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Evaluation of lymphocyte populations in dogs naturally infected by Ehrlichia canis with and without clinical signs
Ticks and Tick-borne Diseases 3 (2012) 278–281
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Evaluation of lymphocyte populations in dogs naturally infected by Ehrlichia canis with and without clinical signs Alejandra Villaescusa a,∗ , Miguel Angel Tesouro b , Mercedes García-Sancho a , Tania Ayllón a , Fernando Rodríguez-Franco a , Angel Sainz a a b
Dept. of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Complutense University of Madrid, Avda Puerta de Hierro s/n, 28040 Madrid, Spain Dept. of Veterinary Medicine, Surgery and Anatomy, Faculty of Veterinary Medicine, University of León, Campus de Vegazana s/n, 24071 León, Spain
a r t i c l e Keywords: Ehrlichia Tick-borne disease Dog Flow cytometry Lymphocyte Immune response
i n f o
a b s t r a c t Immune response elicited by the host during ehrlichial infections could influence the clinical signs and laboratory and pathological findings. Twenty-eight dogs naturally infected by Ehrlichia canis were included in this study. Twenty of them presented only laboratory findings traditionally associated with canine monocytic ehrlichiosis (CME), whilst 8 dogs also showed clinical signs classically associated with CME (pale mucous membranes, fever, lymphadenopathy, weight loss, anorexia, lethargy or signs attributable to bleeding tendencies). A multiparametric flow cytometric study was performed to analyze the distribution of the main lymphocyte subsets (T, Th, Tc, B, and those that express MHC class II) in the peripheral blood. Statistically significant differences between dogs naturally infected by E. canis in a clinical or subclinical stage were not detected when evaluating lymphocyte subsets in peripheral blood samples. Dogs with clinical signs showed lower relative and absolute values of B lymphocytes than dogs without clinical signs, although the differences were not statistically significant. © 2012 Elsevier GmbH. All rights reserved.
Introduction Ehrlichia canis is the main aetiological agent of canine monocytic ehrlichiosis (CME). After an incubation period of 8–20 days, the course of CME has been classically divided into acute, subclinical, and chronic phases (Harrus et al., 1999). The acute phase of the disease begins approximately 10 days post infection and lasts 2–4 weeks. During this phase, dogs usually present thrombocytopenia, leukopenia, fever, depression, and anorexia. Most dogs recover from the acute disease after adequate therapy (Neer and Harrus, 2006), but in some cases, the acute phase is followed by a subclinical phase that can last from months to years (Waner et al., 1997; Harrus et al., 1998). The most consistent finding during the subclinical stage in experimental infections is thrombocytopenia, but hyperglobulinaemia, anaemia, and leukopenia have also been described (Codner and Farris-Smith, 1986; Harrus et al., 1997). Some dogs suffering from the subclinical phase of CME can develop the chronic phase of the disease, which may be mild to severe. Clinical signs described during this phase are numerous and include multisystemic, ocular, neuromuscular, and articular signs (Neer and Harrus, 2006). Usually dogs chronically infected develop bi- or pancytopenia, lymphocytosis, hypoalbuminaemia, hyperglobulinaemia, and elevated
∗ Corresponding author. Tel.: +34 91 3943807; fax: +34 91 3943808. E-mail address: [email protected] (A. Villaescusa). 1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.034
serum ALT and ALP activities (Harrus et al., 1997; Mylonakis et al., 2004; Neer and Harrus, 2006). The conditions that cause a subclinically infected dog to develop clinical signs are not fully understood. Nevertheless, it may be related to the strain of the parasite, the breed of the dog, the immune status of the animal, stress conditions, coinfections with other parasites, geographical location, and/or persistent reinfections (Hegarty et al., 1997; Breitschwerdt et al., 1998; Harrus et al., 1999). Specifically, it has been suggested that the immune response elicited by the host during an infection could influence pathogenesis, clinical signs, laboratory and pathological findings (Harrus et al., 1999; de Castro et al., 2004; Neer and Harrus, 2006). The aim of the present study was to evaluate the peripheral blood lymphocyte subsets in dogs naturally infected by E. canis with or without clinical signs of the disease. Materials and methods Animals Twenty-eight dogs naturally infected by E. canis, 12 males and 16 females, with a mean age of 5.7 ± 1.1 years, were included in the study. All dogs followed a protocol approved by the Animal Experimentation Committee of the Complutense University of Madrid and were diagnosed of CME using indirect immunofluorescent antibody (IFA) test and/or PCR, based on clinical suspicion. Besides,
A. Villaescusa et al. / Ticks and Tick-borne Diseases 3 (2012) 278–281
dogs included in the study had to present clinical signs and/or laboratory abnormalities compatible with CME. Exclusion criteria were: positivity to Anaplasma spp., Neorickettsia spp., or Leishmania spp. (using serology or PCR), the diagnosis of other concurrent disease or coinfections, or a previous therapy with tetracyclines or steroids. Two groups of animals were evaluated: - dogs with subclinical infection (n = 20), without clinical signs of CME, but with laboratory findings traditionally associated with CME (thrombocytopenia, anaemia, and/or hyperproteinaemia) - dogs with clinical disease (n = 8), with clinical signs classically associated with CME (pale mucous membranes, fever, lymphadenopathy, weight loss, anorexia, lethargy or signs attributable to bleeding tendencies). Blood collection Blood was collected from each dog by cephalic or jugular venipuncture in EDTA- and heparin-anticoagulant tubes for haematology, biochemical analysis, serology, and PCR against
279
Ehrlichia/Anaplasma/Neoricketttsia spp. and Leishmania spp., and flow cytometry. Samples for biochemical analysis were centrifuged at 2500 rpm for 10 min before analysis. IFA test Samples were screened for IgG against E. canis (strain Madrid), A. phagocytophilum (Protatek, St. Paul, Minnesota), N. risticii (kindly donated by Dr. I. Kakoma, University of Illinois) and L. infantum (L-75), as previously described (Amusategui et al., 2008). Cut-off was established at an antibody titre of 1:80 for E. canis, 1:40 for A. phagocytophilum and N. risticii, and 1:100 for L. infantum. PCR DNA was extracted from 200 l of blood in EDTA using a commercial kit (UltraCleanTM DNA BloodSpin Kit, Mo Bio Laboratories, Inc., Carlsbad, California, USA). The final eluted volume of DNA extracted was 200 l per sample. DNA concentration was quantified by spectrophotometry (NanoDropTM , Thermo Scientific,
Table 1 Haematology and blood chemistry values in dogs naturally infected by E. canis in a clinical or subclinical stage of the disease. Parameter RBC (×106 /l) HGB (g/dl) PCV (%) Mean cell volume (MCV) (fl) Mean cell haemoglobin (MCH) (pg) Mean cell haemoglobin concentration (MCHC) (g/dl) Platelets × 10 /l 3
White blood cells × 103 /l Neutrophils/l Lymphocytes/l Monocytes/l Eosinophils/l Basophils/l Glucose (mg/dl) Urea (mg/dl) Creatinine (mg/dl) ALT (IU/l) Total protein (g/dl) Albumin (g/dl) Globulins (g/dl) ␣1-Globulins (g/dl) ␣2-Globulins (g/dl) -Globulins (g/dl) ␥-Globulins (g/dl) Albumin/globulins ratio
CME stage
Average
Standard deviation
Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical
5.07 6.75 11.65 15.44 36.5 48.4 71.45 71.90 22.83 22.88 32.15 31.96 160.63 182.38 10.721 10.549 5367.83 6508.65 4167 2740.18 641.56 443.32 245.66 695.3 10.11 19.7 97.17 89.41 26.04 25.47 0.82 0.88 117.14 38.45 8.14 7.06 2.12 2.95 6.67 4.06 0.22 0.28 0.48 0.62 1.94 1.82 4 1.31 0.33 0.77
1.13 1.01 3.08 2.29 10.12 7.05 6.93 5.77 1.59 1.38 3.15 2.42 129.06 90.69 6.123 6.688 2911.33 5814.74 4858.91 1317.81 498.6 317.19 219.1 533.69 15.52 21.57 28.25 61.09 19.51 14.78 0.23 0.31 230.66 25.92 1.89 0.94 0.35 0.47 1.76 0.98 0.05 0.1 0.13 0.19 0.48 0.42 2.14 0.96 0.11 0.26
p-Value <0.001 0.001 <0.001 0.852 0.932 0.851 0.595 0.948 0.598 0.437 0.178 0.002 0.178 0.765 0.932 0.665 0.402 0.189 0.001 0.028 0.207 0.145 0.592 0.048 0.001
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Table 2 Relative and absolute counts of different lymphocyte subset populations in dogs with CME in a clinical or subclinical stage of the disease. Parameter Lymphocytes T (CD3+) (%) Lymphocytes T (CD3+) (/l) Lymphocytes Th (CD3+CD4+) (%) Lymphocytes Th (CD3+CD4+) (/l) Lymphocytes Tc (CD3+CD8+) (%) Lymphocytes Tc (CD3+CD8+) (/l) Lymphocytes B (CD21+) (%) Lymphocytes B (CD21+) (/l) Lymphocytes CMH II+ (%) Lymphocytes CMH II+ (/l) Lymphocytes CD3−CD21− (%) Lymphocytes CD3−CD21− (/l) CD4/CD8 ratio
CME stage
Average
Standard deviation
Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical
73.27 71.98 2332.91 2096.38 29.08 33.87 1323.6 927.63 30.26 27.41 1985.39 900.58 10.43 15.13 251.34 386.25 91.41 92.05 3993.14 2656.76 28.13 11.47 377.81 292.11 2.41 1.68
12.54 8.38 2277.64 1155.54 13.21 8.28 1934.91 448.15 25.77 15.06 2772.09 834.52 10.87 6.6 221.94 239.36 5.74 5.12 4685.83 1384.51 26.35 7.23 194.45 197.69 3.6 0.98
p-Value 0.757 0.71 0.390 0.610 0.703 0.344 0.140 0.165 0.763 0.451 0.147 0.313 0.613
Delaware, USA). Presence of PCR inhibitors was evaluated by the amplification of a fragment of the constitutive gene for the GAPDH protein (Birkenheuer et al., 2003). Generic PCR for detection of Ehrlichia/Anaplasma/Neorickettsia spp. was performed using the GEP-s and GEP-as, as previously described (Eddlestone et al., 2007). Specific amplification of E. canis was obtained using the primers E. canis-s and GEP-as (Beall et al., 2008). PCR for amplification of L. infantum DNA was performed using the primers LSH-1 and LSH-2 (Lachaud et al., 2002). Positivity was confirmed in every case by analysis of sequence of the amplified bands.
cell volume (PCV), haemoglobin (HGB), eosinophils, albumin, and albumin/globulins ratio than dogs with subclinical infection (Table 1). Besides, dogs with clinical signs presented higher levels of globulins (specifically gamma-globulins) than dogs without clinical signs (Table 1). Statistically significant differences were not detected when evaluating lymphocyte subsets in peripheral blood samples in both groups of animals (Table 2). Dogs with clinical signs showed lower relative and absolute values of CD21+ B lymphocytes than dogs without clinical signs, although the differences were not statistically significant (p = 0.140 and p = 0.165, respectively).
Flow cytometry
Discussion
A multiparametric flow cytometric study using a FACSCalibur flow cytometer was performed to analyze the distribution of the main lymphocyte subsets (T, Th, Tc, B, and those that express MHC class II) in each sample. Monoclonal antibodies were supplied by AbD Serotec and included: anti-canine CD3 (clone CA17.2A12) conjugated with FITC, anti-canine CD4 (clone YKIX302.9) conjugated with PE, anti-canine CD8 (clone YCATE55.9) conjugated with AlexaFluor 647, anti-canine CD21 (clone CA2.1D6) conjugated with PE, and anti-canine MHC class II (clone YKIX334.2) conjugated with FITC.
Severity of disease and organ pathology can depend on the host immune response type, taking into account different studies performed in murine models (Ismail et al., 2010; Ghose et al., 2011). Immune response of the dog during ehrlichial infection also seems to play an essential role in the pathogenesis of the disease and in the presence of different clinical signs and laboratory and pathological findings (Harrus et al., 1999; Neer and Harrus, 2006). The course of CME in dogs experimentally infected with E. canis has been investigated extensively (Waner et al., 1997; Harrus et al., 1999; de Castro et al., 2004; Neer and Harrus, 2006). Alteration of the host immune system by using cyclophosphamide and antilymphocyte serum has proven to alter the pathologic and clinical manifestations of experimental E. canis infection (Reardon and Pierce, 1981). However, data on immunopathogenesis of canine ehrlichiosis in natural infections is scanty, probably due to the difficulties to perform an accurate staging of the disease in practice (Neer and Harrus, 2006) and to the common possibility of concurrent infections or diseases. Some authors consider that E. canis-seropositive dogs, with bior pancytopenia associated with bone marrow hypoplasia could satisfy the diagnostic criteria for the chronic phase of the disease (Harrus et al., 1997; Mylonakis et al., 2004). Most of the dogs with clinical signs included in the present study were assumed to present a mild chronic ehrlichiosis, taking into account the time of diagnosis (in seasons where ticks are absent), the favourable follow-up
Analysis of data Statistical analysis was performed using the Statgraphics (Centurion XVI version) software. Student’s t-test or Wilcoxon test were performed, considering a level of significance of p < 0.05. Results Clinical signs in dogs with clinical disease included weight loss (n = 5), bleeding tendencies (n = 5), depression (n = 4), lethargy (n = 4), fever (n = 4), and anorexia (n = 3). Alterations in haematology, blood biochemistry, and protein electrophoresis were commonly detected in dogs from both groups. Dogs in a clinical stage of CME showed lower values of red blood cells (RBC), packed
A. Villaescusa et al. / Ticks and Tick-borne Diseases 3 (2012) 278–281
after therapy, and some laboratory abnormalities detected in these cases (especially non-regenerative anaemia, bi- or pancytopenia, and medullary hypoplasia). In the authors’ experience, the severe form of chronic ehrlichiosis is not very common in the area of study, and most infected dogs recover after appropriate therapy. Results obtained when analyzing the immune response in dogs with severe chronic ehrlichiosis could differ from the values obtained in the present study. Dogs with clinical infection showed more pronounced laboratory abnormalities than dogs in a subclinical stage. In agreement with these results, some authors consider that alterations in the globulin levels can be related with the length of the infection (Woody and Hoskins, 1991). Results of immunophenotype obtained in dogs with subclinical ehrlichiosis from the present study are concordant with those from previous studies (Lorente et al., 2008). Although there were no significant differences in the lymphocyte subsets analyzed between dogs with clinical and subclinical infection, dogs with clinical signs tends to present lower absolute and relative CD21+ B lymphocytes. Similarly, differences in lymphocyte subsets depending on the presence or absence of clinical signs have been described in canine diseases caused by other intracellular pathogens. Specifically, a higher percentage of CD8+ T lymphocytes in peripheral blood is associated with subclinical disease and a low parasite load in canine leishmaniasis (Reis et al., 2006). The role of B lymphocytes in ehrlichial infections could be related with a protective role, probably mediated by the secretion of different cytokines and by the stimulation of Ehrlichia-specific CD4+ T lymphocytes (Yager et al., 2005; Bitsaktsis et al., 2007). However, it is also possible that these lower values of B cells in peripheral blood in dogs with clinical signs could be associated with a higher presence of these lymphocytes and plasma cells in kidney, spleen, and bone marrow in clinical phases of CME (Codner et al., 1992; Neer and Harrus, 2006). Further studies are needed in order to elucidate the mechanisms involved in cell-mediated and humoral-mediated immune responses in dogs naturally infected by E. canis. References Amusategui, I., Tesouro, M.A., Kakoma, I., Sainz, A., 2008. Serological reactivity to Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii, Borrelia burgdorferi and Rickettsia conorii in dogs from northwestern Spain. Vector Borne Zoonotic Dis. 8, 797–803. Beall, M.J., Chandrashekar, R., Eberts, M.D., Cyr, K.E., Diniz, P.P., Mainville, C., Hegarty, B.C., Crawford, J.M., Breitschwerdt, E.B., 2008. Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector Borne Zoonotic Dis. 8, 455–464. Birkenheuer, A.J., Levy, M.G., Breitschwerdt, E.B., 2003. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. J. Clin. Microbiol. 41, 4172–4177.
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Bitsaktsis, C., Nandi, B., Racine, R., MacNamara, K.C., Winslow, G., 2007. Tcell-independent humoral immunity is sufficient for protection against fatal intracellular ehrlichia infection. Infect. Immun. 75, 4933–4941. Breitschwerdt, E.B., Hegarty, B.C., Hancock, S.I., 1998. Doxycycline hyclate treatment of experimental canine ehrlichiosis followed by challenge inoculation with two Ehrlichia canis strains. J. Vet. Intern. Med. 42, 362–368. Codner, E.C., Farris-Smith, L.L., 1986. Characterization of the subclinical phase of ehrlichiosis in dogs. J. Am. Vet. Med. Assoc. 189, 47–50. Codner, E.C., Caceci, T., Saunders, G.K., Smith, C.A., Robertson, J.L., Martin, R.A., Troy, G.C., 1992. Investigation of glomerular lesions in dogs with acute experimentally induced Ehrlichia canis infection. Am. J. Vet. Res. 53, 2286–2291. de Castro, M.B., Machado, R.Z., de Aquino, L.P., Alessi, A.C., Costa, M.T., 2004. Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings. Vet. Parasitol. 119, 73–86. Eddlestone, S.M., Diniz, P.P., Neer, T.M., Gaunt, S.D., Corstvet, R., Cho, D., Hosgood, G., Hegarty, B., Breitschwerdt, E.B., 2007. Doxycycline clearance of experimentally induced chronic Ehrlichia canis infection in dogs. J. Vet. Intern. Med. 21, 1237–1242. Ghose, P., Ali, A.Q., Fang, R., Forbes, D., Ballard, B., Ismail, N., 2011. The interaction between IL-18 and IL-18 receptor limits the magnitude of protective immunity and enhances pathogenic responses following infection with intracellular bacteria. J. Immunol. 187, 1333–1346. Harrus, S., Bark, H., Waner, T., 1997. Canine monocytic ehrlichiosis: an update. Compend. Contin. Educ. Pract. Vet. 19, 431–444. Harrus, S., Waner, T., Aizenberg, I., Foley, J.E., Poland, A.M., Bark, H., 1998. Amplification of ehrlichial DNA from dogs 34 months after infection with Ehrlichia canis. J. Clin. Microbiol. 36, 73–76. Harrus, S., Waner, T., Bark, H., Jongejan, F., Cornelissen, A.W., 1999. Recent advances in determining the pathogenesis of canine monocytic ehrlichiosis. J. Clin. Microbiol. 37, 2745–2749. Hegarty, B.C., Levy, M.G., Gager, R.F., Breitschwerdt, E.B., 1997. Immunoblot analysis of the immunoglobulin G response to Ehrlichia canis in dogs: an international survey. J. Vet. Diagn. Invest. 9, 32–38. Ismail, N.M.D., Bloch, K.C., McBride, J.C., 2010. Human ehrlichiosis and anaplasmosis. Clin. Lab. Med. 30, 261–292. Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J.P., Bastien, P., 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40, 210–215. Lorente, C., Sainz, A., Tesoro, M.A., 2008. Immunophenotype of dogs with subclinical ehrlichiosis. Ann. N.Y. Acad. Sci. 1149, 114–117. Mylonakis, M.E., Koutinas, A.F., Breitschwerdt, E.B., Hegarty, B.C., Billinis, C.D., Leontides, L.S., Kontos, V.S., 2004. Chronic canine ehrlichiosis (Ehrlichia canis): a retrospective study of 19 natural cases. J. Am. Anim. Hosp. Assoc. 40, 174–184. Neer, T.M., Harrus, S., 2006. Canine monocytotropic ehrlichiosis and neorickettsiosis (E. canis, E. chaffeensis, E. ruminantium, N. sennetsu, and N. risticii infections). In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat. Saunders Elsevier, St. Louis, pp. 203–216. Reardon, M.J., Pierce, K.R., 1981. Acute experimental canine ehrlichiosis. II. Sequential reaction of the hemic and lymphoreticular system of selectively immunosuppressed dogs. Vet. Pathol. 18, 384–395. Reis, A.B., Teixeira-Carvalho, A., Giunchetti, R.C., Guerra, L.L., Carvalho, M.G., Mayrink, W., Genaro, O., Correa-Oliveira, R., Martins-Filho, O.A., 2006. Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi. Clin. Exp. Immunol. 146, 303–311. Waner, T., Harrus, S., Bark, H., Bogin, E., Avidar, Y., Keysary, A., 1997. Characterization of the subclinical phase of canine ehrlichiosis in experimentally infected beagle dogs. Vet. Parasitol. 69, 307–317. Woody, B.J., Hoskins, J.D., 1991. Ehrlichial diseases of dogs. Vet. Clin. North Am. Small Anim. Pract. 21, 75–98. Yager, E., Bitsaktsis, C., Nandi, B., McBride, J.W., Winslow, G., 2005. Essential role for humoral immunity during Ehrlichia infection in immunocompetent mice. Infect. Immun. 73, 8009–8016.
Contents lists available at SciVerse ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Evaluation of lymphocyte populations in dogs naturally infected by Ehrlichia canis with and without clinical signs Alejandra Villaescusa a,∗ , Miguel Angel Tesouro b , Mercedes García-Sancho a , Tania Ayllón a , Fernando Rodríguez-Franco a , Angel Sainz a a b
Dept. of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Complutense University of Madrid, Avda Puerta de Hierro s/n, 28040 Madrid, Spain Dept. of Veterinary Medicine, Surgery and Anatomy, Faculty of Veterinary Medicine, University of León, Campus de Vegazana s/n, 24071 León, Spain
a r t i c l e Keywords: Ehrlichia Tick-borne disease Dog Flow cytometry Lymphocyte Immune response
i n f o
a b s t r a c t Immune response elicited by the host during ehrlichial infections could influence the clinical signs and laboratory and pathological findings. Twenty-eight dogs naturally infected by Ehrlichia canis were included in this study. Twenty of them presented only laboratory findings traditionally associated with canine monocytic ehrlichiosis (CME), whilst 8 dogs also showed clinical signs classically associated with CME (pale mucous membranes, fever, lymphadenopathy, weight loss, anorexia, lethargy or signs attributable to bleeding tendencies). A multiparametric flow cytometric study was performed to analyze the distribution of the main lymphocyte subsets (T, Th, Tc, B, and those that express MHC class II) in the peripheral blood. Statistically significant differences between dogs naturally infected by E. canis in a clinical or subclinical stage were not detected when evaluating lymphocyte subsets in peripheral blood samples. Dogs with clinical signs showed lower relative and absolute values of B lymphocytes than dogs without clinical signs, although the differences were not statistically significant. © 2012 Elsevier GmbH. All rights reserved.
Introduction Ehrlichia canis is the main aetiological agent of canine monocytic ehrlichiosis (CME). After an incubation period of 8–20 days, the course of CME has been classically divided into acute, subclinical, and chronic phases (Harrus et al., 1999). The acute phase of the disease begins approximately 10 days post infection and lasts 2–4 weeks. During this phase, dogs usually present thrombocytopenia, leukopenia, fever, depression, and anorexia. Most dogs recover from the acute disease after adequate therapy (Neer and Harrus, 2006), but in some cases, the acute phase is followed by a subclinical phase that can last from months to years (Waner et al., 1997; Harrus et al., 1998). The most consistent finding during the subclinical stage in experimental infections is thrombocytopenia, but hyperglobulinaemia, anaemia, and leukopenia have also been described (Codner and Farris-Smith, 1986; Harrus et al., 1997). Some dogs suffering from the subclinical phase of CME can develop the chronic phase of the disease, which may be mild to severe. Clinical signs described during this phase are numerous and include multisystemic, ocular, neuromuscular, and articular signs (Neer and Harrus, 2006). Usually dogs chronically infected develop bi- or pancytopenia, lymphocytosis, hypoalbuminaemia, hyperglobulinaemia, and elevated
∗ Corresponding author. Tel.: +34 91 3943807; fax: +34 91 3943808. E-mail address: [email protected] (A. Villaescusa). 1877-959X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2012.10.034
serum ALT and ALP activities (Harrus et al., 1997; Mylonakis et al., 2004; Neer and Harrus, 2006). The conditions that cause a subclinically infected dog to develop clinical signs are not fully understood. Nevertheless, it may be related to the strain of the parasite, the breed of the dog, the immune status of the animal, stress conditions, coinfections with other parasites, geographical location, and/or persistent reinfections (Hegarty et al., 1997; Breitschwerdt et al., 1998; Harrus et al., 1999). Specifically, it has been suggested that the immune response elicited by the host during an infection could influence pathogenesis, clinical signs, laboratory and pathological findings (Harrus et al., 1999; de Castro et al., 2004; Neer and Harrus, 2006). The aim of the present study was to evaluate the peripheral blood lymphocyte subsets in dogs naturally infected by E. canis with or without clinical signs of the disease. Materials and methods Animals Twenty-eight dogs naturally infected by E. canis, 12 males and 16 females, with a mean age of 5.7 ± 1.1 years, were included in the study. All dogs followed a protocol approved by the Animal Experimentation Committee of the Complutense University of Madrid and were diagnosed of CME using indirect immunofluorescent antibody (IFA) test and/or PCR, based on clinical suspicion. Besides,
A. Villaescusa et al. / Ticks and Tick-borne Diseases 3 (2012) 278–281
dogs included in the study had to present clinical signs and/or laboratory abnormalities compatible with CME. Exclusion criteria were: positivity to Anaplasma spp., Neorickettsia spp., or Leishmania spp. (using serology or PCR), the diagnosis of other concurrent disease or coinfections, or a previous therapy with tetracyclines or steroids. Two groups of animals were evaluated: - dogs with subclinical infection (n = 20), without clinical signs of CME, but with laboratory findings traditionally associated with CME (thrombocytopenia, anaemia, and/or hyperproteinaemia) - dogs with clinical disease (n = 8), with clinical signs classically associated with CME (pale mucous membranes, fever, lymphadenopathy, weight loss, anorexia, lethargy or signs attributable to bleeding tendencies). Blood collection Blood was collected from each dog by cephalic or jugular venipuncture in EDTA- and heparin-anticoagulant tubes for haematology, biochemical analysis, serology, and PCR against
279
Ehrlichia/Anaplasma/Neoricketttsia spp. and Leishmania spp., and flow cytometry. Samples for biochemical analysis were centrifuged at 2500 rpm for 10 min before analysis. IFA test Samples were screened for IgG against E. canis (strain Madrid), A. phagocytophilum (Protatek, St. Paul, Minnesota), N. risticii (kindly donated by Dr. I. Kakoma, University of Illinois) and L. infantum (L-75), as previously described (Amusategui et al., 2008). Cut-off was established at an antibody titre of 1:80 for E. canis, 1:40 for A. phagocytophilum and N. risticii, and 1:100 for L. infantum. PCR DNA was extracted from 200 l of blood in EDTA using a commercial kit (UltraCleanTM DNA BloodSpin Kit, Mo Bio Laboratories, Inc., Carlsbad, California, USA). The final eluted volume of DNA extracted was 200 l per sample. DNA concentration was quantified by spectrophotometry (NanoDropTM , Thermo Scientific,
Table 1 Haematology and blood chemistry values in dogs naturally infected by E. canis in a clinical or subclinical stage of the disease. Parameter RBC (×106 /l) HGB (g/dl) PCV (%) Mean cell volume (MCV) (fl) Mean cell haemoglobin (MCH) (pg) Mean cell haemoglobin concentration (MCHC) (g/dl) Platelets × 10 /l 3
White blood cells × 103 /l Neutrophils/l Lymphocytes/l Monocytes/l Eosinophils/l Basophils/l Glucose (mg/dl) Urea (mg/dl) Creatinine (mg/dl) ALT (IU/l) Total protein (g/dl) Albumin (g/dl) Globulins (g/dl) ␣1-Globulins (g/dl) ␣2-Globulins (g/dl) -Globulins (g/dl) ␥-Globulins (g/dl) Albumin/globulins ratio
CME stage
Average
Standard deviation
Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical
5.07 6.75 11.65 15.44 36.5 48.4 71.45 71.90 22.83 22.88 32.15 31.96 160.63 182.38 10.721 10.549 5367.83 6508.65 4167 2740.18 641.56 443.32 245.66 695.3 10.11 19.7 97.17 89.41 26.04 25.47 0.82 0.88 117.14 38.45 8.14 7.06 2.12 2.95 6.67 4.06 0.22 0.28 0.48 0.62 1.94 1.82 4 1.31 0.33 0.77
1.13 1.01 3.08 2.29 10.12 7.05 6.93 5.77 1.59 1.38 3.15 2.42 129.06 90.69 6.123 6.688 2911.33 5814.74 4858.91 1317.81 498.6 317.19 219.1 533.69 15.52 21.57 28.25 61.09 19.51 14.78 0.23 0.31 230.66 25.92 1.89 0.94 0.35 0.47 1.76 0.98 0.05 0.1 0.13 0.19 0.48 0.42 2.14 0.96 0.11 0.26
p-Value <0.001 0.001 <0.001 0.852 0.932 0.851 0.595 0.948 0.598 0.437 0.178 0.002 0.178 0.765 0.932 0.665 0.402 0.189 0.001 0.028 0.207 0.145 0.592 0.048 0.001
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Table 2 Relative and absolute counts of different lymphocyte subset populations in dogs with CME in a clinical or subclinical stage of the disease. Parameter Lymphocytes T (CD3+) (%) Lymphocytes T (CD3+) (/l) Lymphocytes Th (CD3+CD4+) (%) Lymphocytes Th (CD3+CD4+) (/l) Lymphocytes Tc (CD3+CD8+) (%) Lymphocytes Tc (CD3+CD8+) (/l) Lymphocytes B (CD21+) (%) Lymphocytes B (CD21+) (/l) Lymphocytes CMH II+ (%) Lymphocytes CMH II+ (/l) Lymphocytes CD3−CD21− (%) Lymphocytes CD3−CD21− (/l) CD4/CD8 ratio
CME stage
Average
Standard deviation
Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical Clinical Subclinical
73.27 71.98 2332.91 2096.38 29.08 33.87 1323.6 927.63 30.26 27.41 1985.39 900.58 10.43 15.13 251.34 386.25 91.41 92.05 3993.14 2656.76 28.13 11.47 377.81 292.11 2.41 1.68
12.54 8.38 2277.64 1155.54 13.21 8.28 1934.91 448.15 25.77 15.06 2772.09 834.52 10.87 6.6 221.94 239.36 5.74 5.12 4685.83 1384.51 26.35 7.23 194.45 197.69 3.6 0.98
p-Value 0.757 0.71 0.390 0.610 0.703 0.344 0.140 0.165 0.763 0.451 0.147 0.313 0.613
Delaware, USA). Presence of PCR inhibitors was evaluated by the amplification of a fragment of the constitutive gene for the GAPDH protein (Birkenheuer et al., 2003). Generic PCR for detection of Ehrlichia/Anaplasma/Neorickettsia spp. was performed using the GEP-s and GEP-as, as previously described (Eddlestone et al., 2007). Specific amplification of E. canis was obtained using the primers E. canis-s and GEP-as (Beall et al., 2008). PCR for amplification of L. infantum DNA was performed using the primers LSH-1 and LSH-2 (Lachaud et al., 2002). Positivity was confirmed in every case by analysis of sequence of the amplified bands.
cell volume (PCV), haemoglobin (HGB), eosinophils, albumin, and albumin/globulins ratio than dogs with subclinical infection (Table 1). Besides, dogs with clinical signs presented higher levels of globulins (specifically gamma-globulins) than dogs without clinical signs (Table 1). Statistically significant differences were not detected when evaluating lymphocyte subsets in peripheral blood samples in both groups of animals (Table 2). Dogs with clinical signs showed lower relative and absolute values of CD21+ B lymphocytes than dogs without clinical signs, although the differences were not statistically significant (p = 0.140 and p = 0.165, respectively).
Flow cytometry
Discussion
A multiparametric flow cytometric study using a FACSCalibur flow cytometer was performed to analyze the distribution of the main lymphocyte subsets (T, Th, Tc, B, and those that express MHC class II) in each sample. Monoclonal antibodies were supplied by AbD Serotec and included: anti-canine CD3 (clone CA17.2A12) conjugated with FITC, anti-canine CD4 (clone YKIX302.9) conjugated with PE, anti-canine CD8 (clone YCATE55.9) conjugated with AlexaFluor 647, anti-canine CD21 (clone CA2.1D6) conjugated with PE, and anti-canine MHC class II (clone YKIX334.2) conjugated with FITC.
Severity of disease and organ pathology can depend on the host immune response type, taking into account different studies performed in murine models (Ismail et al., 2010; Ghose et al., 2011). Immune response of the dog during ehrlichial infection also seems to play an essential role in the pathogenesis of the disease and in the presence of different clinical signs and laboratory and pathological findings (Harrus et al., 1999; Neer and Harrus, 2006). The course of CME in dogs experimentally infected with E. canis has been investigated extensively (Waner et al., 1997; Harrus et al., 1999; de Castro et al., 2004; Neer and Harrus, 2006). Alteration of the host immune system by using cyclophosphamide and antilymphocyte serum has proven to alter the pathologic and clinical manifestations of experimental E. canis infection (Reardon and Pierce, 1981). However, data on immunopathogenesis of canine ehrlichiosis in natural infections is scanty, probably due to the difficulties to perform an accurate staging of the disease in practice (Neer and Harrus, 2006) and to the common possibility of concurrent infections or diseases. Some authors consider that E. canis-seropositive dogs, with bior pancytopenia associated with bone marrow hypoplasia could satisfy the diagnostic criteria for the chronic phase of the disease (Harrus et al., 1997; Mylonakis et al., 2004). Most of the dogs with clinical signs included in the present study were assumed to present a mild chronic ehrlichiosis, taking into account the time of diagnosis (in seasons where ticks are absent), the favourable follow-up
Analysis of data Statistical analysis was performed using the Statgraphics (Centurion XVI version) software. Student’s t-test or Wilcoxon test were performed, considering a level of significance of p < 0.05. Results Clinical signs in dogs with clinical disease included weight loss (n = 5), bleeding tendencies (n = 5), depression (n = 4), lethargy (n = 4), fever (n = 4), and anorexia (n = 3). Alterations in haematology, blood biochemistry, and protein electrophoresis were commonly detected in dogs from both groups. Dogs in a clinical stage of CME showed lower values of red blood cells (RBC), packed
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after therapy, and some laboratory abnormalities detected in these cases (especially non-regenerative anaemia, bi- or pancytopenia, and medullary hypoplasia). In the authors’ experience, the severe form of chronic ehrlichiosis is not very common in the area of study, and most infected dogs recover after appropriate therapy. Results obtained when analyzing the immune response in dogs with severe chronic ehrlichiosis could differ from the values obtained in the present study. Dogs with clinical infection showed more pronounced laboratory abnormalities than dogs in a subclinical stage. In agreement with these results, some authors consider that alterations in the globulin levels can be related with the length of the infection (Woody and Hoskins, 1991). Results of immunophenotype obtained in dogs with subclinical ehrlichiosis from the present study are concordant with those from previous studies (Lorente et al., 2008). Although there were no significant differences in the lymphocyte subsets analyzed between dogs with clinical and subclinical infection, dogs with clinical signs tends to present lower absolute and relative CD21+ B lymphocytes. Similarly, differences in lymphocyte subsets depending on the presence or absence of clinical signs have been described in canine diseases caused by other intracellular pathogens. Specifically, a higher percentage of CD8+ T lymphocytes in peripheral blood is associated with subclinical disease and a low parasite load in canine leishmaniasis (Reis et al., 2006). The role of B lymphocytes in ehrlichial infections could be related with a protective role, probably mediated by the secretion of different cytokines and by the stimulation of Ehrlichia-specific CD4+ T lymphocytes (Yager et al., 2005; Bitsaktsis et al., 2007). However, it is also possible that these lower values of B cells in peripheral blood in dogs with clinical signs could be associated with a higher presence of these lymphocytes and plasma cells in kidney, spleen, and bone marrow in clinical phases of CME (Codner et al., 1992; Neer and Harrus, 2006). Further studies are needed in order to elucidate the mechanisms involved in cell-mediated and humoral-mediated immune responses in dogs naturally infected by E. canis. References Amusategui, I., Tesouro, M.A., Kakoma, I., Sainz, A., 2008. Serological reactivity to Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii, Borrelia burgdorferi and Rickettsia conorii in dogs from northwestern Spain. Vector Borne Zoonotic Dis. 8, 797–803. Beall, M.J., Chandrashekar, R., Eberts, M.D., Cyr, K.E., Diniz, P.P., Mainville, C., Hegarty, B.C., Crawford, J.M., Breitschwerdt, E.B., 2008. Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector Borne Zoonotic Dis. 8, 455–464. Birkenheuer, A.J., Levy, M.G., Breitschwerdt, E.B., 2003. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. J. Clin. Microbiol. 41, 4172–4177.
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