Tropical Animal Health and Production, 36 (2004) 307^320 # 2004 Kluwer Academic Publishers. Printed in the Netherlands
A Classical Live Attenuated Vaccine for Sheep Pox V. Bhanuprakash1*, B.K. Indrani2, R. Hegde3, M.M. Kumar3 and A.R.S. Moorthy4 Department of Veterinary Microbiology, Veterinary College, University of Agricultural Sciences, HA Farm, Hebbal, Bangalore, Karnataka, India; 1ERV and Clinical Virology Laboratories, Division of Virology, IVRI, Mukteshwar 263138, Nainital (Distt.), Uttaranchal, India; 2Rallis Research Centre, Peenya Industrial Area, Bangalore, Karnataka, India; 3Institute of Animal Health and Veterinary Biologicals, HA Farm, Hebbal, Bangalore, Karnataka, India; 4Department of Veterinary Microbiology, Veterinary College, University of Agricultural Sciences, HA Farm, Hebbal, Bangalore, Karnataka, India *Correspondence: E-mail:
[email protected] Bhanuprakash, V., Indrani, B.K., Hegde, R., Kumar, M.M. and Moorthy, A.R.S., 2004. A classical live attenuated vaccine for sheep pox. Tropical Animal Health and Production, 36(4), 307^320 ABSTRACT A classical live attenuated sheep pox vaccine was prepared using the Ranipet strain of sheep pox virus (SPV) at the 50th passage in a secondary lamb testicular cell system. The TCID50 and RD50 were 109.63/ ml and 109.51/ml, respectively. The SID50 of SPV challenge virus was 105/ml. The vaccine was found to have no adverse e¡ects in laboratory animals, and was safe and e¡ective in SPV seronegative lambs. In the ¢eld, 660 sheep were vaccinated with an immunizing dose containing 16102 TCID50. Randomly selected vaccinated sheep mounted good cell-mediated immunity and humoral responses as measured by glucose utilization test and serum neutralization test, respectively, for the study period of 6 months. Keywords: attenuated virus, Ranipet strain, sheep pox, vaccine Abbreviations: AGPT, agar gel precipitation test; CMI, cell-mediated immunity; CPE, cytopathic e¡ect; GUT, glucose utilization test; PLT, primary lamb testicular cells; RD50, reactive dose 50%; SID50, skin infective dose 50%; SLT, secondary lamb testicular cells; SN, serum neutralization; SPV, sheep pox virus; SPV-R, sheep pox virus Ranipet strain; TCID50, tissue culture infective dose 50
INTRODUCTION Sheep pox is a highly contagious and devastating disease of sheep that occurs in Asia and Africa but has not been reported from the Americas and Australia. The disease has been eradicated from most European countries (Sathe, 1931). The virus belongs to the viral genus Capripox of the subfamily Chordopoxvirinae of the Poxviridae family. In the ¢eld, the virus is transmitted by the aerosol route, close contact with infected animals and, mechanically, by biting (stable) £ies (Carn, 1993). Among the pox viral diseases of domestic animals, sheep pox is highly infectious with considerable morbidity and mortality (10^50%). Sheep are the primary host; all age groups are susceptible, but death occurs mainly in young lambs, occasionally in 307
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yearlings and rarely in adults. Severe economic losses are due to high mortality, abortion and mastitis, skin condemnation and loss of wool and mutton, besides restricting the export potential of the meat, wool and skin (Ramyar, 1965). The disease is endemic in India and outbreaks have been reported regularly from almost all the states including Karnataka. Karnataka is one of the major sheep-rearing states in India with a population of more than 7.5 million sheep, kept mainly by small and marginal farmers. Sheep farming is an important source of family income but it is substantially compromised by this disease (Anon., 1998^1991). At present, an inactivated vaccine is in use in most of the states, but this su¡ers problems such as short duration of immunity and di¤culty in large-scale production, handling, transportation and storage (Pandey and Nanda, 1988). The aim of the present study was to produce and study the e¤cacy of a live attenuated sheep pox vaccine made from the sheep pox virus (SPV) Ranipet strain for the prevention and eradication of infection. MATERIALS AND METHODS Animals Twenty lambs, 3^4 months old and seronegative to SPV, were used. Cells Secondary lamb testicular cells (SLT) were used for the propagation of SPV. Vaccine virus The live attenuated sheep pox virus Ranipet strain (SPV-R) at 50th passage was obtained from the Institute of Animal Health and Veterinary Biologicals, Bangalore, Karnataka, India. The virus was propagated in SLT cells grown in Dulbecco's minimum essential medium (D-MEM) containing 10% neonatal calf serum. Challenge virus A virulent SPV challenge virus in the form of ampoules of freeze-dried materials was procured from the Institute of Veterinary Preventive Medicine, Ranipet, Tamil Nadu, India.
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Vaccine The SLT cells were prepared from primary lamb testicular (PLT) cells (Awati, 1992). The SLT cells were infected with SPV-R and incubated at 378C for 7 days with 2% neonatal calf serum. When the cytopathic e¡ect (CPE) was 80%, the inoculated cells were frozen and thawed three times. The viral suspension was clari¢ed by centrifugation at 600g for 10 min. The supernatant was tested for bacterial, mycoplasmal and fungal contamination. SNT and AGPT con¢rmed the presence of SPV in the supernatant. The supernatant was mixed with an equal quantity of stabilizer (2.5% lactalbumin hydrolysate, 5% sucrose in D-MEM, pH 7.6), distributed in 1 ml aliquots, freeze-dried, labelled and stored at ^408C. TCID50 of vaccine The freeze-dried vaccine was titrated in 20-well cell culture plate with an SLT cell system. Serial 10-fold dilutions from 10^1 to 10^12 of the vaccine were made in maintenance medium. Five wells were infected with 1 ml of each viral dilution. Five uninfected SLT wells served as cell controls. The plates were incubated at 378C with 5% carbon dioxide for a period of 7 days. The plates were examined daily for the appearance of CPE. The ¢nal results were recorded as positive for the presence of CPE and negative for the absence of CPE in the titration chart on day 7. TCID50 was then calculated (Reed and Muench, 1938). Reactive dose 50% (RD50) of the vaccine Two male lambs, 3^5 months of age, that had not been vaccinated against sheep pox or exposed to any outbreak of sheep pox were used. The lambs were shaved over the abdominal area, which was then thoroughly washed with soap followed by a wash with distilled water. The cleaned area was made moisture-free by wiping with a sterile gauze. Five randomly selected vaccine vials, each reconstituted with 1 ml of phosphatebu¡ered saline, pH 7.4, were pooled and serially diluted from 10^1 to 10^10. A 10^3 dilution of SPV-R vaccine virus was applied to each of the two lambs on the thorax, followed by 10^4, 10^5, 10^6 and 10^7 dilutions and a control of PBS in the £ank region. Two hundred microlitres of each dilution were injected intradermally into the skin and ¢ve sites were used for each dilution. The shaved area of each lamb was covered with double-layered gauze cloth and secured to prevent scratching and soiling of inoculated sites. Temperature was recorded daily to observe any rise and the inoculated sites were examined for the development of local reactions. The ¢nal reading was taken on the 10th day after inoculation. The RD50 was calculated (Reed and Muench, 1938). The experiment was repeated on a third lamb with concentrations of 10^6, 10^7, 10^8, 10^9 amd 10^10 because the end point could not be ascertained from the ¢rst two lambs.
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Propagation of challenge virus Three female lambs, 3^5 months of age, that had not been vaccinated against sheep pox or exposed to the disease were used. Preparation of the abdominal area was done as described above. The freeze-dried contents of vial were reconstituted and a 1:50 dilution was prepared with PBS (pH 7.4). Eight sites, well apart in the shaved area, four on each side, were chosen for inoculation. At each site, 5^6 ml of the viral suspension was inoculated subcutaneously. The swollen area was gently massaged to facilitate spreading of the inoculum. The entire shaved area was then covered with 2^3 layers of sterile gauze cloth and secured. The cloth was changed daily to avoid soiling and contamination of the inoculated sites. Temperature was recorded daily. On day 7, after recording of the temperature, inoculated sites were examined for hyperaemia and pendular appearance due to the accumulation of the £uid as well as tissue reaction. This was the correct time for the harvest of the virus. After exsanguination of the lambs, tissue and gelatinous material were collected from the inoculated sites under aseptic conditions and placed in a sterile beaker in an ice-bath. A 25% suspension was prepared by blending, followed by addition of stabilizer (2.5% lactalbumin hydrolysate and 5% sucrose in D-MEM). The suspension was strained through sterile gauze and later ¢ltered through 0.45 mm ¢lters. Gentamycin at the rate of 30 mg/ml was added to the ¢ltrate, which was dispensed into 1 ml aliquots, freezedried and stored at ^408C. Skin infective dose 50% (SID50) of challenge virus A female lamb aged 3 months that had not been vaccinated against sheep pox was used. The titration of SPV challenge virus was done as described earlier and the ¢nal reading was taken on day 10 after inoculation. The SID50 was calculated (Reed and Muench, 1938). Experimental design Safety test in laboratory animals Five vials of the vaccine were selected at random and each of the vials was reconstituted with 1 ml of sterile, cold PBS and pooled. The vaccine was tested in two guinea pigs and six mice. Each of the guinea pigs was inoculated intradermally and intramuscularly with 0.5 ml of reconstituted vaccine (103 TCID50/dose) and each of the mice was inoculated intraperitoneally with 0.2 ml of the vaccine. The guinea pigs and mice were observed daily for a period of 14 days. The temperature of the guinea pigs was recorded daily and the mice were observed for mortality (Pandey and Nanda, 1988).
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Safety test in lambs Two SPV seronegative lambs were each inoculated with 10 ¢eld doses (one ¢eld dose = 103 TCID50) of the vaccine contained in 1 ml by the subcutaneous route at the abaxial surface of the tail, and the lambs were observed daily for a period of 14 days for the development of any untoward reactions (Pandey and Nanda, 1988). Potency test The potency test was done by vaccinating two groups of four SPV seronegative lambs, each with one ¢eld dose (103 TCID50) and one-tenth of a ¢eld dose (102 TCID50) contained in 0.1 ml by intradermal inoculation in the caudal fold. The lambs were observed daily for 14 days for the development of cutaneous reactions at the site of inoculation. Body temperatures of all the lambs including unvaccinated controls were recorded daily for 14 days (Pandey and Nanda, 1988). Challenge studies Four lambs in each vaccinated group (group I and group II), in the in-contact group (group III) and in the healthy control group (group IV) were challenged on the 28th day after vaccination by subcutaneous inoculation with 104 SID50 of the virulent SPVR contained in 1 ml. The lambs were kept under observation for 2 weeks, during which time the body temperature, local reactions and clinical symptoms, if any, were recorded daily and compared with the in-contact and unvaccinated healthy control groups of lambs (Pandey and Nanda, 1988). Field trials A total of 660 sheep at various farms located in di¡erent agroclimatic regions of the state were vaccinated with SPV-R at an immunizing dose of 102 TCID50 intradermally at the abaxial surface of the tail. Since challenge studies were not permitted at the ¢eld level, the blood and serum samples were collected periodically from approximately 10% of vaccinated and unvaccinated control groups on days 0, 21, 45, 90 and 180 after vaccination. The blood and serum samples were utilized for studying the CMI and humoral responses, respectively. Assay of cell-mediated immunity (CMI) The CMI response was measured by the glucose utilization test (GUT) (Awati, 1992). Blood was collected from vaccinated animals for sensitized lymphocytes and also from unvaccinated, seronegative lambs. One ml of blood was collected aseptically from each
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lamb in a vacutainer tube containing 20 units of heparin. The heparinized blood was transferred to the laboratory and cultured in 10 ml sterile screw-capped tubes. Each tube contained 2 ml of RPMI 1640 (16) and 0.2 ml of blood. Then 0.3 ml of cell culture-adapted SPV-R vaccine with a titre of 105 TCID50/ml was added to each tube. Five tubes were maintained per sample. The contents were mixed well and kept at 378C for 4 days with periodic agitation of the tubes at 12 h intervals. (Five tubes each containing 2 ml of RPMI 1640 and 0.2 ml of blood from unvaccinated control lambs with 0.3 ml of SPV-R vaccine served as controls.) After 4 days, the tubes were centrifuged at 600g for 10 min. The supernatants from ¢ve tubes for each animal as well as ¢ve control tubes were pooled separately. Then 0.2 ml of the supernatant from each group was taken and diluted to 1 ml with distilled water. Phenol (5% in distilled water) in 0.5 ml quantities was added to each tube, ensuring uniform distribution. Later, 5 ml of concentrated sulphuric acid was added to this mixture and allowed to stand for 10 min. The tubes were shaken well and placed in a water-bath maintained at 288C for 15 min. One ml of fresh distilled water served as a blank. The absorbance was measured at 490 nm. Assay of humoral response The humoral immune response was measured by a serum neutralization test (Ramyar and Hessami, 1970). Serial 10-fold dilutions of each serum were mixed with 102 TCID50 of SPV-R strain in 96-well microplates. The plates were held at room temperature for 1 h and 105 SLT cells were then added to each well. The plates were sealed and incubated at 378C for 7 days under 5% CO2. The ¢nal reading was taken on day 7. The titre of each serum was expressed as the reciprocal of the highest dilution of the serum that inhibited the cytopathic e¡ect (CPE). The neutralization index was then calculated (Reed and Muench, 1938). Statistics The GUT and serum neutralization index values were analysed by Student's t-test; statistically signi¢cant di¡erences are indicated in tables by superscripts (Snedecor and Cochran, 1967). RESULTS TCID50 of the vaccine strain The titre of the SPV-R at the 50th passage in SLT cells was 109.63 TCID50/ml after freeze-drying.
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RD50 of the vaccine virus The body temperature in all three lambs increased gradually and reached a maximum of 40.7 to 41.78C on the 6th to 7th day after inoculation. Reactions appeared at the inoculation spots in all the lambs on the 4th day. In the ¢rst two lambs, the reactions appeared at all of the inoculated spots at all of the dilutions (10^4 to 10^7). In the third lamb, reactions appeared at all the spots at 10^6 and 10^7 dilutions; there were three reactions out of ¢ve inoculated spots at the 10^8 dilution and only two reactions out of ¢ve inoculated spots at the 10^9 dilution. Areas inoculated with PBS as control did not show any in£ammatory signs (Figure 1). The RD50 of SPV-R vaccine strain was found to be 109.51/ml.
Figure 1. Lamb exhibiting local reactions at the sites of inoculation with SPV-R strain during RD50 titration
Propagation of challenge SPV There was a gradual increase in temperature in all the three lambs inoculated with the virulent strain of SPV-R 3 days after inoculation, and the highest temperature was recorded on days 7 (40.88C) and 8 (40.98C) after inoculation. Reactions started appearing on day 5 after inoculation in all the three lambs and they became hyperaemic and pendulous on days 6 and 7 owing to tissue reactions and accumulation of £uids.
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SID50 of virulent SPV The inoculated lamb showed a gradual rise in body temperature (39.68C) from the 4th day onwards and reached a peak of 41.18C on the 8th day. The temperature decreased to normal on the 13th day. Reactions appeared on the 5th day after inoculation for 10^3 and 10^4 dilutions and on the 6th day for 10^5 dilution. The area inoculated with PBS did not exhibit any reactions. The ¢nal reading was taken on day 10 after inoculation. The SID50 of the challenge SPV was 105/ml. Safety test in laboratory animals Guinea pigs and mice inoculated with live attenuated SPV-R vaccine showed neither temperature changes or local reactions and remained healthy and normal during the 14-day observation period. Safety test in lambs Both lambs showed a gradual rise in body temperature and attained a peak of 40.3^ 41.28C by the 6th and 8th days after vaccination, which subsided by the 11th day. The lambs exhibited distinct swellings at the sites of inoculation, these measuring about 0.5 cm on day 5 after inoculation. The reactions were 0.5 cm on day 5 and 0.6 cm on day 6, and subsided from the 12th day onwards in both lambs. There were no local necrotic lesions or generalized reactions in either lamb. Potency test Each of the four lambs in groups I and II showed a rise in body temperature by the 5th and 6th days (40.58C), which subsided gradually from day 7 onwards and reached normal on the 9th day. The unvaccinated in-contact lambs (group III) did not exhibit any rise in temperature. Reactions appeared at the sites of vaccination in all the lambs of group I and group II on the 4th day and faded on the 10th day with the formation of small scabs at the site of inoculation (Figure 2). The unvaccinated, in-contact lambs (group III) did not exhibit any clinical signs or local reactions. Challenge studies Vaccinated lambs in group I and group II did not show any rise in temperature after challenge with virulent SPV-R during the observation period, whereas the in-contact (group III) and healthy control lambs (group IV) exhibited severe temperature increase (Figure 3), extensive local reactions and other associated clinical signs of sheep pox.
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Figure 2. Lamb exhibiting mild local reactions on the abaxial surface of the tail after vaccination with SPV-R strain on the 5th day
Figure 3. Average temperatures in lambs vaccinated with live attenuated SPV-R vaccine and control lambs after challenge with virulent SPV
21 1.30b+0.10 (15) 1.62 +0.01 (4)
0 1.57a+0.065 (15) 1.63 +0.06 (4)
Signi¢cantly di¡erent (p40.05)
1.34b+0.07 (15) 1.63 +0.10 (4)
45
a^c
Signi¢cantly di¡erent (p40.05)
n, number of samples; SE, standard error
Vaccinated sheep (n) Control sheep (n)
Type of samples ^ (15) ^ (4)
0
45 2.07b+0.34 (15) ^ (4)
21 1.46a+0.13 (15) ^ (4)
Days after vaccination
TABLE II Mean+SE of SN indices of sheep vaccinated with live attenuated SPV-R vaccine and control sheep
a^d
n, number of samples; SE, standard error
Vaccinated sheep (n) Control sheep (n)
Type of sample
Days after vaccination
TABLE I Mean+SE of OD (490 nm) values of GUT of SPV-R-vaccinated and control sheep
1.87b+0.25 (15) ^ (4)
90
1.08c+0.19 (15) 1.48 +0.02 (4)
90
2.49c+0.26 (15) ^ (4)
180
1.38d+0.07 (15) 1.52 +0.02 (4)
180
316
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Field trials Among 660 sheep vaccinated during the ¢eld trials with SPV-R cell culture vaccine, 165 sheep were closely monitored for a period of 10 days after vaccination for temperature and local reactions. There was no discernible rise of temperature. Local reactions at the site of vaccination appeared 2^4 days after inoculation and subsided by the 8th day. The feeding behaviour of the sheep was normal and no speci¢c signs of sheep pox were observed. CMI response A signi¢cant di¡erenc in OD values by GUT was observed among the randomly selected vaccinated and unvaccinated control sheep at 0, 21, 45, 90 and 180 days after vaccination. The di¡erences were statistically signi¢cant (p40.05) (Table I). Humoral response The serum neutralization indices of the vaccinated sheep on days 0, 21, 45, 90 and 180 days after vaccination were statistically signi¢cant (p40.05). The control sheep were negative for sheep pox antibody (Table II). DISCUSSION In the present study, variations in virus titres were observed between in vitro and in vivo models as observed by various workers with di¡erent strains of SPV (Jassim and Hawa, 1981; Hegde, 1988; Kalpana, 1993). Thus, it appears that these variations were in£uenced by various viral factors (strain, pathogenicity, virulence, number of passages), cell systems (type, availability of receptors and ¢broblasts) and animal factors (age, sex, breed, nutritional and immune status). Therefore, it was found to be necessary to include both in vivo and in vitro models for assaying the viral titres. Further, it was reported in earlier work (Ramesh, 1980) and noticed in the present study that viral titres were relatively high in lamb testes compared to other cell systems, and it is possible that SLT cell cultures are best suited for large-scale production of SPV. In the present study, the challenge SPV had a considerable titre in in vivo models, as reported by earlier workers (Martin et al., 1973), and this showed that the virulent strains had a high pathogenic potential for lambs. The di¡erence in titres between the present study and earlier studies could be due to the factors mentioned above. The vaccine was innocuous in experimental animals as found by earlier workers with other strains of SPV (Pandey and Nanda, 1988). The SPV-R vaccine strain at the 50th passage was found safe and su¤ciently attenuated for use as vaccine in susceptible sheep in both laboratory and ¢eld. Other workers have used various cell culture systems for propagation and adaptation of di¡erent strains of SPV (Martin et al., 1973; Jassim, 1979; Ramesh, 1980).
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The vaccine produced mild reactions con¢ned to the sites of vaccination in in vivo models. This showed complete attenuation of the virus. All the in-contact animals were normal throughout the observation period, indicating non-shedding of virus from the vaccinates. Most of the earlier workers (Jassim, 1979; Ramesh, 1980; Awati, 1992) opted for the abaxial surface of the tail for vaccination, as in the present study. No untoward e¡ects were observed among the vaccinates in the present study, indicating that the virus was su¤ciently attenuated to be used as a vaccine at the ¢eld level. Compared to inactivated vaccines, live vaccines are more reliable and produce better immunity. Attenuation of virulent virus by various means has become a popular method of production of live vaccines as it is more economical, dependable and reproducible (Shome, 1988). As reported by earlier workers (Ramesh, 1980; Awati, 1992), challenge study was the method of choice to assess the protection o¡ered by SPV vaccines and in the present experiments all animals were protected, whereas controls succumbed to challenge. Field trials were conducted in 660 sheep from di¡erent sheep farms situated in di¡erent agroclimatic zones to ascertain the e¤cacy of SPV-R vaccine. All the sheep exhibited mild reactions and a slight rise in temperature during the observation period. One notable feature of this vaccine was that an immunizing dose of 102 TCID50 could be used. Such a vaccine is more e¡ective and economical for large-scale immunization at ¢eld level. No untoward e¡ects were reported even 6 months after vaccination and no outbreaks of sheep pox occurred in the £ocks vaccinated, although outbreaks did occur in neighbouring unvaccinated £ocks during the observation period. Although no challenge studies at the ¢eld level were permitted, periodic seromonitoring was done for CMI and humoral responses. GUT indicated a signi¢cant di¡erence in immunity between the vaccinated and control sheep for CMI for a period of 6 months. Similar results have been reported by other workers with various strains of SPV (Kalpana, 1993). There was rise in neutralizing index from the 21st day after vaccination. No reports were available to study the SN indices for a 6 month period. Earlier studies (Rafyi and Chamsy, 1956; Shome, 1988) also indicated high levels of neutralizing antibodies by 21 days after vaccination. The present study showed that the live attenuated SPV-R was innocuous, safe and immunogenic and protected against virulent challenge. In addition, the vaccine was found to be e¡ective in a ¢eld study and induced appropriate immune responses during a 6 month observation period. This suggests that the vaccine could be a suitable alternative to the conventional inactivated SPV vaccines. ACKNOWLEDGEMENTS The author thanks the Director and Joint Director, IVRI, and Dr B.Y. Shridhar, IAH and VB for the encouragement and technical support rendered by them. This work is part of a PhD thesis submitted by V. Bhanuprakash to the University of Agricultural Sciences, Bangalore, Karnataka in December 2001.
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REFERENCES Anon., 1998^1999. Report on integrated sample survey for estimation of production of milk, eggs, wool and meat for the year 1998^99. Annual Report, (Directorate of Animal Husbandry and Veterinary Services, Karnataka) Awati, B., 1992. Safety and potency tests of Kenyan strain of sheep pox virus vaccine in goats, (MVSc thesis, University of Agricultural Sciences, Bangalore, Karnataka, India) Carn, V.M., 1993. Control of capripox virus infections. Vaccine, 11, 1275^1279 Hegde, R., 1988. Studies on antigenic analysis of Ranipet strain of sheep pox virus, (MVSc thesis, University of Agricultural Sciences, Bangalore, Karnataka, India) Jassim, F.A., 1979. Biological properties of Ranipet strain of sheep pox virus, (MVSc thesis, University of Agricultural Sciences, Bangalore, Karnataka, India) Jassim, F.A. and Hawa, L.H., 1981. Adaptation and attenuation of local strain of sheep pox virus in lamb testicle monolayers system. Indian Veterinary Journal, 58, 757^762 Kalpana, G., 1993. Evaluation of immunity conferred by Kenyan strain of Capripox virus against goatpox infection and the antigenic relationship between the two viruses, (MVSc thesis, University of Agricultural Sciences, Bangalore, Karnataka, India) Martin, W.B., Ergin, H. and Koylu, A., 1973. Tests in sheep of attenuated sheep pox vaccines. Research in Veterinary Science, 14, 53^61 Pandey, A.K. and Nanda, Y.P., 1988. Laboratory Manual for Tissue Culture Sheep Pox Vaccine Production, (IVRI, Izatnagar, UP, India) Rafyi, A. and Chamsy, M.H., 1956. Seven years' control of sheep pox in Iran with an adsorbed tissue vaccine on aluminium gel. British Veterinary Journal, 112, 541^547 Ramesh, K.G., 1980. Immunological studies on the Ranipet strain of sheep pox virus propagated in lamb testes cell culture, (MVSc thesis, University of Agricultural Sciences, Bangalore, Karnataka, India) Ramyar, H., 1965. Studies on the immunogenic properties of tissue culture sheep pox virus. Zentallblatt fu«r Veterinarmedizin, 12B, 537^540 Ramyar, H. and Hessami, M., 1970. Studies on the duration of immunity conferred by a live modi¢ed sheep pox tissue culture virus vaccine. Zentralblatt fu«r Veterinarmedizin, 17B, 869^874 Reed, L.J. and Muench, H., 1938. Simple method of estimating 50% end points. American Journal of Hygiene, 27, 493^497 Sathe, R.G., 1931. Pox in sheep. Indian Veterinary Journal, 8, 118^121 Shome, B.R., 1988. Comparative biochemical and immunological studies on polypeptides of ¢eld and cell culture adapted Ranipet isolate of sheep pox virus, (MVSc thesis, University of Agricultural Sciences, Bangalore, Karnataka, India) Snedecor, G.W. and Cochran, A.W., 1967. Statistical Methods, (IBH Publications, Calcutta) (Accepted: 17 February 2003) Vaccin classique vivant atte¨nue¨ de protection contre la variole du mouton Re¨sume¨ ^ Un vaccin classique, vivant, atte¨nue¨ de protection contre la variole du mouton a e¨te¨ pre¨pare¨ en utilisant la souche Ranipet du virus de la varicelle du mouton (SVP pour sheep pox virus) au 50e passage dans le syste©me secondaire de cellules testiculaires du mouton. La dose infective telle que de¨rive¨e d'une culture de tissu TCID50 et la di¡e¨rence de taux RD50 ont e¨te¨ de 109,63/ ml et de 109,51/ml respectivement. La dose infective du mouton SID50 du virus de provocation SPV a e¨te¨ de 105/ml. Le vaccin s'est ave¨re¨ n'avoir aucun e¡et inde¨sirable chez les animaux de laboratoire et a e¨te¨ ino¡ensif et e¤cace chez les agneaux se¨rone¨gatifs. 660 moutons ont e¨te¨ vaccine¨s dans les champs avec une dose d'immunisation contenant 16102 de TCID50. Des moutons vaccine¨s se¨lectionne¨s ont manifeste¨ une bonne immunite¨ a© me¨diation cellulaire et des re¨ponses humorales telles que mesure¨es par le test d'utilisation du glucose et le test de neutralisation du se¨rum, respectivement, durant la pe¨riode d'e¨tude de 6 mois. Vacuna cla¨sica de virus vivos atenuados para la viruela ovina Resumen ^ Se preparo¨ una vacuna cla¨sica de virus vivos atenuados para la viruela ovina utilizando la cepa Ranipet del virus de la viruela ovina (VVO o SPV en ingle¨s), en su 50 pasaje por un sistema de cultivo
320 celular testicular secundario de cordero. Las dosis infecciosas medias de cultivo de tejido (TCID50 en ingle¨s) y RD50 fueron de 109,63/ml y 109,51/ml, respectivamente. SID50 del VVO era 105/ml. Se encontro¨ que la vacuna no ten|¨ a efectos secundarios en los animales de laboratorio, y resultaba segura y efectiva en corderos seronegativos para VVO. En el campo, se vacunaron a 660 ovejas con una dosis inmunizadora que conten|¨ a 16102 TCID50. Las ovejas vacunadas que se seleccionaron al azar alcanzaron buena inmunidad celular y respuestas humorales como quedo¨ medido por el test de utilizacio¨n de la glucosa y el test de neutralizacio¨n serolo¨gica respectivamente, durante el periodo de estudio de 6 meses.
