14. Virus shedding ceased by Day 35 post infection in all of the FIV infected and in 19/20 of the non-FIV infected cats (one non-FIV infected cat did not cease shedding until after Day 42 post infection) (Table 2). The titers of virus present in ocular and oral secretions were essentially identical in the two groups of animals for the duration of illness, with more virus present in ocular compared with oral secretions (Figs. 2 (a) and 2 (b)). Virus shedding peaked at Day 7 post infection, dropped slightly, and then levelled offuntil Day 28 post infection. Virus shedding ceased abruptly after that time. Shedding of FHV- 1 was never observed in the two non-FHV- 1 infected control groups. Antibody responses to FHV-1

Antibodies that neutralized FHV-1 in vitro were detectable as early as 14 days post infection in both FIV infected and non-FIV infected groups of cats. The mean FHV-1 neutralizing antibody titers were significantly lower ( P < 0.05) in FIV infected eats for the first 21 days post infection and then reached levels similar to those of non-FIV infected animals (Fig. 3 (a)). The peak of the FHV-1 neutralizing antibody responses occurred in both FHV-1 exposed groups of cats around 8 weeks post infection. FHV-1 neutralizing antibodies were never detectable in sera from the two non-FHV-I infected control groups. Immunofluorescence antibodies to FHV-1 that were of both IgM and IgG classes appeared in the serum 14 days post infection. The mean IgG antibody response against FHV-1 was not significantly different between FIV infected and non-FIV infected cats (Fig. 3 ( b ) ) . However, the FHV-1 specific IgM antibody response was significantly depressed in FIV infected compared with non-FIV infected cats during the entire 2 month post infection observation period (Fig. 3 ( c ) ) . Antibodies to whole FHV-1 were not observed in sera collected from the two non-FHV- 1 control groups of cats. Antibody responses to F I V

IgG specific antibodies to FIV were detected in sera from all FIV infected cats. Mean FIV antibody titers in sera from FIV alone infected cats ranged from 1 : 893 to 1:960 (Fig. 4 (a)). In the FIV/FHV-1 infected cats, mean FIV antibody titers were 1 : 1355 on Day 0, 1:733 on Week 2, 1:725 on Week 3,

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and 1:836 on Week 4 post FHV-1 infection (Fig. 4(a) ).There was no statistically significant difference in the mean titers of the two groups at any time point.

Hematologicchanges The mean preinfection blood levels of neutrophils were lower in the two FIV infected than in the two non-FIV infected groups of cats, but the decrease was only significant for the FIV infected group that was subsequently infected with FHV-1 (Fig. 5 (a)). Neutrophilia was observed on Day 7 following FHV1 infection in both FIV infected and non-FIV infected animals. There was little increase in band neutrophils, with no difference in their numbers found in circulation during this neutrophilia. Toxic changes were found in neutrophils of the sickest cats in both groups. The ratio of the absolute blood neutrophil count to total clinical score on Day 7 post infection was significantly lower in FIV/FHV- 1 infected than in non-FIV infected cats ( P = 0.008). The ratio for the FIV/FHV-1 infected cats was 0.26_+0.03 vs 0.39_+0.04

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(mean + SEM) for the non-FIV infected cats. The mean blood neutrophil counts returned to their respective baseline levels in both FIV infected and non-FIV infected group by Days 21-28 following FHV-1 exposure. The mean blood neutrophil counts remained unchanged during the entire course of the study for the two non-FHV- 1 inoculated control groups of cats. Preinfection mean blood lymphocyte counts were similar in naive, FIV alone and FIV/FHV-1 infection groups (Fig. 5 ( b ) ) . However, cats in the FHV- 1 infection group had significantly higher mean preinfection blood lymphocyte counts. A pronounced blood lymphopenia was observed on Day 7

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1 10

G.H. REUBEL ET AL.

following FHV- 1 infection in both FIV and non-FIV infected cats. The mean blood lymphocyte counts returned to pre-exposure levels in both groups of cats by Day 14 post infection.There were no alterations in the mean blood lymphocyte counts in the two non-FHV-1 infected control groups during the period of the study. The mean blood levels of CD4 + T-lymphocytes, and the CD4 + / C D 8 + T-lymphocyte ratios, were significantly lower prior to the start of the experiment in the two groups of FIV infected cats compared with the two groups of non-FIV infected animals (Fig. 6(a) and 6 ( b ) ) . The mean blood levels of CD8 + T-lymphocytes were not significantly different in all four experimental groups at the start of the study (Fig. 6 (b)). Infection with FHV- 1 caused an acute drop in both CD4 + and CD8 + T-lymphocytes in both FIV infected and non-FIV infected cats (Figs. 6 (a) and 6 ( b ) ) . This decrease paralleled the lymphopenia and was the result of a proportional drop in the numbers of both CD4 + and CD8 + T-lymphocytes as indicated by a lack of change in the CD4 + / C D 8 + T-lymphocyte ratios during this time. The C D 4 + / C D 8 + T-lymphocyte ratios decreased at Day 14 following FHV- 1 infection in both FIV infected and non-FIV infected cats (Fig. 6 (c) ). This was a consequence of an absolute increase in the numbers of CD8 + Tlymphocytes without a concomitant change in the numbers of CD4 + T-lymphocytes (Figs. 6 (a) and 6 ( b ) ) . The absolute mean blood levels of the lymphocyte subsets and their ratios returned to preinfection baseline values by 3-4 weeks following exposure to FHV-1 in both FIV infected and non-FIV infected cats.

Specific and non-specific mitogen induced lymphocyte proliferation FHV- 1 infection caused an increased spontaneous uptake of [ 3H ] thymidine by lymphocytes in the blood as evidenced by control cultures that were not treated with either specific (inactivated whole FHV-1 ) or non-specific (Con A and PWM ) mitogens ( Fig. 7 (a) ). Spontaneous blastogenic activity peaked at Day 7 following FHV-1 inoculation in both FIV infected and non-FIV infected cats and then eventually returned to preinfection levels. Spontaneous in vitro lymphocyte blastogenesis remained elevated longer in the FIV/FHV1 infected cats than in the cats infected with FHV-1 alone. No spontaneous lymphocyte blastogenesis was observed during the study in the two non-FHV1 exposed control groups. Blood lymphocyte blastogenesis responses to specific FHV-1 antigen appeared shortly after FHV-1 infection in both FIV infected and non-FIV infected cats (Fig. 7 ( b ) ) . The FHV-1 specific antigen responses tended to parallel the spontaneous blastogenic responses. However, the FIV infected cats responded at levels twice that of spontaneous blastogenesis and sustained these levels for a longer period of time. Although the FIV infected cats appeared to

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respond better to the specific antigen, the differences were of borderline significance (P= 0.052). Blood lymphocytes from the two non-FHV- 1 infected control groups failed to respond to specific antigen during the course of the study. The in vitro blastogenesis response of blood lymphocytes to Con A and

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P W M was highest before FHV-1 infection in the naive control group of cats. The two groups of cats with pre-existent FIV infections had significantly lower responses to P W M prior to infection with FHV-1 and throughout the entire experimental period (Fig. 8 (a)). Responses to Con A tended to be more similar among the four groups of cats.

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Blood lymphocyte blastogenesis responses to the non-specific mitogens Con A and PWM were markedly decreased at Day 7 following FHV-1 exposure in both FIV infected and non-FIV infected groups and rapidly returned to baseline levels (Figs. 8 (a) and 8 (b)). The lymphocyte responsiveness to Con A was significantly lower for the FIV infected compared with non-FIV infected cats only at Day 7 following inoculation, while PWM responsiveness of the FIV infected animals remained significantly depressed compared with nonFIV infected cats for 3 weeks. DISCUSSION

Cats with pre-existing and outwardly asymptomatic FIV infection became significantly more ill after FHV-1 infection than cats that were not FIV infected. The results of this study confirmed those of similar experiments with different common feline pathogens. Cats that were in the primary stage of experimental FIV infection became more ill after being artificially infected with feline calicivirus and had diminished antibody responses (Dawson et al., 1991 ). FIV infected cats also became ticker after being infected with Chlamydia psittaci, vat. felis than non-FIV infected cats (O'Dair ct al., 1991 ). Although FIV infected cats developed a more severe disease following FHV1 infection, the pattern of illness was different than what was expected. It was anticipated that FIV infected cats would not only become more ill, but would remain sicker for a longer period of time, shed more FHV-1, have an extended period of FHV-1 carriage, and develop some of the chronic manifestations of FHV-1-related disease, e.g. chronic rhinitis, chronic conjunctivitis or chronic keratitis. However, most of the differences in FHV-1-related illness between FIV infected and non-FIV infected cats were observed in the acute rather than chronic stages of the illness, and manifestations of chronic FHV-1 related signs were not observed at all. Reasons for the increased severity of the acute FHV-1 induced disease in the FIV infected cats were not precisely determined. However, several hematologic and immunologic differences were noted between FIV infected and non-FIV infected animals. These differences appeared in neutrophil and lymphocyte numbers, lymphocyte subsets, lymphocyte blastogenesis responses, and primary antibody responses to FHV-1. FIV infected cats had significantly lower blood neutrophil counts prior to FHV-1 infection compared with non-FIV infected animals. Neutropenia is documented in clinical reports of people infected with HIV (Costello, 1988; Mir et al., 1989) and of cats infected with FIV (Shelton et al., 1989, 1990), but sequential neutrophil counts in response to FIV infection have not been monitored so far. A pronounced neutrophilia was observed at Day 7 following FHV-1 infection. Although the FIV infected cats started out with lower mean blood neutrophils, they were able to mount a mean neutrophil response

1 14

G.H. REUBEL ET AL.

of equal magnitude to that of the non-FIV infected cats. However, individual FIV infected cats that developed the most severe clinical signs of FHV-l induced illness were usually the cats that had the lowest neutrophil counts before FHV-1 infection, the cats that were least able to m o u n t a neutrophilia during infection, and the cats that produced the lowest titers of neutralizing antibodies. Evidently the intrinsic factors that decrease blood neutrophil counts in FIV infected cats do not necessarily prevent increased production a n d / o r release of neutrophils during times of inflammation. After the neutrophilia subsided, the mean blood neutrophil counts of cats with pre-existing FIV infection declined to their lower than normal preinfection levels. FIV infected cats had significantly lower total blood lymphocyte counts than non-FIV infected cats prior to infection with FHV-1. An absolute lymphopenia occurred following infection with FHV-1. However, the decrease in lymphocyte numbers in FIV infected and non-FIV infected cats paralleled each other and at the lowest point both groups were equally lymphopenic. The marked drop in lymphocyte counts of the cats infected with FHV-1 on Day 7 post infection may have reflected the stress of disease or, more likely, a recruitment of lymphocytes from the blood to lymphoid organs as a result of generalized i m m u n e response (Hall and Morris, 1963 ). The FIV infected cats had significantly lower CD4 + T-lymphocytes at the start of the infection than did non-FIV infected cats, consistent with the CD4 + T-lymphocyte depletion in people with HIV infection (Costello, 1988 ) and in FIV infected cats (Barlough et al., 1991; Torten et al., 1991 ). Infection with FHV-1 induced an acute drop in the numbers of both CD4 + and CD8 ÷ T-lymphocytes in both FIV infected and non-FIV infected cats that was most apparent on Day 7 post infection, coinciding with the absolute lymphopenia. The CD4 + / C D 8 ÷ T-lymphocyte ratios were unchanged at this time indicating that the lymphopenia was the result of an equal loss of both CD4 + and C D 8 ÷ T-lymphocytes from the blood. On Day 14 post infection, when the total blood lymphocyte count had returned to normal, there was a significant but transient drop in the C D 4 + / C D 8 ÷ T-lymphocyte ratios in both FIV infected and non-FIV infected cats. The decrease in the ratios was a consequence of an approximate twofold increase over preinfection levels of CD8 + T-lymphocytes; CD4 ÷ T-lymphocytes had returned to baseline values. Day 14 of the infection was just prior to the time that humoral i m m u n e responses were levelling off, and it would be tempting to attribute the absolute increase in CD8 ÷ T-lymphocytes at this time to normal immunoregulatory mechanisms. The absolute C D 4 + and CD8 + T-lymphocyte counts and the C D 4 + / CD8 ÷ T-lymphocyte ratios at the end of the study in both FIV infected and non-FIV infected groups of cats were not significantly different from their preinfection levels. Thus, it appeared that FHV-1 infection did not have a long-term deleterious effect on the levels of C D 4 + T-lymphocytes in FIV infected cats.

FELINE HERPES AND IMMUNODEFICIENCY VIRUS INFECTIONS

1 15

Long-term experimentally FIV infected cats develop marked cellular immune dysfunctions such as loss of absolute CD4 + T-lymphocyte numbers and suppression of cell mediated immunity as measured by mitogenic lymphocyte proliferation (Taniguchi et al., 1990; Barlough et al., 1991; Torten et al., 1991 ). A similar reduction of PWM and Con A responsiveness was observed in both FIV infected groups prior to the herpesvirus challenge indicating that the FIV infected cats in the study were already becoming immunocompromised. Transient perturbations of mitogenic lymphocyte blastogenesis occur frequently in infectious diseases of normal cats (Gaskell and Povey, 1982; Tham and Studdert, 1987 ). The intensity and duration of this immune dysfunction, however, were significantly more prominent in the FIV infected than in the non-FIV infected cats. FHV-1 infection appeared to cause an increase in activated blood mononuclear cells, as evidenced by the peak of spontaneous lymphocyte proliferation observed in non-mitogen treated lymphocyte cultures taken on post infection Day 7. This spontaneous proliferation was more prominent in the FIV infected than non-FIV infected cats. In contrast to the suppressed non-specific mitogen response, antigen specific lymphocyte reactivity was higher in the FIV infected than in the nonFIV infected cats, although it was not flamboyant in either group. The relatively poor FHV-1 antigen specific response in both groups exposed to FHV1 is in agreement with previous reports (Cocker et al., 1986; Tham and Studdert, 1987) and paralleled the spontaneous proliferation. FIV infected cats also have had a greater Toxoplasma gondii antigen specific blastogenesis response than non-FIV infected cats (Lappin et al., 1991 ). The most pronounced immunologic differences between FIV infected and non-FIV infected cats were in primary virus specific antibody responses. The IgM antibody responses to FHV-1 were significantly lower in FIV infected cats, while the FHV-1 specific IgG responses were unaffected. Subtle abnormalities in primary humoral immune responses to synthetic antigens have already been recognized in experimentally FIV infected cats (Torten et al., 1991 ). This antibody defect involves mainly T-lymphocyte dependent antigens and becomes more pronounced with time after infection and CD4 + Tlymphocyte losses. Although chronic FIV infection enhanced the acute FHV-1 induced disease, there was no evidence that the FHV-1 infection or immunity affected the underlying FIV infection. The acute FHV-1 infection did not change the antibody response to FIV. This could be considered as an indirect evidence that the generalized immunologic stimulation brought about by acute FHV-1 infection did not enhance replication of FIV. This was surprising because it is known that coinfection with FHV-1 will upregulate chloramphenicol acetyltransferase (CAT) expression by a FIV-LTR-CAT construct in an in vitro cell transfection assay (E.E. Sparger, Davis, personal communication, 1991 ).

116

G.H. REUBEL ET AL.

Human herpesvirus genes will also upregulate HIV in vitro (Laurence, 1990). However, preliminary studies failed to show upregulation of either FIV or FHV-1 replication by coinfection of cells with the two viruses in vitro (E.E. Sparger, personal communication, 1991 ). This corroborated what was observed in vivo in this study. There is no evidence up to this point that common feline infectious diseases accelerate the demise of the immune system in cats chronically infected with FIV. Non-specific immune activation has been theorized to switch on virus production in latently infected lymphocytes and to lead to cell death; increased lymphocyte destruction would then lead to an accelerated decline in specific subsets of T-lymphocytes and increased immunodeficiency (Zagury et al., 1986; Margolick et al., 1987 ). In spite of a vigorous clinical course of disease, and an apparent pronounced immune response to the causative agent, the immune system of the cats infected with FIV for 18 months still performed adequately in a qualitative, if not quantitative, manner. The degree of CD4 + T-lymphocyte depletion was also not enhanced by FHV-1 infection. However, the cats reported in this study have only experienced two sequential infections, toxoplasmosis and herpesvirus infection. It is thus possible that more numerous, more frequent, or more severe immune stimuli could accelerate the normal, time related decline in C D 4 + T-lymphocyte numbers or other immune functions that have already been observed in cats kept isolated from all pathogens (Barlough et al., 1991; Torten et al., 1991 ). ACKNOWLEDGMENTS

This work was supported by Public Health Service grants A1-25802-05 and CA-50179-03 to N.C.P. and 2RO 1 AI-26120-04 to C.K.G. from the National Institutes of Health. We thank Pro-Visions, Checkerboard Square, St. Louis, MO, for the supply of cat food. We are very grateful to Nancy Delemus, Kim Floyd-Hawkins, Jenni Del Carlo, Diane Hoffmann, Renan Acevedo, Peter Hill and Dr. Dorette Reubel for expert technical assistance. We also thank Dr. Gordon Theilen, Department of Surgery, School of Veterinary Medicine, University of California, Davis, in whose laboratory a portion of this research was performed.

REFERENCES Ackley, C.D., Hoover, E.A. and Cooper, M.D., 1990. Identification ofa CD4 homologue in the cat. Tissue Antigens, 35: 92-98. Armitage, P. and Berry, G., 1987. Statistical Methods in Medical Research. Blackwell Scientific Publications, Oxford, pp. 94-100, 411-416. Barlough, J.E., Ackley, C.D., George, J.W., Levy, N., Acevedo, R., Moore, P.F., Rideout, B.A.,

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Cooper, M.D. and Pedersen, N.C., 1991. Acquired immune dysfunction in cats with experimentally induced feline immunodeficiency virus infection: comparison of short-term and long-term infections. J. AIDS, 4(3): 219-227. Baskin, G.B., Martin, L.N., Rangan, S.R.S., Gormus, B.J., Murphey-Corb, M., Wolf, R.H. and Soike, K.F., 1988. Transmissible lymphoma and simian acquired immunodeficiency syndrome in rhesus monkeys. J. Natl. Cancer Inst., 77: 127-139. Carrigan, D.R., Knox, K.K. and Tapper, M.A., 1990. Suppression of human immunodeficiency virus type l replication by human herpesvirus-6. J. Infect. Dis., 162(4): 844-851. Cocker, F.M., Newby, T.J., Gaskell, R.M., Evans, P.A., Gaskell, C.J., Stokes, C.R., Harbour, D.A. and Bourne, J.F., 1986. Responses of cats to nasal vaccination with a live, modified feline herpesvirus type 1. Res. Vet. Sci., 41: 323-330. Costello, C., 1988. Haematological abnormalities in human immunodeficiency virus infection. J. Clin. Pathol., 41:711-715. Dawson, S., Smyth, N.R., Bennett, M., Gaskell, R.M., McCracken, C.M., Brown, A. and GaskeU, C.J., 199 I. Effect of primary-stage feline immunodeficiency virus infection on subsequent feline calicivirus vaccination and challenge in cats. AIDS, 5: 747-750. Eyster, M.E., Gail, M.H., Ballard, J.O., A1-Mondhiri, H. and Goedert, J.J., 1987. Natural history of human immunodeficiency virus infection in hemophiliacs: effects of T cell subsets, platelet counts, and age. Ann. Intern. Med., 107: 1-6. Gaskell, R.M. and Goddard, L.E., 1984. The epizootiology of feline viral rhinotracheitis with particular reference of the nature and role of the carrier state. In: G.I. Wittmann, R.M. Gaskell and H.J. Rziha (Editors), Latent Herpesvirus Infections in Veterinary Medicine. Martinus Nijhoff, Brussels, pp. 337-349. Gaskell, R.M. and Povey, R.C., 1982. Transmission of feline rhinotracheitis. Vet. Rec., I 11: 359-362. Gregory, C.R., Taylor, N.J., Willits, N.H. and Theilen, G.H., 1987. Response to isoantigens and mitogens in the cat: effects of cyclosporin A. Am. J. Vet. Res., 48 ( l ): 126-130. Hall, J.G. and Morris, B., 1963. The lymph-borne cells of the immune response. Q. J. Exp. Physiol., 48: 235-247. Jain, N.C., 1986. Schalm's Veterinary Hematology. Lea & Febiger. Philadelphia. Jason, J., Kung-Jong, L., Ragni, M.V., Hessol, N.A. and Darrow, W.W., 1989. Risk of developing AIDS in HIV-infected cohorts of hemophiliac and homosexual men. J. Am. Med. Assoc., 261: 725-727. Klotz, F.W. and Cooper, M.D., 1986. A feline thymocyte antigen defined by a monoclonal antibody (FT2) identifies a subpopulation of non-helper cells capable of specific cytotoxicity. J. Immunol., 136: 2510-2514. Lappin, M.R., George, J.W., Pedersen, N.C., Badough, J.E. and Murphy, C., 1991. Experimental induction of toxoplasmosis in cats chronically infected with feline immunodeficiency virus. First Int. Conf. FIV Res., 4-7 September 1991, Davis, USA: 29. Laurence, J., 1990. Molecular interactions among herpesviruses and human immunodeficiency viruses. J. Infect. Dis., 162(2): 338-346. Mann, D.L., Murray, C., Yarchoan, R., Blattner, W.A. and Goedert, J.J., 1988. HLA antigen frequencies in HIV-seropositive disease-free individuals and patients with AIDS. J. AIDS, l: 13-17. Margolick, J.B., Volkman, D.J., Folks, T.M. and Fauci, A.S., 1987. Amplification of HTLV-III/ LAV infection by antigen-induced activation of T cells and direct suppression by virus of lymphocyte blastogenic responses. J. Immunol., 138:1719-1723. Mir, N., Costello, C., Luckit, J. and Lindley, R., 1989. HIV-disease and bone marrow changes: a study of 60 cases. Eur. J. Haematol., 42: 339-343. O'Connor, T.P.J., Tanguay, S., Steinman, R., Smith, R., Barr, M.C., Yamamoto, J.K., Pedersen, N.C., Andersen, P.R. and Tonelli, Q.J., 1989. Development and evaluation of immunoassay

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for detection of antibodies to the feline T-lymphotropic lentivirus (feline immunodeficiency virus). J. Clin. Microbiol., 27 ( 3 ): 474-479. O'Dair, H. A., Gruffyd-Jones, T. J. and Hopper, C. D., 1991. Co-infection of cats infected with feline immunodeficiency virus (FIV) with Chlamydia psittaci. First Int. Conf. FIV Res., 47 September 1991, Davis, USA: 28. Okubo, S. and Yasunaga, K., 1990. Significance of viral coinfections by HIV, HTLV-I, EpsteinBarr virus, and cytomegalovirus for immunological abnormalities in hemophiliacs. Cancer Detect. Prev., 14(3): 343-346. Ostrove J.M., Leonard, J., Weck, K.E., Rabson, A.B. and Gendelman, H.E., 1987. Activation of the human immunodeficiency virus by herpes simplex virus type I. J. Virol., 61 (12): 3726-3732. Pauly, J.L., Sokal, J.E. and Han, T., 1973. Whole-blood culture technique for functional studies of lymphocyte reactivity to mitogens, antigens and homologous lymphocytes. J. Lab. Clin. Med., 82: 500-512. Pedersen, N.C., 1988. Feline Infectious Diseases. American Veterinary Publications, Goleta, CA. Pedersen, N.C., Yamamoto, J.K., Ishida, T. and Hansen, H., 1989. Feline immunodeficiency virus infection. Vet. Immunol. Immunopathol., 21 ( 1): 111-129. Pedersen, N.C., Torten, M., Rideout, B., Sparger, E., Tonachini, T., Luciw, P.A., Ackley, C., Levy, N. and Yamamoto, J., 1990. Feline leukemia virus infection as a potentiating cofactor for the primary and secondary stages of experimentally induced feline immunodeficiency virus infection. J. Virol., 64 (2): 598-606. Povey, R.C., 1979. A review of feline rhinotracheitis (feline herpesvirus 1 infection). Comp. Immunol. Microbiol. Infect. Dis., 2: 373-387. Povey, R.C., Koonse, H. and Hays, M.B., 1980. Immunogenicity and safety of an inactivated vaccine for the prevention of rhinotracheitis, caliciviral disease, and panleukopenia in cats. J. Am. Vet. Med., 177(4): 347-350. Quinn, T.C., Piot, P., McCormick, J.B., Feinsod, F.M., Taelman, H., Kapita, B., Stevens, W. and Fauci, A.S., 1987. Serologic and immunologic studies in patients with AIDS in North America and Africa. The potential role of infectious agents in human immunodeficiency virus infection. J. Am. Med. Assoc., 257:2617-2621. Reed, L.J. and Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg., 27: 493-497. Schechter, M.T., Boyko, W.J., Jeffries, E., Willoughby, B., Nitz, R., Constance, P., Weaver, M., Wiggs, B. and O'Shaugnessy, M., 1985. The Vancouver lymphadenopathy-AIDS study: 4. Effects of exposure factors, cofactors and HTLV-III seropositivity on number of helper T cells. Can. Med. Assoc. J., 133: 286-292. Shelton, G.H., Abkowitz, J.L., Linenberger, M.L., Russell, R.G. and Grant, C.K., 1989. Chronic leukopenia associated with feline immunodeficiency virus infection in a cat. J. Am. Vet. Assoc., 194: 253-255. Shelton, G.H., Linenberger, M.L., Grant, C.K. and Abkowitz, J.L., 1990. Hematologic manifestations of feline immunodeficiency virus infection. Blood, 76:1104-1109. Taniguchi, A., Ishida, T., Konno, A., Washizu, T, and Tomoda, I., 1990. Altered mitogen response of peripheral blood lymphocytes in different stages of feline immunodeficiency virus infection. Jpn. J. Vet. Sci., 52(3): 513-518. Tham, K.M. and Studdert, M.J., 1987. Antibody and cell-mediated immune responses to feline herpesvirus 1 following inactivated vaccine and challenge. J. Vet. Med. B., 34(8): 585-597. Torten, M., Franchini, M., Barlough, J.E., George, J.W., Mozes, E., Lutz, H. and Pedersen, N.C., 1991. Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus. J. Virol., 65 (5): 2225-2230. Tremblay, M., Gornitsky, M. and Wainberg, M.A., 1989. Active replication of human immu-

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nodeficiency virus type 1 by peripheral blood mononuclear cells following coincubation with herpes viruses. J. Med. Virol., 29(2): 109-114. Watkins, J.D., Conway-Welch, C., Creedon, J.J., Crenshaw, T.L., DeVos, R.M., Gebbie, K.M., Lee, B.J. III., Lilly, F., O'Connor, J.C. and Primm, B.J., 1988. Interim Report of the Presidential Commission on the Human Immunodeficiency Virus Epidemic: Chairman's recommendations--Part 1. J. AIDS, 1 ( 1 ): 69-103. Webster, A., 1991. Cytomegalovirus as a possible cofactor in HIV disease progression. J. AIDS, 4 (suppl. 1 ): 47-52. Winkelstein, W.J., Lyman, D.M., Padian, N., Grant, R., Samuel, M., Wiley, J.A., Anderson, R.E., Lang, W., Riggs, J. and Levy, J.A., 1987. Sexual practices and risk of infection by the human immunodeficiency virus. J. Am. Med. Assoc., 257: 321-325. Yamamoto, J.K., Hansen, H., Ho, E.W., Morishita, T.Y., Okuda, T., Sawa, T.R., Nakamura, R.M. and Pedersen, N.C., 1989. Epidemiologic and clinical aspects of feline immunodeficiency virus infection in cats from the continental United States and Canada and possible mode of transmission. J. Am. Vet. Med. Assoc., 194 (2): 213-220. Zagury, D., Bernard, J., Leonard, R., Cheynier, R., Feldman, M., Sarin, P.S. and Gallo, R.C., 1986. Long term cultures of HTLV-III-infected cells: a model of cytopathology of T-cell depletion in AIDS. Science, 231: 850-853.

Veterinary Immunology and Immunopathology, 35 (1992) 121-131

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Elsevier Science Publishers B.V., Amsterdam

Effect of primary phase feline immunodeficiency virus infection on cats with chronic toxoplasmosis Michael R. Lappin a, Peter W. Gasper b, Barbara J. Roseb and Cynthia C. Powella aDepartment of Clinical Sciences, College of VeterinaryMedicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA bDepartment of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA

ABSTRACT Lappin, M.R., Gasper, P.W., Rose, B.J. and Powell, C.C., 1992. Effect of primary phase feline immunodeficiency virus infection on cats with chronic toxoplasmosis. Vet. Immunol. Immunopathol., 35: 121-131. The effect of primary phase feline immunodeficiency virus (FIV)infection on clinical signs, hematological values, Toxoplasma gondii oocyst shedding, T. gondii-specific serology, T. gondii-specific cell-mediated immune responses, non-specific cell-mediated immune responses, and lymphocyte subpopulations from cats with experimentally induced chronic toxoplasmosis was studied. No significant clinical or hematologic abnormalities were noted following inoculation with FIV. T. gondii-specific IgM was significantly increased, concanavalin A, T. gondii tachyzoite antigen and T. gondii secretory antigen induction of lymphocyte transformation were significantly suppressed, and CD4 + cell numbers were significantly decreased following inoculation with FIV. The changes were attributed to FIV effects on the immune system and resultant activated toxoplasmosis. ABBREVIATIONS Con A, concanavalin A; FeLV, feline leukemia virus; FIV, feline immunodeficiency virus; HAG, host cell antigen; IL-2, interleukin-2; PI, post inoculation; SAG, secretory antigens; TAG, tachyzoite antigens; WBC, white blood cells, leukocytes.

INTRODUCTION

Experimentally induced feline immunodeficiency virus infection (FIV) of cats induces a distinct primary phase of infection characterized by transient Correspondence to: M.R. Lappin, Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

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fever, neutropenia, and lymphadenopathy and leads chronically to progressive immune dysfunction (Yamamoto et al., 1988; Barlough et al., 1991; Torten et al., 1991 ). Clinical disease induced by opportunistic infections may be common in cats immunosuppressed by FIV (Yamamoto et al., 1989 ). Toxoplasma gondii and FIV coinfections have been documented in cats (Witt et al., 1989; Lappin et al., 1989a, 1991 a; Heidel et al., 1990; O'Neil et al., 1991 ). Some cats have shown clinical signs of activated toxoplasmosis and have responded to anti-Toxoplasma drugs (Lappin et al., 1989a; O'Neil et al., 1991 ). Activated toxoplasmosis resulting in severe clinical disease is commonly diagnosed in people with the acquired immunodeficiency syndrome (Holland et al., 1988; Grant et al., 1990). Cats are the only known definitive host of T. gondii (Dubey, 1986). Cats that have completed the enteroepithelial cycle of infection can have oocyst shedding induced for a second time following the administration of immunosuppressive doses of glucocorticoids (Dubey and Frenkel, 1974). It is unknown whether the immunosuppression induced by primary phase FIV infection is sufficient to induce oocyst shedding in cats with chronic toxoplasmosis. Feline leukemia virus (FeLV) infection was shown to be a potentiating cofactor for FIV infection (Pedersen et al., 1990). Cats previously infected with FeLV developed more severe clinical, hematologic, and immunologic abnormalities when infected with FIV than cats with FeLV or FIV infections alone (Pedersen et al., 1990). Toxoplasma gondii infection of mice was associated with T-lymphocyte dysfunction (Chanet al., 1986 ). Recent T. gondii infection in cats was shown to be immunosuppressive (Lappin et al., 199 lb ). Whether previous infection with T. gondii will potentiate FIV infection is undetermined. Toxoplasma gondii-specific humoral and cell-mediated immune responses of naturally infected cats with serologic evidence of T. gondii and FIV coinfection are often different than those of T. gondii-infected, FIV-naive cats (Lappin et al., 1989a; Witt et al., 1989; O'Neil et al., 1991 ). Toxoplasma gondii-infected, FIV-infected cats commonly have persistent T. gondii-specific IgM titers, elevated T. gondii-specific IgM titers, and either increased (Witt et al., 1989 ) or decreased T. gondii-specific IgG titers when compared with T. gondii-infected, FIV-naive cats (Lappin et al., 1991a; O'Neil et al., 1991 ). These changes were hypothesized to be the result of FIV effects on immune responses. The purpose of this study was to define the effects of primary phase FIV infection on clinical signs, hematologic values, T. gondii oocyst shedding, T. gondii-specific serology, T. gondii-specific cell-mediated immune responses, non-specific cell-mediated immune responses, and lymphocyte subpopulations from cats with experimentally induced chronic toxoplasmosis.

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MATERIALS AND METHODS

Experimental animals Specific pathogen free domestic shorthair cats were purchased and housed in infectious disease isolation facilities. Animal maintenance was in accordance with current federal guidelines and experimental protocols approved by a campus-wide research committee. Each cat was shown to be seronegative for antibodies against T. gondii, FIV (CiteRCombo; IDEXX, Portland, ME), and FeLV antigen p27 (CiteR Combo; IDEXX) prior to induction of experimental infection.

Experimentally induced T. gondii infection Strain CF-1 mice were inoculated subcutaneously with 1 × 10 4 T. gondii strain ME49 sporulated oocysts (J.P. Dubey, Animal Parasitology Institute, BeRsville, MD). At 6 weeks post inoculation (PI), the brains were collected from the mice following the induction of anesthesia and euthanasia by cervical dislocation. Brain tissue was homogenized in sterile 0.9% NaC1 by repeatedly drawing the tissue through an 18 gauge needle. Tissue cysts were quantitated using a hemocytometer. After an overnight fast, each cat was administered 1.0 × 103 tissue cysts orally.

Experimentally induced FIV infection Cats were infected with the Petaluma isolate of FIV by intravenous administration of 0.5 ml heparinized whole blood collected from a chronically infected cat (N.C. Pedersen, School of Veterinary Medicine, Davis, CA). While clinically normal, this cat was shown to have persistent leukopenia and an inverted CD4 + / C D 8 + T-lymphocyte ratio. Infection by FIV was confirmed by demonstration of IgG antibodies against FIV in the serum of all experimentally infected cats by Week 4 PI.

Experimental design A total of ten age-matched cats were identified for study: one FIV-naive T.

gondii-naive cat; one FIV-infected T. gondii-naive cat; one FIV-naive T. gondii-infected cat; seven FIV-infected T. gondii-infected cats. Toxoplasma gondii-infected cats had been seropositive for 6 months prior to the initiation of this study. Fecal samples were collected for analysis by sugar centrifugation (Dubey, 1973) daily for 30 days PI and then once or twice weekly for the duration of the project. Blood was obtained by jugular venipuncture prior to infection by FIV and on Weeks 4, 8, 12, and 16 PI. The cats were evaluated

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daily for clinical signs of anorexia or depression. Each cat was evaluated for ocular manifestations of disease by slit lamp biomicroscopy and indirect ophthalmoscopy prior to infection with FIV and on Weeks 4, 8, 12, and 16 PI.

Complete blood cell counts Whole blood was placed immediately into sterile EDTA tubes ( 1.5 ml draw) following collection and mixed thoroughly. Evaluations of blood obtained prior to inoculation with FIV and on Weeks 4, 8, and 12 PI included electronic total leukocyte (WBC), erythrocyte, and platelet counts (Coulter S Plus IV System), microhematocrit determination, and WBC differential from Wright-Geimsa-stained blood smears. T. gondii serology Serum collected prior to inoculation with FIV and on Weeks 4, 8, 12, and 16 PI was assayed for T. gondii-specific IgM and T. gondii-specific IgG using enzyme-linked immunosorbent assays (ELISA) as previously described (Lappin et al., 1989b) with the following adaptations. Positive control serum, negative control serum, and serum from experimental animals were diluted 1:64 in 0.01 M phosphate-buffered saline (pH 7.2) plus 0.05% Tween 20. Each diluted serum sample was pipetted into triplicate wells (50/tl per well) of a microELISA plate and the IgM ELISA and IgG ELISA performed as described by Lappin et al. (1989b). The mean absorbance of each diluted serum sample was calculated. The mean absorbance results of the suspect serum samples were converted to %ELISA using the results of the negative control serum as the test blank (Waltman et al., 1984). Chromagen and enzyme-control wells were included on each plate.

T. gondii-specific lymphocyte stimulation microassay Whole blood collected prior to inoculation with FIV and on Weeks 4, 8, and 12 PI was placed into heparin tubes immediately following collection and mixed thoroughly. Lymphocyte stimulation microassays using concanavalin A (Con A), T. gondii sonicated tachyzoite antigens (TAG), T. gondii secretory antigens (SAG), and host cell antigen (HAG) were performed as previously described (Lappin et al., 1991a, 1992 ) with the following adaptations. Lymphocyte separation was performed using a Histopaque (Sigma Chemical Co., St. Louis, MO) concentration gradient. Lymphocyte numbers were adjusted to 2× 106 cells m1-1 and 0.1 ml of the cell suspensions was pipetted into each culture well. Triplicate cultures of each sample were made and stim-

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ulated by Con A and both TAG and SAG as follows. Con A was used at a concentration of 10 #g ml-1. TAG was assessed at concentrations of 2.5 ag m l - 1, 5.0 #g m l - 1, and 10/tg m l - 1. SAG was assessed at concentrations of 40 /tg m1-1, 80 #g m1-1, and 160 pg m1-1. Unstimulated cultures were used as the control for the tritiated thymidine. Lymphoblast transformation in response to the mitogen and antigens was calculated using the mean change in counts m i n - 1 between the stimulated cultures and the unstimulated cultures.

Lymphocyte subset enumeration C D 4 + and CD8 + lymphocytes were enumerated using monoclonal antibodies to feline CD4 + and CD8 + lymphocytes and fluorescence-activated cytometry (Dean et al., 1991 ).

Interleuldn-2 quantitation Intedeukin-2 (IL-2) concentrations were calculated using an adaptation of the tetrazolium salt method (Hansen et al., 1989). Lymphocytes were isolated as described above for the lymphocyte stimulation microassay. Lymphocytes were concentrated to 2 X 106 cells ml-1 and incubated with 1 ml of Con A (10/tg m1-1 ) for 24 h at 37°C in 5°/0 C O 2. The lymphocyte suspensions were then centrifuged at 400 × g for l 0 rain followed by removal of the supernatants which were stored frozen at - 7 0 °C until assayed. Serial dilutions of stock murine IL-2 (250 U m l - l ; recombinant murine interleukin-2; Genzyme Corporation, Cambridge, MA) and each supernate collected as above were made in triplicate in a 96 well plate (Falcon; Becton Dickinson, Lincoln Park, N J). Mouse IL-2-dependent cytotoxic T-cells (CTLL-2; American Type Culture Collection, Rockville, M D ) were diluted in CTLL growth medium (2 ml L-glutamine, 1 ml sodium pyruvate, 2 ml penicillin-streptomycin in 100 ml of X-vivo; Whittaker Bioproducts, Walkersville, MD) and 2 × 104 cells were pipetted into each well. Following a 20 h incubation at 37°C in 5% CO2, 50/d o f a 5 mg ml-1 concentration of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemicals, St. Louis, MO) was pipetted into each well. Following a 2 h incubation at 37°C in 5% CO2, 100 #l of lysing buffer (20% sodium dodecyl sulfate dissolved in 50% N,N-dimethylformamide and 50% distilled H20, pH adjusted to 4.7 with 2.5% of 80% acetic acid and 2.5% 1 N hydrochloric acid; Sigma) was pipetted into each well. Following overnight incubation at 37 °C in 5% CO2 the plate was read with a microELISA reader (Titertek Multiskan; ICN Laboratories, Costa Mesa, CA) equipped with a 690 nm reference filter and a 560 nm test filter. The absorbance values of the IL-2 standards were plotted

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as a standard curve and the IL-2 levels in the unknown samples were determined from this standard curve.

Statistical evaluation Group mean results were calculated and compared statistically with the twotailed Student's t-test using the FIV preinoculation group mean from FIVinfected T. gondii-infected cats as the expected mean. Significance was defined as P < 0.05. In each case of statistical significance using these criteria, the results were also compared with individual control cat values and found to be significant. RESULTS Overt clinical illness did not develop in any cat following inoculation with FIV. Significant changes in hematologic parameters were not noted in any cat during the study period. Each cat was serologically negative for FIV and T. gondii prior to inoculation with T. gondii. The oocyst shedding period following primary exposure to T. gondii was from 4 to 11 days PI; oocysts were detected in the feces of six cats. Recurrence of oocyst shedding was not detected following inoculation with FIV. Antibodies against FIV were detected in the serum of each inoculated cat by Week 4 PI. Each cat developed detectable T. gondii-specific IgM and IgG antibody titers following inoculation by T. gondii. The group mean ELISA IgM %ELISA 25

20

15

10

4

8

12

16

Weeks post-inoculation Toxo+/FIV+

~

Toxo+/FIV-

~

Toxo-/FIV-

--X-- Toxo-/FIV+

Fig. 1. Sequential T. gondii-specificIgM %ELISAresults from control cats and cats experimentally inoculatedwith T. gondii (6 months previously) and feline immunodeficiencyvirus (Week 0). *Statisticallysignificant.

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IgG %ELISA 100

80:

I

60' 40

20

0

4

8

12

16

W e e k s post-inoculation --g'- Toxo + IF|V +

+

Toxo +/FIV-

-~-

Toxo-IFIV-

--X-- Toxo-/FIV-

Fig. 2. Sequential T. gondii-specific IgG %ELISA results from control cats and cats experimentally inoculated with T. gondii (6 months previously) and feline immunodeficiency virus (Week

0). Counts per minute-(thousands) 1210864-

200

4

8

12

Weeks post-inoculation ~1

ConcanavalinA

~

TAG

~

SAG

Fig. 3. Sequential mitogen and T. gondii antigen-specific lymphocyte responses from cats experimentally inoculated with T. gondii (6 months previously) and feline immunodeficiency virus (Week 0). *Statistically significant. TAG, tachyzoite antigens; SAG, secretory antigens.

results were high for T. gondii-specific IgG (%ELISA= 82.6) and low for T. gondii,specific IgM (%ELISA = 0.9 ) at the time the cats were inoculated with FIV. A significant rise in T. gondii-specific IgM occurred on Week 4 (t = 4.402; P<0.01 ) and Week 8 (t--2.899; P < 0 . 0 5 ) following inoculation with FIV with return to baseline levels by Weeks 12 and 16 PI (Fig. 1 ). Significant changes in T. gondii-specific IgG did not occur during the study (Fig. 2 ).

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T-helper lymphocyte numbers/mcl 2000

1500

lOO0

500 0

~ 4

8

12

Weeks post-inoculation +

Toxo + / F I V +

Fig. 4. Sequential T-helper lymphocyte numbers (CD4 + ) from cats experimentally inoculated with T. gondii (6 months previously) and FIV (Week 0). *Statistically significant.

Group mean lymphocyte responses to Con A were suppressed on Week 4 (t=3.859; P<0.01 ) and Week 8 ( t = 5.067; P<0.01 ). Group mean lymphocyte responses to TAG were suppressed on Week 8 ( t = 5.343; P < 0.01 ). Group mean lymphocyte responses to JAG were suppressed on Week 8 ( t = 13.177; P < 0.005; Fig. 3 ). Group mean absolute CD4 + lymphocyte numbers were suppressed on Week 12 PI (t=5.534; P<0.01; Fig. 4). The group mean C D 4 + / C D 8 + ratio was decreased on Week 8 PI ( P < 0 . 1 ) and Week 12 ( P < 0.1 ), but the changes were not significant. No significant changes in group mean IL-2 levels were noted following inoculation with FIV. DISCUSSION

Increased group mean T. gondii-specific IgM ELISA results following inoculation with FIV is suggestive of activated toxoplasmosis. Similar results occurred in cats with chronic toxoplasmosis following the administration of methylprednisolone acetate (Lappin et al., 1991 b). It is likely that the effects of primary phase FIV infection on the immune system allows for transient increased proliferation of T. gondii bradyzoites encysted in tissues leading to increased antigen production. Increased circulating T. gondii antigens stimulate virgin B-lymphocytes to produce T. gondii-specific IgM in a T-independent fashion. Alternatively, the effect of FIV on antibody-producing cells may lead to increased levels of specific antibodies. B-lymphocyte activation is documented in HIV-1 infected patients (Amadori and Chieco-Bianchi, 1990; Ascher and Sheppard, 1990). It recently was shown that FIV directly infects B-lymphocytes (English et al., 1991 ). If B-lymphocyte activation occurs in FIV-infected cats, it is possible that some cells may produce specific antibodies against certain antigens.

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When compared with preinoculation values, significant suppression of Con A (Weeks 4 and 8), TAG (Week 8) and SAG (Week 8) lymphocyte responses were noted following inoculation with FIV. These abnormalities occurred independent of significant CD4 + cell number changes, CD4 +/CD8 + ratio changes, or decreases in IL-2 levels, and had normalized by Week 12 PI. Thus, this apparent non-specific immunosuppression appears to be transient and independent of FIV effects on lymphocyte subpopulation numbers or IL2 production capabilities. However, the early effects of FIV on CD4 + cells may be qualitative (Bishop et al., 1991 ). Deficits associated with IL-2 could also be secondary to decreased IL-2 receptor expression or decreased receptor affinity for IL-2. Alternatively, specific and non-specific suppression of lymphocyte transformation may be a manifestation of activated toxoplasmosis. Recent T. gondii infection in mice (Chan et al., 1986) and people (Sklenar et al., 1986; Luft et al., 1987) was shown to be immunosuppressive. Immunosuppression appears to be mediated by both macrophages and T-lymphocytes. Cats with recent toxoplasmosis (8 weeks) that received 5 mg methylprednisoloneacetate kg- 1 weekly developed significant suppression of Con A induced lymphocyte transformation whereas cats with chronic toxoplasmosis ( 14 months) that received 5 mg methylprednisoloneacetate kg- 1weekly did not (Lappin et al., 199 lb). These results suggest that recent T. gondii infection in cats can be immunosuppressive. Thus, the changes noted in lymphocyte responsiveness in the cats described herein may have resulted from activated toxoplasmosis induced by FIV coinfection. It appears that the degree of immunosuppression or activation of T. gondii induced by this model was insufficient for the induction of clinical toxoplasmosis, recurrence of oocyst shedding, or potentiation of the primary phase of FIV. These findings may be caused by the strain or infective dosage of T. gondii and FIV used or the relative chronicity of T. gondii infection prior to inoculation with FIV. It is possible that small numbers of oocysts may have been missed because of the relative insensitivity of fecal flotation techniques. Bioassay would have been a more sensitive technique. The cats may also have cleared T. gondii from the tissues prior to inoculation with FIV. This seems unlikely since each cat had exacerbation of T. gondii-specific IgM, suggesting activation of infection as discussed above. Failure to document hematologic changes may have been related to sample procurement timing. Cats with chronic FIV infection develop short-term ( < 4 weeks) neutropenia following inoculation with T. gondii (Lappin et al., 1991c). ACKNOWLEDGMENTS

Supported in part by a grant from an anonymous pet animal research foundation and PHS grant R29-CA46371. The authors thank Christi Cooper for

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technical assistance and Dr. G.A. Dean for performing the fluorescence-activated cytometry.

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