Experimental transmission with ovine rinderpest virus

Small Ruminant Research ELSEVIER

Small Ruminant Research 16 (1995) 277-282

Experimental transmission with ovine rinderpest virus K. Sripad,

N.G. Ramesh Babu, M.M. Babu, T. Gopal*, B.S. Keshavamurthy Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore-

024, India

Accepted 1 I May 1994

Abstract The behaviour of ovine rinderpest virus in experimentally infected crossbred calves and indigenous sheep was studied in two phases. In the first phase, clinical manifestations were more pronounced in calves than in sheep. Leukopenia was significantly higher. However, there were no differences in duration of leukopenia between the two species. There was a positive correlation of leukopenia with fever in calves, which showed predominant lymphopenia. No mortality was noticed in sheep, whereas one calf died on Day 10 post-infection. In the second phase of the experiment, virus recovered from the reactor calf was subinoculated into another calf and sheep. The calf showed classical signs of tinderpest. In sheep, anorexia and diarrhoea were the only two signs noticed. Duration of leukopenia associated with reduced lymphocytes was 3 days in sheep whereas in calves for 8 days. Comparative responses of the two species suggest that calves may serve as a better indicator host than sheep in in vivo characterization of rinderpest isolates of ovine origin. Keywords: Rinderpest; Sheep; Experimental transmission

1. Introduction Rinderpest (RP) continues to be prevalent in several states of India on a reduced scale, despite annual prophylactic vaccination of bovine populations at 60 X 1O6 doses. An alarming aspect of its epizootiology is the persistence of the virus in small ruminants in the southern peninsula. There are several reports in the Indian and African Veterinary literature highlighting circumstantial evidence of the spread of bovine virus to sheep (Achar and Srikantaiah, 1934), sheep and goats (Beaton, 1930); and similarly virus of sheep origin to bovine populations (Zwart and Macadam, 1967; Ramani, 1973). There are also several references regarding the occurrence of the ovine disease in some regions without * Correspondingauthor. Elsevier Science B.V. SSD10921-4488(95)00620-6

any preceeding or concomitant overt manifestation of rinderpest in bovines (D’costa, 1936; Khera, 1979). There have been field observations of concurrent visitations involving sheep and cattle (Gajapathy, 1973; Ananth, 1980). In our investigations of several outbreaks of rinderpest in Karnataka State, a striking epidemiological feature was detected on several occasions, Even in regularly vaccinated cattle populations small number of susceptibles, particularly young ones, have been attacked and these attacks were traced almost in every instance to the introduction of unvaccinated nomadic sheep. Clinical disease in ovines is often vague and confusing. Some early reports characterized it as a febrile syndrome dominated by hyperthermia, rhinorrhoea, conjunctivitis, buccal and vulvoanal erosions and severe diarrhoea. Skin lesions, abortions and primary

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K. Sripad et al. /Small Ruminant Research 16 (1995) 277-282

respiratory involvement occur on a lesser scale (Scott et al., 1967). Likewise, experimentally induced disease in sheep has also been characterized (Lall, 1947; Dhanda and Manjrekar, 1952; Johnson, 1958). Information on haematological changes in natural and experimental ovine rinderpest is scanty. Leukopenia has been consistently observed (Maurer et al., 1956). Changes affecting the other cell constituents were not regarded as significant (Krishnan and Vishwanathan, 1965; Krishnan and Ranga Rao, 1972). The present investigation studied behaviour of rinderpest virus of ovine origin in experimentally infected crossbred calves and indigenous sheep. The virus recovered from a reactor calf was subinoculated into a calf and sheep to ascertain differences in its pathogenicity. These were monitored clinically, haematologitally and histopathologically. Results of clinical and haematological studies are presented in this paper.

2. Materials and methods Znoculum: A field isolate of the virulent rinderp-est virus of sheep origin from this Institute was used in the transmission studies. The infectivity of the isolate was first established by inoculating primary bovine kidney(PBK) cells with a pooled 1% suspension in PBS (pH 7.2) of lymph node and spleen tissues of the affected sheep. The suspension was clarified and filtered before inoculation. A 10% suspension of the same filtrate was used for animal inoculation. 2.1. Animal inoculations 2.1.1. Phase 1 Six crossbred cattle calves of both sexes, about one year old and three adult female sheep (2-3 years approximately) which did not have any demonstrable levels of RP antibodies were used in the study. Two calves and two sheep received the inoculum intravenously and subcutaneously, the dose being 5 ml per animal in each route. Two other calves which were vaccinated three weeks earlier with 100 doses of tissue culture rinderpest vaccine, also received a similar dose of inoculum by similar routes. A third group of two calves and one sheep served as controls. These three groups of animals were maintained separately.

2.1.2. Phase 2 The blood collected at the height of pyrexia from reactor calf was sub-inoculated intravenously into another calf ( 15 ml) and a sheep (5 ml), both free of rinderpest antibodies. 2.2. Clinical observations The inoculated calves and sheep were monitored for clinical signs and temperature reactions. An elevation of 1°C above the pre-inoculation mean values was considered as hyperthermia. 2.3. Serology The presence of specific viral precipitinogens in the buffy coats of the reactor calves and sheep was tested by Agar Gel Immunodiffusion (AGID) and Counterimmunoelectrophoresis (CIB) tests (Mangapathi Rao, 1987) daily from Day 8 to Day 30, post-infection (P.I.) and thereafter at weekly intervals till the experiment ended. 2.4. Hematology Samples of blood in EDTA collected daily from Day 8 to Day 30 from the inoculated animals were analysed for hemoglobin (Hb), total erythrocyte count (TEC) , packed cell volume (PCV) and total leukocyte counts (TLC) as per standard procedures (Nemi and Jain, 1986). Differential leukocyte counts were also estimated to ascertain whether changes in TLC could be related to changes in particular cell populations. Analysis of clinicopathological data was according to standard statistical methods (Snedecor and Co&ran, 1968).

3. Results 3.1. Phase 1 3.1.1. Clinical observations 3.1.1.1. Calves. Clinical reactions were manifested only in the unvaccinated animals. These consisted of marginal pyrexia on Days 6 and 8 post inoculation respectively, and temperature increases of 0.8 and

K. Sripadef al. /Small Ruminant Research 16 (1995) 277-282

gestion. The animals uneventfully.

Fig. 1. Fever curves in inoculated

calves.

1.3”C above mean preinoculation values. Peak temperatures were 41.1 and 41.2”C which correspond to increases of 2.1 and 2.1”C, respectively, and occurred on Days 2 and 4 of fever. Fever durations were 3-4 days, respectively (Fig. 1) . Nonspecific febrile signs characterized by dullness and anorexia were observed on Day 1 or 2 of fever, along with suggestive signs of tinder-pest showing lachrymation and erosions on the buccal mucosa. Rhinorrhoea set in on Day 10 P.I. Mucosal erosions and nasolachrymal discharges persisted until death in one and other on Day 15 P.I. The latter developed diarrhoea on Day 15 of fever and persisted for 4 days. Recovery was complete by Day 18 after inoculation. Necropsy of calf- 1 revealed changes associated with mild abomasitis and enteritis. Both mucosae were studded with petechiae and edematous. Other significant features included severe pneumonia, multiple haemorrhages on the endocardium and enlargement of the mesenteric lymph nodes and spleen due to oedema and hyperaemia. Specific viral antigens were detected in the spleen and and mesenteric Iymph nodes by AGID and CE. Vaccinated calves did not react overtly to virus inoculation. 3.1.1.2. Sheep. The inoculated sheep developed a lowgrade thermal response on Days 6 and 10, respectively, the increase in temperature was 0.5 and 0.6”C. Peak levels were reached 4 and 8 days later, but these were no more than 0.1 and 0.7”C above preinoculation values. There were no other reactions of clinical significance except for mild granular vaginitis with mucosal con-

279

had recovered

by Day 16 P.I.

3.1.2. Haemogram There were no significant haematological changes in calf- 1, and sheep- 1 and -2 consequent to the virus infection (Table 1). Calf- 1 had significant reduction in Hb and TEE. However, there were no striking alterations in PCV values. The preinoculation TLC, in calf-l and -2 were 11 950 and 6700 per mm3 respectively, based on two observations each. The experimental disease in these animals was associated with significant leukopenia (PI 0.05) occurring on Days 2 and 3 of fever, respectively (Table 1). Leukopenia was more impressive in calf-l and persisted till death which occurred on Day 5 of fever (Fig. 1) . In the other animal the the duration was 6 days. Table 1 Haematological values in experimentally (Phases 1and 2)

infected cattle and sheep

Animal

Parameter

Pre-inoculation Mean

Post-inoculation mean

t

Calf 1

PCV Hb TEC

43.0 11.0 8.9

35 *4.3 1.3 f 0.3 4.5 f 0.2

0.40 4.96” 4.31*

Calf-2

PCV Hb TEC

31.5 9.0 6.3

31.9k1.9 8.OiO.3 5.7 f 0.2

0.01 0.34 0.23

Calf-3

PCV Hb TEC

33.7k2.8 8.9kO.7 6.1

32.4+0.6 7.8 f 0.2 6.2kO.3

0.51 0.59 0.21

Sheep-l

PCV Hb TEC

39.0 10.25 7.53

30.1 kO.9 8.2 kO.2 7.4kO.l

0.86 0.95 0.09

Sheep-2

PCV Hb TEC

41.5 12.5 10.9

38.4rt1.4 9.9 f 0.5 8.3 f0.8

0.88 0.54 0.34

Sheep-3

PCV Hb TEC

21.8 6.3 6.0 + 0.5

19.3 f 0.8 5.1*0.1 3.8 f 0.3

0.54 0.78 1.39

*Significant (P10.05). PCV, packed cell volume (percentage). Hb, Haemoglobin (g%) TEC, total erythrocyte count ( lo6 per cu mm).

K. Sripad et al. /Small Ruminant Research 16 (1995) 277-282

280

Table 2 Total leucocyte count (TLC) and lymphocyte count (LC), (x f SE) in infected cattle and sheep (Phases-l and -2) Animal

Parameter Pre-inoculation xrtSE

Calf- 1

TLC= LCb

11.9 62.5

6.5 40.53 56.643.14

Calf-2

TLC LC

6.1 61.5

5.0~0.07 65.0 4 0.94

6.3 f 0.66 54.2 f 2.32

TLC LC

g7*0.45 62.6k1.19

6.4 f 0.45 56.2 f 0.96

7.1*0.79 61.0f0.79

Sheep- 1

TLC LC

9.8 62.0

6.9 f 0.40 53.oi1.06

Sheep-2

TLC LC

10.3 59.0

6.8 f 0.93 58.7 f 0.98

Sheep-3

TLC LC

10.1 f 1.67 61.2k0.66

9.9 f 1.02 57.7 io.41

Calf-3

Post-inoculation Fever days XfSE

Post fever day xfSE

TLC, in thousands. bLC, in percentage.

Leukopenia was highlighted by lymphopenia (Table 1). This was more obvious in calf- 1 on Day 5 of fever when the animal died. Lymphopenia was relatively mild in calf-2. One of the vaccinated calves died of other causes. In the other, there was a transient leukocytosis, but this did not seem related to virus inoculation as there were no changes in lymphocyte counts. Inoculated sheep also showed significant leukopenia from Day 8 P.I. in the absence of marked pyrexia (P 5 0.05)) whereas in sheep- 1 leukopenia persisted for 4 days (Table 2), in the other it was biphasic. The first peak was on Day 8 P.I., the values having gradually declined by Day 10. A second lower peak was observed on Day 12 P.I. and continued for one more day. Leukopenia values were significantly higher in calves (PI 0.01)) but no differences existed in the duration of leukopenia between the two species (P < 0.1) . Leukopenia in calves was significantly correlated to fever (P I 0.05)) but not in sheep. 3.2. Phase 2 Calf-3 that received infective material from calf-2 (Phase 1) developed a distinct hyperthermia on Day 4

39

Fig.

0

4

5

6

7 8 DAYS ?I.

9

2. Correlation between temperature

m

11

iz

and leulcopenia.

P.I. Fever lasted for 6 days and the peak temperature was noted on Day 4 of fever (Fig. 2). Anorexia, depression and erosive lesions on the oral mucosa were manifested on Day 2 of fever. One day later there were hyperemic eruptions on the genital mucosa. The inoculated sheep (sheep-3) was anorexic and depressed from Day 2 P.I. However, no febrile reactions ensued. Diarrhoea set in on Day 4 and persisted till death, which occurred on Day 8. Postmortem examination revealed severe abomasitis, enteritis and pneumonia. There were no gross changes of importance in the mesenteric lymph nodes and spleen, although viral antigens could be demonstrated. There were no detectable haematological changes of interest in calf-3 and sheep-3 in the post-inoculation period (Table 1). Mean preinoculation total leukocyte counts were 7700 and 12 850 per mm3 respectively. Following inoculation both developed leukopenia on Day 4, whereas the duration of leukopenia in sheep was 3 days and lasted for 8 days in the calf. There was a depression in total lymphocyte values in both the animals.

4. Discussion An intriguing feature of ovine rinderpest in India is its restriction to the southern peninsula. As underlying causes of this phenomenon are not fully understood, it is logical to assume that apart from large congregations of unvaccinated sheep and their uncontrolled movements, it is the practice of mixed husbandry which facilitates close contact between cattle and sheep that may be a factor of significance. This type of husbandry

K. Sripad et al. /Small Ruminant Research 16 (1995) 277-282

practice is probably not prevalent in other parts of the country. A successful effort was made to characterize the ovine RP and to demonstrate the viral antigens in the infected issues using ACID and CIE. Similarly, the virus was isolated in cell cultures( Shaila et al., 1990). However, as these approaches lead only to a presumptive diagnosis, we sought to establish the pathogenic behaviour of the isolate in the biological test system using both calves and sheep as indicator hosts. Differences in responses between the two species were marked. In sheep, pyrexia was marginal and inconsistent, a finding which was in agreement with an African study (Plowright, 1952) but was in marked contrast to significant hyperthermia reported by Indian workers (Ramani et al., 1974; Manohar Rao et al., 1974). However, the absence of mucosal erosions noted in the present study was similar to their observations. It is logical to assume that sheep used in our studies probably had a higher innate resistance. Differences in response of Indian breeds of sheep to rinderpest have been documented (Lall, 1947; Sharma and Tulsa Ram, 1955; Krishnamacharyalu and Gangadhara Rao, 1980). However, this may be considered with caution as our observations were restricted to three animals. Leukopenia was a finding in both the inoculated calves and sheep and the values were significantly higher in the latter. However, there were no differences between the two species in duration of leukopenia. The leukopenia in both species affected the lymphocytes. A significant correlation between leukopenia and hyperthermia was observed in calves but not in sheep. The occurrence of leukopenia without significant fever in sheep was reminiscent of report from European pigs by Scott et al. ( 1959). Leukopenia apart, there were no haematological changes of significance in the two species. This was in agreement with findings of other workers (Maurer et al., 1956; Krishnan and Vishwanathan, 1965). Significant oligocythemia and reduction in haemoglobin values noticed in one of the calves in our study are of interest and warrant further investigation. Oligocythemia may be incidental to increased fragility of the erythrocytes as observed by Sen and Roy ( 1933). No apparent differences in the severity and duration of diarrhoea were noticed in the inoculated calves and sheep. Mortality rates were similar, one of the three inoculated in each species succumbing to the infection.

281

Comparative responses of the two species suggest that calves may serve as a better indicator host than sheep in studies designed to characterize rinderpest isolates of ovine origin. The behaviour of rinderpest virus in small ruminants and their role in maintenance and transmission of the virus to cattle needs further investigation. Sheep appeared to be refractory to rinderpest and showed varying clinical responses, although our observations were from a small number of animals.

Acknowledgements The authors wish to acknowledge Dr. S. Ramachandran, Centre for Tropical Veterinary Medicine (CTVM), University of Edinburgh, for his guidance during the study, and the Indian Veterinary Research Institute (IVRI), Bangalore for providing facilities.

References Achar, SC. and Srikantaiah, G.N., 1934. The control of rinderpest with special reference to goat adapted virus as a vaccine. J. Mysore Agric. Exp. Union. 14: 5761. Ananth, M., 1980. Epidemiological aspects of the incidence of rinderpest in Karnataka State with special reference to sheep and goats. Proceedings of the State Level Seminar on Rinderpest, College of Vet. Sci. Thimpathi, India. Beaton, W.G., 1930. Rinderpestingoats in Nigeria. J. Comp. Pathol., 43: 301-307. D’costa., 1936. Rinderpest - Its symptoms. Indian Vet. J., 13: 716. Dhanda, M.R. and Manjrekar, S.L., 1952. Observations on rinderpest among sheep and goats in the state of Bombay, Indian Vet. J., 28: 306319. Gajapathy, VS., 1973. Proceedings of the 26th Meeting of Central Rinderpest Control Committee, Udaipur, Rajasthan, India. Jain, V., 1986. Schalm’s Veterinary Hematology. Lea and Febiger, Philadelphia, USA, 4th edn., pp. 2048. Johnson, R.H., 1958. An outbreak of rinderpest involving cattle and Sheep. Vet. Rec., 70; 457-461. Khera., 1979. Rinderpest Eradication Programme in India. Proceedings of the End International Symposium on Veterinary Epidemiology and Economics, New Delhi, India. Krishnamacharyalu, E. and Gangadhara Rao, Y.V.B., 1980. A short note on the outbreak of rinderpest in sheep at A.I.C.R.P. on sheep for mutton, Palamner, Andra Pradesh, India. Proceedings of the State Level Seminar on Rinderpest, College of Vet. Sci., Thirupathi, India. Krishnan, R. and Vishwanathan, S., 1965. Tissue changes in goats used for rinderpest vaccine production. Indian Vet. J., 42: 480483.

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Krishnan, R. and Ranga Rao, D.V., 1972. Experimental rinderpest in sheep. Cheiron, 1: 1-17. Lall, H.K., 1947. Some observations on the immunization of sheep and goats against rinderpest. Indian, J. Vet. Sci., 17: 4-l 1. Mangapathi Rao, V., 1987. Nitrocellulose enzyme linked immunosorbent assay (NC-ELISA) for the demonstration of rinderpest virus antigen. Thesis, University of Agricultural Sciences, Bangalore, India. Manohar Rao., Indira Devi, T., Ramachandran, S. and Scott, G.R., 1974. Rinderpest in sheep in Andhra Pradesh and its control by vaccination. Indian Vet. J., 51: 439-450. Maurer, F.D., Fuicher, M.G., Gillans, W.J., Meyer, K. and Parks, SE., 1956. Diseases of cattle. Am. Vet. Publications, Evanston, 111, p. 624. Plowright, W., 1952. Observations on the behaviour of rinderpest virus in indigenous African sheep. Br. Vet. J., 108: 450-457. Ramani, K., 1973. Rinderpest in Sheep. Proceedings of the Ninth Rinderpest Officers’ Conference, Gauhati, India. Ramani, K., Samuel, C.Y. and Ramachandran, S., 1974. Further studies on rinderpest virus of sheep origin. Indian Vet. J., 51: 129-138.

Scott, G.R., Taylor, W.P. and Rossitier, P., 1967. Diagnosis of rinderpest, FAO Monograph Serial 7 1, FAO, Rome. Scott, G.R., De Tray, D.E. and White, G., 1959. A preliminary note on the susceptibility of pigs of European origin to rinderpest, Bull. Off. Int. Epizoot., 51: 694-698. Sen, KC. and Roy, A.C., 1933. Serum proteins of healthy hill bulls and their variations during rinderpest and after recovery. Indian J. Anim. Sci., 3: 39-64. Shaila, M.S., Deepa, A.B., Gopal, T., RameshBabu, N.G. and Sripad, K., 1990. Preliminary observations on the isolation of rinderpest virus from an ovine outbreak in Karnataka State. Indian Vet. J., 67: 383-384. Sharma, R.M. and TulsaR., 1955. Scarification versus sub cutaneous method of vaccination against rinderpest in goats and sheep. Indian J. Vet. Sci., 25: 129-142. Snedecor, G.W. and Cochran, W.G., 1968. Statistical Methods, Oxford and IBH, Bombay, India, 5th edn., pp. 59-65. Zwart, D. and Macadam, I., 1967. Transmission of rinderpest by contact from cattle to sheep and goats II. Observations on rinderpest in sheep and goats and transmission to cattle. Res. Vet. Sci., 8: 3748.