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Experimental studies on immunosuppressive effects of peste des petits ruminants (PPR) virus in goats
Comparative Immunology, Microbiology & Infectious Diseases 28 (2005) 287–296 www.elsevier.com/locate/cimid
Experimental studies on immunosuppressive effects of peste des petits ruminants (PPR) virus in goats K.K. Rajak, B.P. Sreenivasa, M. Hosamani*, R.P. Singh, S.K. Singh, R.K. Singh, S.K. Bandyopadhyay Division of Virology, Indian Veterinary Research Institute (IVRI), Mukteswar Campus, Nainital (Uttaranchal) 263138, India Accepted 27 July 2005
Abstract Effect of virulent and attenuated peste des petits ruminants (PPR) virus on the immune response to nonspecific antigen (ovalbumin) was investigated. Clinical and serological responses were monitored in goats administered with ovalbumin concurrently with either PPR vaccine or virulent virus. Study showed that PPR virulent virus causes marked immunosuppression as evidenced by leukopenia, lymphopenia, and reduced early antibody response to both specific and nonspecific antigen. These observations were predominant particularly during acute phase of disease (4–10 days post-infection). On the other hand, the vaccine virus induced only a transient lymphopenia without significantly affecting the immune response to nonspecific antigen or to itself during this period. Further, the antibody levels to ovalbumin in the group administered with virulent PPRV increased significantly between days 28 and 35 post-infection in comparison to the titers in other two groups given with either ovalbumin alone or in combination with vaccine. q 2005 Elsevier Ltd. All rights reserved. Re´sume´ L’effet du virus virulent et du virus atte´nue´ de la peste des petits ruminants sur la re´ponse immune a` e´te´ e´tudie´. Les re´ponses se´rologiques et cliniques ont e´te´ enregistre´es chez les che`vres qui ont rec¸u
Abbreviations p.v., post-vaccination; p.i., post-infection; PPR, peste des petits ruminants; RPV, rinderpest virus; VNT, virus neutralization test; c-ELISA, competitive ELISA; s-ELISA, sandwich ELISA; TLC, total leukocyte count; DLC, differential leukocyte count. * Corresponding author. Tel.: C91 5942 286348; fax: C91 5942 286347. E-mail address: [email protected] (M. Hosamani). 0147-9571/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2005.08.002
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de l’ovalbumine en meˆme temps que le vaccin contre la peste des petits rumiants ou le virus virulent lui-meˆne. Les etudes ont montre´ que le virus virulent de las peste des petits ruminants provoque une immunode´pression marquee caracte´rise´e par une leucope´nie, une lymphope´nie et une reduction de la re´ponse en anticorps a` la fois via-a`-vis de l’antige`ne spe´cifique et de l’antige`ne non-spe´cifique. Ces faits ont e´te´ observes surtout pendant la aigue¨ de la maladie (4 a`10 jours apre`s l’infection). Par ailleurs, le virus vaccin induit seulement une leucope´nie transitoire sans affecter significativement la re´ponse immune a` l’antige`ne non spe´cifique ou a` l’antige`ne spe´cifique Durant cette pe´riode. En outre´, le taux des anticorps antiovalbumine dans le groupe administer avec le virus virulent augmente sifnificativement entre les jours 28 et 35 apre`s l’infection en comparaison avec les titres obtenus dans les deux groupes, celui avec l’albumine seule ou celui avec l’albumine et al vaccin, q 2005 Elsevier Ltd. All rights reserved.
1. Introduction Peste des petits ruminants (PPR) is an acute, highly contagious viral disease in small ruminants causing huge economic loss in the endemic regions. Clinically, PPR is characterized by pyrexia, necrotic stomatitis, catarrhal inflammation of the ocular and nasal mucosa, enteritis and pneumonia followed by death or recovery from the disease. The disease is often associated with high morbidity and mortality [1] and is considered to be one of the main constraints in improving small ruminant productivity [2]. PPR is now endemic in India [3,4]. The etiological agent, PPR virus has been classified under genus Morbillivirus in the family Paramyxoviridae along with other members including rinderpest virus (RPV), measles virus (MV), canine distemper virus (CDV) and morbilliviruses of marine mammals [5,6]. PPR virus is antigenically closely related to RPV. For the control of PPR, tissue culture rinderpest (TCRP) vaccine was earlier used in sheep and goats in India. However, use of TCRP vaccine was discontinued in view of the interference of the virus in the ongoing serosurveillance programme launched by Government of India under National Project on Rinderpest Eradication (NPRE). A live attenuated homologous PPR vaccine has recently been developed at Indian Veterinary Research Institute (IVRI), Mukteswar (India) from an Indian isolate of PPR virus [7,8]. The vaccine has been tested extensively both in-house and field trials and has been found to be highly efficacious, safe and potent in small ruminants. Morbilliviruses including MV and RPV are known to cause immunosuppression [9–12]. However, there is meager information on the immunosuppressive effects of PPR virus and resulting interference in development of immunity to other antigens. Therefore, the present investigation was undertaken to study the effect of virus on the immune status of goats either on vaccination with attenuated PPR virus or on experimental infection with virulent PPR virus. 2. Materials and methods 2.1. Vero cell line Vero cells (ATCC CCL-13) were propagated in growth medium containing Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS,
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Sigma, St Louis, MI, USA). These cells were maintained in EMEM supplemented with 2% FBS. This cell line was used for virus neutralization test (VNT). 2.2. Vaccine virus The live attenuated vaccine was developed at National Morbillivirus Referral Laboratory, Division of Virology, Indian Veterinary Research Institute (IVRI), Mukteswar, using an indigenous isolate of PPR virus (PPRV Sungri/96) belonging to lineage IV [13]. 2.3. PPR virulent virus A highly virulent PPR virus (PPRV Izatnagar/94) isolated from an outbreak in Uttar Pradesh, India in 1994 was used for challenge experiments [14,15]. This virus is being maintained by animal-to-animal passage in the laboratory. A single subcutaneous inoculation of 2 ml of 10% splenic suspension of PPR virulent virus was used as Challenge virus. 2.4. Experimental animals Apparently healthy female hill goats between 1 and 2 years of age were used in the present study. These goats were screened for the absence of PPR virus antibody using a monoclonal antibody based competitive ELISA (c-ELISA) and VNT. For detection and titration of PPR antibodies, c-ELISA described by Singh et al. [16] was employed. Animals having titre of !40% inhibition (PI) value in c-ELISA test and neutralization titre of !1:8 in VNT were considered sero-negative for PPR. Twelve goats were randomly divided into three groups (A to C) of four animals each. Group A received ovalbumin in combination with 105TCID50 of PPR vaccine virus (approximately 2log10 higher than the prescribed dose). Ovalbumin (Sigma, St Louis, MI, USA) emulsified with equal volume of Freund’s incomplete adjuvant (Sigma) was used for inoculation of goats in different groups. Each animal received 2 mg of ovalbumin in 2 ml inoculum, by subcutaneous and intramuscular routes divided in equal doses. Goats in group B were injected with adjuvanted ovalbumin and 2 ml virulent PPR virus as splenic cell suspension by subcutaneous route. Group C received adjuvanted ovalbumin only, as control group. Animals in each group were housed separately in isolation sheds. 2.5. Clinical monitoring of animals Goats were clinically monitored daily after immunization for PPR specific symptoms. Rectal temperature was recorded daily and heparinized blood, ocular and nasal swabs and sera samples were collected at regular intervals. 2.6. Anti-ovalbumin antibody response Indirect ELISA was optimized for measuring anti-ovalbumin antibodies by checkerboard method. Ovalbumin was coated in flat-bottom 96-well plate (Nunc, Maxisorp) at
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4 mg per well in 100 ml carbonate bicarbonate buffer by incubation at 37 oC for 1 h. Unbound sites in the wells were blocked by addition of blocking solution (5% skim milk powder in PBS) for 1 h at 37 oC. Sera at optimized dilution (1:1000) were added in duplicate (100 ml in each well) and incubated for 1 h at 37 oC under constant shaking. Anti-goat HRPO Conjugate (Sigma, St Louis, MI, USA) was added at a dilution of 1:30, 000 in PBS to each well (100 ml) and incubated for 1 h at 37 oC under constant shaking. The plates were washed three-times with PBS (pre-warmed to 37 8C) containing 0.05% tween-20 between each incubation step. Color was developed by addition of 100 ml OPD substrate (Sigma, St Louis, MI, USA) after incubating at 37 8C without shaking for 10 min and optical density (OD) was taken in the ELISA reader at 492 nm (TECAN, Austria). 2.7. Total leukocyte count (TLC) and differential leukocyte count (DLC) Heparinized blood samples were collected for carrying out TLC and DLC as described by Jain [17] with little modification. For DLC, blood films were stained by the Giemsa’s method and a total of 100 cells were counted by following Battlement method of counting. 2.8. Detection of PPR virus Sandwich-ELISA test as described by Singh et al. [18] was employed for detection of PPR virus in clinical samples (ocular and nasal swabs) from PPR infected animals. The test used a polyclonal antiboby as capture antibody and monoclonal antibody to ‘N’ protein of PPRV as detection antibody. 2.9. Measurement of PPRV antibody response Detection of PPRV antibodies in the sera samples collected from PPR infected and vaccinated animals was carried out using monoclonal antibody (directed to H protein) based competitive ELISA (c-ELISA) test [16]. 3. Results 3.1. Clinical monitoring of animals on immunization and challenge Goats in group A remained apparently normal following vaccination. Expectedly, group B that received virulent virus with ovalbumin developed a clinical disease characteristic of PPR infection. These animals exhibited signs of pyrexia (Fig. 1), dullness and anorexia from day 4 p.i. Diarrhea, respiratory distress accompanied with coughing and ocular and nasal discharges were noticed between days 5 and 13 p.c. PPR viral antigen could be detected in nasal and ocular discharges collected from all the animals of group B between days 7 and 13 p.i. using sandwich-ELISA (Fig. 2a and b), whereas similar swabs collected from group A or group C did not show the presence of PPR antigen (data not shown). Animals of group C remained healthy throughout the study period.
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Fig. 1. Thermal reaction following infection with PPR virus: Group A (ovalbuminCPPR vaccine); Group B (ovalbuminCPPR challenge); Group C (ovalbumin alone). Each value represents the mean of four animals in each group.
3.2. Total leukocyte count (TLC) and differential leukocyte count (DLC) Vaccinated animals in group A showed a normal TLC picture following immunization (Fig. 3a). However, DLC picture indicated a transient but mild lymphopenia on day 6 p.v. (Fig. 3b). Group B which was inoculated with both ovalbumin and virulent PPR virus concurrently, showed a significant leukopenia from days 6 to 10 p.c. (Fig. 3a). A gradual and moderate decrease in lymphocyte count was also evident in this group from days 6 to 8 p.c. (Fig. 3b). In the ovalbumin control group, TLC and lymphocyte count remained within the normal limits.
Fig. 2. Detection of PPR antigen in (a) ocular and (b) nasal swabs in different groups using sandwich-ELISA. The antigen could be detected between days 7 and 13 post-infection in group B.
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Fig. 3. (a) Total leukocyte count (b) lymphocyte count in different groups of animals. A significant decline in TLC was observed with virulent virus (group B) between days 6 and 10, which was not evident in other groups. A transient decline in lymphocyte count was noticed on day 6 in group A and days 6–8 in group B animals.
3.3. Humoral immune response against PPR virus Development of PPRV specific antibodies in experimental animals was monitored by c-ELISA. There was a steady increase of antibody titre in group A till 14 days p.v. However, animals inoculated with virulent virus did not show any specific antibody response till day 7, which coincided with the peak rectal temperature. On the other hand, PPRV antibody response followed a normal trend both in groups A and B from day 14 post-inoculation. No PPRV antibodies could be detected in animals inoculated with ovalbumin (Fig. 4).
Fig. 4. Antibody response against PPR virus using competitive ELISA in different groups of animals. Kinetics of antibody response in different groups against PPRV showed a steady increase in antibody till day 14. Group B animals did not show any specific antibody response till day 7.
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Fig. 5. Antibody response against ovalbumin measured by indirect ELISA in different groups. Early antibody response against ovalbumin indicated a sharp increase on day 14 post-inoculation in group C, a moderate increase in group A, while titres were undetectable in group B.
3.4. Humoral immune response against ovalbumin Antibody response against ovalbumin in various groups of animals at weekly intervals was studied using indirect-ELISA. The kinetics of early antibody response revealed a sharp increase in ovalbumin control group from day 7 and a moderate rise in vaccinated group on day 14. However, in group B, the rise was not detectable until day 14 postinfection as depicted in Fig. 5. Further, a peak antibody titer was observed in the challenge group on day 35; whereas in rest of the groups, peak response was observed on day 21 post-inoculation followed by a plateau. When antibody responses among various groups were statistically compared, the response to ovalbumin on day 14 was found to be significantly lower in both the group A (P!0.05) as well as group B (P!0.01) as compared to the group C. Further on day 21 also, the antibody levels in group B remained significantly lower (P!0.05) as compared to other groups. Interestingly, antibody response in group B was remarkably high during days 28–35 p.c. in comparison to other two groups. Nevertheless, antibody responses against ovalbumin showed no significant difference among all groups by day 42 p.v.
4. Discussion Vaccination is the most ideal approach for the control of PPR in India where the disease is causing widespread outbreaks round the year. Tissue Culture Rinderpest (TCRP) vaccine was earlier used in sheep and goats for control of PPR, until homologous vaccine was developed at IVRI [7,8]. The vaccine has undergone extensive field trials with nearly more than half a million doses being used across the country without any adverse reactions. This Vero cell derived vaccine has an ability to induce
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strong humoral response in sheep and goats. However, in an isolated case, concurrent coccidial outbreak in an organized flock following PPR vaccination lead to speculation among the end users that the vaccine may induce immunosuppression giving rise to secondary infections. Although the vaccine under investigation has been proved to be completely attenuated [8] and apparently safe in target population including pregnant ones [19], the present study was undertaken to investigate whether the PPR vaccine virus causes immunosuppression. Morbilliviruses are known to induce strong humoral antibody response on vaccination or infection. In the present study, early antibody response during first 2 weeks using PPR vaccine and virulent challenge virus showed a marked difference. In group B, which received the challenge virus, no detectable level of antibodies could be observed during the first week. In comparison, the vaccinated animals (group A) showed a sharp increase in antibody response by day 7 p.v. This showed that the challenge virus delayed the induction of immune response against PPRV, suggestive of transient impairment of host immune responses. Hampered early antibody response to virulent virus coincided with an active phase of clinical disease that lasted for about 10 days. All the animals of group B exhibited clinical signs such as pyrexia, off feeding and dullness between days 4 and 10 p.i. Excretion of antigen in nasal and ocular secretions was also demonstrable between days 7 and 13 p.i. in this group. This observation was consistent with earlier studies carried out by Singh et al. [20] in this laboratory. Antibody response to PPRV returned to a normal trend in both the groups after 14 days. Immune response to egg albumin has been used to study the nutrient modulation of immune responses [21,22]. In this experiment, effect of vaccination and experimental challenge infection on the immune response to ovalbumin was assessed. The antiovalbumin antibody response in goats was monitored after inoculation of ovalbumin along with either PPR vaccine or PPR challenge virus. Antibody response on day 14 was significantly lower in animals of both the groups A (P!0.05) and B (P!0.01) in comparison to group C animals. Challenge virus used in group B caused a profound inhibitory effect on antibody response to an unrelated antigen (ovalbumin). This effect persisted in group B even up to day 21 p.c. (P%0.05). On the other hand, PPR vaccine appeared to reduce the development of early antibody response transiently. Data on titration of antibody response every 2/3 days would have been more comprehensive. However it cannot be ruled out that concurrent immunization of a nonreplicating antigen with other live viral vaccines can affect the antibody response to the former. Nevertheless, after 28 days, a plateau was observed in groups A and C without significant difference among them, while the titres in group B increased significantly high between days 28 and 35 post-inoculation. PPR virus has got strong affinity for epithelial cells of gastrointestinal tract and lymphoid tissue [23]. The total and differential leukocyte counts serve as indicators for studying pathogenesis of lymphotropic viruses like PPRV. Lymphopenia is one of the important indicators of virus induced immune suppression in both RPV [24] and PPRV [25,26]. In the present study, mean TLC in the vaccinated group appeared to be in normal range although a slight decline was evident in early stage of vaccination. The results are partially in agreement with a similar report on vaccine against RPV [9]. A transient lymphopenia on day 6 p.v. was also observed in group A animals. Lymphopenia can be
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attributed to lymphocyte trapping in the lymphoid tissues that may occur early during induction of immune response following local replication of virus. This may also be attributed to the high dose of vaccine virus used, which was 100 times more than the recommended field dose of vaccine. In group B, marked decline in TLC was observed between 6–10 days following inoculation of challenge virus in goats. A moderate lymphodepletion was also evident between days 4 and 6 post-infection, which coincided with severity of clinical symptoms. Anderson et al. [27] have also reported profound leukopenia with virulent rinderpest virus, which was later linked to marked immunosuppression caused by this virus [9]. From the present investigation, it could be concluded that PPR vaccine may transiently suppress the generation of immune response to other antigens. However, it remains to be seen if this property is associated with other live viral vaccines as well. Further, it cannot be inferred from the data that vaccination may lead to impairment of overall immune responses against nonspecific antigens including replicating ones. The transient reduction of immune responses may not be significant enough to cause secondary infections. Recent studies involving RBOK strain of Rinderpest vaccine also showed that the vaccine strain may not cause biologically significant immune suppression [9]. Further, it could be demonstrated that the virulent PPR virus causes significant immunosuppression as indicated by reduced humoral antibody response to PPR virus, ovalbumin antigen as well as marked leukopenia and lymphopenia during active phase of infection.
Acknowledgements Authors acknowledge the Director, IVRI, Izatnagar for providing the necessary facilities for carrying out the work. This work was financially supported from NPRE, Government of India. We are grateful to Dr. Srini Kaveri for providing the French translation of the abstract.
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[8] Sreenivasa BP, Dhar P, Singh RP, Bandyopadhyay SK. Evaluation of an indigenously developed homologous live attenuated cell culture vaccine against peste des petits ruminants infection of small ruminants. In: proceedings of the XX annual conference of indian association of veterinary microbiologists, immunologists and specialists in infectious diseases and national symposium on trends in vaccinology for animal diseases, (College of veterinary Science, G.B.Pant University of Agriculture and Technology, Pantnagar, Uttaranchal, India); 2000, pp. 84. [9] Lund TB, Tiwari A, Galbraith S, Baron MD, Morrison WI, Barrett T. Vaccination of cattle with attenuated rinderpest virus stimulates CD4CT-cell responses with broad viral antigen specificity. J Gen Virol 2000;81: 2137–46. [10] Wohlsein P, Wamwayi HM, Troutwein G, Pohlenz J, Liess B, Barrett T. Pathomorphological and immunohistological findings in cattle experimentally infected with rinderpest virus isolates of different pathogenicity. Vet Microbiol 1995;44:141–7. [11] Yanagi Y, Cubitt BA, Oldstone MBA. Measles virus inhibits mitogen-induced T-cell proliferation but does not directly perturb the T cell activation process inside the cell. Virology 1992;187:280–9. [12] Jeggo MH, Wardley RC, Cortevn AH. A reassessment of the dual vaccine against rinderpest and contagious bovine pleuropneumonia. Vet Rec 1987;120:131–5. [13] Dhar P, Sreenivasa BP, Barrett T, Corteyn M, Singh RP, Bandyopadhyay SK. Recent epidemiology of peste des petits ruminants virus (PPRV). Vet Microbiol 2002;88:153–9. [14] Kumar P, Tripathi BN, Sharma AK, Kumar Ram, Sreenivasa BP, Singh RP, et al. Pathological and immunohistochemical study of experimental peste des petits ruminants (PPR) virus infection in goats. J Vet Med Series B 2004;51:153–9. [15] Sreenivasa BP, Dhar P, Singh RP, Bandyopadhyay SK. Development of peste des petits ruminants (PPR) challenge virus from a field isolate. In: presented at XIV annual conference and national seminar on management of viral diseases with emphasis on global trade and WTO regime of Indian virological society. January 18–20, (IVRI, Hebbal, Bangalore, India); 2002, pp. 25. [16] Singh RP, Sreenivasa BP, Dhar P, Shah LC, Bandyopadhyay SK. Development of a monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants (PPR) virus. Vet Microbiol 2004;98:3–15. [17] Jain NC. Schalm’s veterinary haematology. 4th ed. Philadelphia, USA: Lea and Febiger; 1986 pp. 225–39. [18] Singh RP, Sreenivasa BP, Dhar P, Bandyopadhyay SK. A sandwich-ELISA for the diagnosis of Peste des petits ruminants (PPR) infection in small ruminants using anti-nucleocapsid protein monoclonal antibody. Arch Virol 2004;149:2155–70. [19] Singh SK, Sreenivas BP, Hosamani M, Singh RP, Bera AK, Ramakrishna C, Rathore R, Bandyopadhyay SK. Evaluation of an indigenously developed live attenuated peste des petits ruminants (PPR) vaccine during pregnancy in goats. In: proceedings of the 4 th Indian veterinary congress and XI annual convention and national symposium of IAAVR. (IVRI, Izatnagar); 2004, p. 39. [20] Singh RP. Production and characterization of monoclonal antibodies to peste des petits ruminants (PPR) virus. PhD Thesis Indian Veterinary Research Institute (Deemed University), Izatnagar, India; 2002. [21] Chang GX, Mallard BA, Mowat DN, Gallo GF. Effect of supplemented chromium on antibody responses of newly arrived feeder calves to vaccines and ovalbumin. Canadian J Vet Res 1996;60:140–4. [22] Rao TVS, Anandan D, Dey A, Nandi S, Harbola PC, Asgola DS. Effect of dietary sulfur on antibody titer response of pashmina goats to egg albumen. Small Rum Res 1998;31:19–22. [23] Fraser CM, Bergeron JA, Mays A, Aiello SE. The merck veterinary manual. 7th ed. NJ, USA: Merck & Co. Inc. Whitehouse Station; 1991. [24] Scott GR. Rinderpest and peste des petits ruminants, in virus diseases of food animals. In: Gibbs EPJ, editor. Disease monographs, Vol-II. NY, London: Academic press; 1981. p. 71. [25] Kumar A, Singh SV, Rana R, Vaid RK, Misri J, Vihan VS. PPR outbreak in goats: Epidemiological and therapeutic studies. Indian J Anim Sci 2001;71:815–8. [26] Raghavendra L, Setty DRL, Raghavan R. Haematological changes in sheep and goats experimentally infected with Vero cell adapted peste des petits ruminants (PPR) virus. Indian J Anim Sci 1997;12:77–8. [27] Anderson J, McKay JA. The detection of antibodies against peste des petits ruminants virus in cattle, sheep and goats and the possible implication to rinderpest control programme. Epidemiol Infect 1994;112:225–31.
Experimental studies on immunosuppressive effects of peste des petits ruminants (PPR) virus in goats K.K. Rajak, B.P. Sreenivasa, M. Hosamani*, R.P. Singh, S.K. Singh, R.K. Singh, S.K. Bandyopadhyay Division of Virology, Indian Veterinary Research Institute (IVRI), Mukteswar Campus, Nainital (Uttaranchal) 263138, India Accepted 27 July 2005
Abstract Effect of virulent and attenuated peste des petits ruminants (PPR) virus on the immune response to nonspecific antigen (ovalbumin) was investigated. Clinical and serological responses were monitored in goats administered with ovalbumin concurrently with either PPR vaccine or virulent virus. Study showed that PPR virulent virus causes marked immunosuppression as evidenced by leukopenia, lymphopenia, and reduced early antibody response to both specific and nonspecific antigen. These observations were predominant particularly during acute phase of disease (4–10 days post-infection). On the other hand, the vaccine virus induced only a transient lymphopenia without significantly affecting the immune response to nonspecific antigen or to itself during this period. Further, the antibody levels to ovalbumin in the group administered with virulent PPRV increased significantly between days 28 and 35 post-infection in comparison to the titers in other two groups given with either ovalbumin alone or in combination with vaccine. q 2005 Elsevier Ltd. All rights reserved. Re´sume´ L’effet du virus virulent et du virus atte´nue´ de la peste des petits ruminants sur la re´ponse immune a` e´te´ e´tudie´. Les re´ponses se´rologiques et cliniques ont e´te´ enregistre´es chez les che`vres qui ont rec¸u
Abbreviations p.v., post-vaccination; p.i., post-infection; PPR, peste des petits ruminants; RPV, rinderpest virus; VNT, virus neutralization test; c-ELISA, competitive ELISA; s-ELISA, sandwich ELISA; TLC, total leukocyte count; DLC, differential leukocyte count. * Corresponding author. Tel.: C91 5942 286348; fax: C91 5942 286347. E-mail address: [email protected] (M. Hosamani). 0147-9571/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2005.08.002
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de l’ovalbumine en meˆme temps que le vaccin contre la peste des petits rumiants ou le virus virulent lui-meˆne. Les etudes ont montre´ que le virus virulent de las peste des petits ruminants provoque une immunode´pression marquee caracte´rise´e par une leucope´nie, une lymphope´nie et une reduction de la re´ponse en anticorps a` la fois via-a`-vis de l’antige`ne spe´cifique et de l’antige`ne non-spe´cifique. Ces faits ont e´te´ observes surtout pendant la aigue¨ de la maladie (4 a`10 jours apre`s l’infection). Par ailleurs, le virus vaccin induit seulement une leucope´nie transitoire sans affecter significativement la re´ponse immune a` l’antige`ne non spe´cifique ou a` l’antige`ne spe´cifique Durant cette pe´riode. En outre´, le taux des anticorps antiovalbumine dans le groupe administer avec le virus virulent augmente sifnificativement entre les jours 28 et 35 apre`s l’infection en comparaison avec les titres obtenus dans les deux groupes, celui avec l’albumine seule ou celui avec l’albumine et al vaccin, q 2005 Elsevier Ltd. All rights reserved.
1. Introduction Peste des petits ruminants (PPR) is an acute, highly contagious viral disease in small ruminants causing huge economic loss in the endemic regions. Clinically, PPR is characterized by pyrexia, necrotic stomatitis, catarrhal inflammation of the ocular and nasal mucosa, enteritis and pneumonia followed by death or recovery from the disease. The disease is often associated with high morbidity and mortality [1] and is considered to be one of the main constraints in improving small ruminant productivity [2]. PPR is now endemic in India [3,4]. The etiological agent, PPR virus has been classified under genus Morbillivirus in the family Paramyxoviridae along with other members including rinderpest virus (RPV), measles virus (MV), canine distemper virus (CDV) and morbilliviruses of marine mammals [5,6]. PPR virus is antigenically closely related to RPV. For the control of PPR, tissue culture rinderpest (TCRP) vaccine was earlier used in sheep and goats in India. However, use of TCRP vaccine was discontinued in view of the interference of the virus in the ongoing serosurveillance programme launched by Government of India under National Project on Rinderpest Eradication (NPRE). A live attenuated homologous PPR vaccine has recently been developed at Indian Veterinary Research Institute (IVRI), Mukteswar (India) from an Indian isolate of PPR virus [7,8]. The vaccine has been tested extensively both in-house and field trials and has been found to be highly efficacious, safe and potent in small ruminants. Morbilliviruses including MV and RPV are known to cause immunosuppression [9–12]. However, there is meager information on the immunosuppressive effects of PPR virus and resulting interference in development of immunity to other antigens. Therefore, the present investigation was undertaken to study the effect of virus on the immune status of goats either on vaccination with attenuated PPR virus or on experimental infection with virulent PPR virus. 2. Materials and methods 2.1. Vero cell line Vero cells (ATCC CCL-13) were propagated in growth medium containing Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS,
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Sigma, St Louis, MI, USA). These cells were maintained in EMEM supplemented with 2% FBS. This cell line was used for virus neutralization test (VNT). 2.2. Vaccine virus The live attenuated vaccine was developed at National Morbillivirus Referral Laboratory, Division of Virology, Indian Veterinary Research Institute (IVRI), Mukteswar, using an indigenous isolate of PPR virus (PPRV Sungri/96) belonging to lineage IV [13]. 2.3. PPR virulent virus A highly virulent PPR virus (PPRV Izatnagar/94) isolated from an outbreak in Uttar Pradesh, India in 1994 was used for challenge experiments [14,15]. This virus is being maintained by animal-to-animal passage in the laboratory. A single subcutaneous inoculation of 2 ml of 10% splenic suspension of PPR virulent virus was used as Challenge virus. 2.4. Experimental animals Apparently healthy female hill goats between 1 and 2 years of age were used in the present study. These goats were screened for the absence of PPR virus antibody using a monoclonal antibody based competitive ELISA (c-ELISA) and VNT. For detection and titration of PPR antibodies, c-ELISA described by Singh et al. [16] was employed. Animals having titre of !40% inhibition (PI) value in c-ELISA test and neutralization titre of !1:8 in VNT were considered sero-negative for PPR. Twelve goats were randomly divided into three groups (A to C) of four animals each. Group A received ovalbumin in combination with 105TCID50 of PPR vaccine virus (approximately 2log10 higher than the prescribed dose). Ovalbumin (Sigma, St Louis, MI, USA) emulsified with equal volume of Freund’s incomplete adjuvant (Sigma) was used for inoculation of goats in different groups. Each animal received 2 mg of ovalbumin in 2 ml inoculum, by subcutaneous and intramuscular routes divided in equal doses. Goats in group B were injected with adjuvanted ovalbumin and 2 ml virulent PPR virus as splenic cell suspension by subcutaneous route. Group C received adjuvanted ovalbumin only, as control group. Animals in each group were housed separately in isolation sheds. 2.5. Clinical monitoring of animals Goats were clinically monitored daily after immunization for PPR specific symptoms. Rectal temperature was recorded daily and heparinized blood, ocular and nasal swabs and sera samples were collected at regular intervals. 2.6. Anti-ovalbumin antibody response Indirect ELISA was optimized for measuring anti-ovalbumin antibodies by checkerboard method. Ovalbumin was coated in flat-bottom 96-well plate (Nunc, Maxisorp) at
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4 mg per well in 100 ml carbonate bicarbonate buffer by incubation at 37 oC for 1 h. Unbound sites in the wells were blocked by addition of blocking solution (5% skim milk powder in PBS) for 1 h at 37 oC. Sera at optimized dilution (1:1000) were added in duplicate (100 ml in each well) and incubated for 1 h at 37 oC under constant shaking. Anti-goat HRPO Conjugate (Sigma, St Louis, MI, USA) was added at a dilution of 1:30, 000 in PBS to each well (100 ml) and incubated for 1 h at 37 oC under constant shaking. The plates were washed three-times with PBS (pre-warmed to 37 8C) containing 0.05% tween-20 between each incubation step. Color was developed by addition of 100 ml OPD substrate (Sigma, St Louis, MI, USA) after incubating at 37 8C without shaking for 10 min and optical density (OD) was taken in the ELISA reader at 492 nm (TECAN, Austria). 2.7. Total leukocyte count (TLC) and differential leukocyte count (DLC) Heparinized blood samples were collected for carrying out TLC and DLC as described by Jain [17] with little modification. For DLC, blood films were stained by the Giemsa’s method and a total of 100 cells were counted by following Battlement method of counting. 2.8. Detection of PPR virus Sandwich-ELISA test as described by Singh et al. [18] was employed for detection of PPR virus in clinical samples (ocular and nasal swabs) from PPR infected animals. The test used a polyclonal antiboby as capture antibody and monoclonal antibody to ‘N’ protein of PPRV as detection antibody. 2.9. Measurement of PPRV antibody response Detection of PPRV antibodies in the sera samples collected from PPR infected and vaccinated animals was carried out using monoclonal antibody (directed to H protein) based competitive ELISA (c-ELISA) test [16]. 3. Results 3.1. Clinical monitoring of animals on immunization and challenge Goats in group A remained apparently normal following vaccination. Expectedly, group B that received virulent virus with ovalbumin developed a clinical disease characteristic of PPR infection. These animals exhibited signs of pyrexia (Fig. 1), dullness and anorexia from day 4 p.i. Diarrhea, respiratory distress accompanied with coughing and ocular and nasal discharges were noticed between days 5 and 13 p.c. PPR viral antigen could be detected in nasal and ocular discharges collected from all the animals of group B between days 7 and 13 p.i. using sandwich-ELISA (Fig. 2a and b), whereas similar swabs collected from group A or group C did not show the presence of PPR antigen (data not shown). Animals of group C remained healthy throughout the study period.
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Fig. 1. Thermal reaction following infection with PPR virus: Group A (ovalbuminCPPR vaccine); Group B (ovalbuminCPPR challenge); Group C (ovalbumin alone). Each value represents the mean of four animals in each group.
3.2. Total leukocyte count (TLC) and differential leukocyte count (DLC) Vaccinated animals in group A showed a normal TLC picture following immunization (Fig. 3a). However, DLC picture indicated a transient but mild lymphopenia on day 6 p.v. (Fig. 3b). Group B which was inoculated with both ovalbumin and virulent PPR virus concurrently, showed a significant leukopenia from days 6 to 10 p.c. (Fig. 3a). A gradual and moderate decrease in lymphocyte count was also evident in this group from days 6 to 8 p.c. (Fig. 3b). In the ovalbumin control group, TLC and lymphocyte count remained within the normal limits.
Fig. 2. Detection of PPR antigen in (a) ocular and (b) nasal swabs in different groups using sandwich-ELISA. The antigen could be detected between days 7 and 13 post-infection in group B.
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Fig. 3. (a) Total leukocyte count (b) lymphocyte count in different groups of animals. A significant decline in TLC was observed with virulent virus (group B) between days 6 and 10, which was not evident in other groups. A transient decline in lymphocyte count was noticed on day 6 in group A and days 6–8 in group B animals.
3.3. Humoral immune response against PPR virus Development of PPRV specific antibodies in experimental animals was monitored by c-ELISA. There was a steady increase of antibody titre in group A till 14 days p.v. However, animals inoculated with virulent virus did not show any specific antibody response till day 7, which coincided with the peak rectal temperature. On the other hand, PPRV antibody response followed a normal trend both in groups A and B from day 14 post-inoculation. No PPRV antibodies could be detected in animals inoculated with ovalbumin (Fig. 4).
Fig. 4. Antibody response against PPR virus using competitive ELISA in different groups of animals. Kinetics of antibody response in different groups against PPRV showed a steady increase in antibody till day 14. Group B animals did not show any specific antibody response till day 7.
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Fig. 5. Antibody response against ovalbumin measured by indirect ELISA in different groups. Early antibody response against ovalbumin indicated a sharp increase on day 14 post-inoculation in group C, a moderate increase in group A, while titres were undetectable in group B.
3.4. Humoral immune response against ovalbumin Antibody response against ovalbumin in various groups of animals at weekly intervals was studied using indirect-ELISA. The kinetics of early antibody response revealed a sharp increase in ovalbumin control group from day 7 and a moderate rise in vaccinated group on day 14. However, in group B, the rise was not detectable until day 14 postinfection as depicted in Fig. 5. Further, a peak antibody titer was observed in the challenge group on day 35; whereas in rest of the groups, peak response was observed on day 21 post-inoculation followed by a plateau. When antibody responses among various groups were statistically compared, the response to ovalbumin on day 14 was found to be significantly lower in both the group A (P!0.05) as well as group B (P!0.01) as compared to the group C. Further on day 21 also, the antibody levels in group B remained significantly lower (P!0.05) as compared to other groups. Interestingly, antibody response in group B was remarkably high during days 28–35 p.c. in comparison to other two groups. Nevertheless, antibody responses against ovalbumin showed no significant difference among all groups by day 42 p.v.
4. Discussion Vaccination is the most ideal approach for the control of PPR in India where the disease is causing widespread outbreaks round the year. Tissue Culture Rinderpest (TCRP) vaccine was earlier used in sheep and goats for control of PPR, until homologous vaccine was developed at IVRI [7,8]. The vaccine has undergone extensive field trials with nearly more than half a million doses being used across the country without any adverse reactions. This Vero cell derived vaccine has an ability to induce
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strong humoral response in sheep and goats. However, in an isolated case, concurrent coccidial outbreak in an organized flock following PPR vaccination lead to speculation among the end users that the vaccine may induce immunosuppression giving rise to secondary infections. Although the vaccine under investigation has been proved to be completely attenuated [8] and apparently safe in target population including pregnant ones [19], the present study was undertaken to investigate whether the PPR vaccine virus causes immunosuppression. Morbilliviruses are known to induce strong humoral antibody response on vaccination or infection. In the present study, early antibody response during first 2 weeks using PPR vaccine and virulent challenge virus showed a marked difference. In group B, which received the challenge virus, no detectable level of antibodies could be observed during the first week. In comparison, the vaccinated animals (group A) showed a sharp increase in antibody response by day 7 p.v. This showed that the challenge virus delayed the induction of immune response against PPRV, suggestive of transient impairment of host immune responses. Hampered early antibody response to virulent virus coincided with an active phase of clinical disease that lasted for about 10 days. All the animals of group B exhibited clinical signs such as pyrexia, off feeding and dullness between days 4 and 10 p.i. Excretion of antigen in nasal and ocular secretions was also demonstrable between days 7 and 13 p.i. in this group. This observation was consistent with earlier studies carried out by Singh et al. [20] in this laboratory. Antibody response to PPRV returned to a normal trend in both the groups after 14 days. Immune response to egg albumin has been used to study the nutrient modulation of immune responses [21,22]. In this experiment, effect of vaccination and experimental challenge infection on the immune response to ovalbumin was assessed. The antiovalbumin antibody response in goats was monitored after inoculation of ovalbumin along with either PPR vaccine or PPR challenge virus. Antibody response on day 14 was significantly lower in animals of both the groups A (P!0.05) and B (P!0.01) in comparison to group C animals. Challenge virus used in group B caused a profound inhibitory effect on antibody response to an unrelated antigen (ovalbumin). This effect persisted in group B even up to day 21 p.c. (P%0.05). On the other hand, PPR vaccine appeared to reduce the development of early antibody response transiently. Data on titration of antibody response every 2/3 days would have been more comprehensive. However it cannot be ruled out that concurrent immunization of a nonreplicating antigen with other live viral vaccines can affect the antibody response to the former. Nevertheless, after 28 days, a plateau was observed in groups A and C without significant difference among them, while the titres in group B increased significantly high between days 28 and 35 post-inoculation. PPR virus has got strong affinity for epithelial cells of gastrointestinal tract and lymphoid tissue [23]. The total and differential leukocyte counts serve as indicators for studying pathogenesis of lymphotropic viruses like PPRV. Lymphopenia is one of the important indicators of virus induced immune suppression in both RPV [24] and PPRV [25,26]. In the present study, mean TLC in the vaccinated group appeared to be in normal range although a slight decline was evident in early stage of vaccination. The results are partially in agreement with a similar report on vaccine against RPV [9]. A transient lymphopenia on day 6 p.v. was also observed in group A animals. Lymphopenia can be
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attributed to lymphocyte trapping in the lymphoid tissues that may occur early during induction of immune response following local replication of virus. This may also be attributed to the high dose of vaccine virus used, which was 100 times more than the recommended field dose of vaccine. In group B, marked decline in TLC was observed between 6–10 days following inoculation of challenge virus in goats. A moderate lymphodepletion was also evident between days 4 and 6 post-infection, which coincided with severity of clinical symptoms. Anderson et al. [27] have also reported profound leukopenia with virulent rinderpest virus, which was later linked to marked immunosuppression caused by this virus [9]. From the present investigation, it could be concluded that PPR vaccine may transiently suppress the generation of immune response to other antigens. However, it remains to be seen if this property is associated with other live viral vaccines as well. Further, it cannot be inferred from the data that vaccination may lead to impairment of overall immune responses against nonspecific antigens including replicating ones. The transient reduction of immune responses may not be significant enough to cause secondary infections. Recent studies involving RBOK strain of Rinderpest vaccine also showed that the vaccine strain may not cause biologically significant immune suppression [9]. Further, it could be demonstrated that the virulent PPR virus causes significant immunosuppression as indicated by reduced humoral antibody response to PPR virus, ovalbumin antigen as well as marked leukopenia and lymphopenia during active phase of infection.
Acknowledgements Authors acknowledge the Director, IVRI, Izatnagar for providing the necessary facilities for carrying out the work. This work was financially supported from NPRE, Government of India. We are grateful to Dr. Srini Kaveri for providing the French translation of the abstract.
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