Long Term Protective Immunity to Goatpox in Goats After Single Immunization with a Live Attenuated Goatpox Vaccine

In this study, duration of immunity following single shot vaccination using an attenuated goatpox vaccine (GTPV/Uttarkashi/1978) was evaluated in sero-negative kids for 52 months. Long term immunity was evaluated by clinical protection upon virulent virus challenge and serum neutralization assay applied for serum samples. Rise in level of GTPV specic antibodies was found to be maximum on 21 days post vaccination, which was maintained between 1 and 2 years of immunization with steady decline. Upon virulent virus challenge on 21 days, 12, 24, 42 and 52 months post vaccination, protection in all vaccinated animals was evident, whereas, control animals developed severe clinical disease. This is for the rst time that long term immunity of a live goatpox vaccine has been investigated up to 52 months of post-vaccination in goats and it has immense potential in controlling and eradicating goatpox from an enzootic situation.


Main Text
The goat population in India is 135.7 million [3]. Goatpox is one of the most important viral infections of goats which affect their productivity considerably. The disease characterized by fever and oculo-nasal discharges is highly contagious. Pox lesions appear on the skin, respiratory and gastro-intestinal mucosae. Goatpox is caused by goatpox virus of the genus Capripoxvirus and the family Poxviridae [17]. Goatpox affects goats of all ages, sex and breeds but it is common in young and old animals, and lactating goats. The disease is currently prevalent throughout the South Western Asia, the Indian subcontinent, and north and central Africa [16]. The disease occurs particularly where small ruminants play an important role in the agriculture economy. An effective vaccine is important to contain the disease. Heterologous vaccination using either sheeppox or goatpox vaccines to protect either have not been very successful [12]. Therefore, it is recommended that homologous vaccine should be used to protect either goatpox or sheeppox [6,18]. A goatpox vaccine using an indigenous strain (Uttarkashi/78) [9] has been developed and commercialized in India [12]. Attenuated capripox vaccines using different sheep and goats strains have been used to control capripoxvirus in several countries [8]. Ability to confer prolonged immunity following vaccination is a key to induce strong immunity in the population towards controlling and eradicating a disease. Protection against virulent virus challenge following immunization using 0240 strain of capripox lasts for three years [17]. However, the duration of immunity produced by other strains should be ascertained by controlled trials. The inactivated capripox vaccines provide short duration of immunity, which may be less than a year [17]. Therefore, in the present study, duration of immunity/protection following single shot vaccination using an indigenous Vero cell adapted goatpox vaccine (GTPV/Uttarkashi/1978) has been evaluated for a period of 52 months 4 years and 4 months).
Vero cells (ATCC, USA) were grown in Eagle's Minimum Essential Medium (EMEM), supplemented with 10% new born calf serum. The cells were used for preparing the goatpox vaccine and virus antigen for neutralization assay. Fifteen goat kids aged between six months and one year of either sex and sero negative to goatpox with no history of vaccination or infection due to natural exposure were immunized each with 0.2ml of vaccine containing 2.5 log 10 TCID 50 of the vaccine virus as per prescribed in OIE manual of standards, intradermally on the caudal-fold. These goats were reared at Laboratory Animal Section, ICAR-IVRI, Mukteswar, India. Five goats, which were sero-negative to goatpox were maintained as in-contact controls.
Three vaccinated goats and in-contact control goats were challenged periodically after 12, 24, 42 and 52 months post immunization with the Mukteswar isolate of virulent GTPV [12]. Animals were challenged with virulent virus with a titre dose of ~2 x 10 6 SRID 50 /ml [skin reactive dose] per animal by intradermal route [19]. All the vaccinated-challenged and control-challenged animals were observed for a period of 14 days post challenge for rectal temperature, the appearance of skin lesions and generalized infection, if any. The serum samples of all the vaccinated animals were collected periodically at 21 days post immunization (dpi) and 12, 24, 42 and 52 months post immunization (mpi). The sera were collected at 7, 14, 21 and 28 days post challenge. The neutralization test was carried out as described earlier [13] with slight modi cation [12]. In brief, a ten-fold dilutions of GTPV against a constant serum dilution were used. Besides safety, potency and broad-spectrum immunity against the prevailing strains/variants of virus, ability of the vaccine to confer long lasting immunity is paramount for the obvious reasons. In general, an ideal vaccine should provide lifelong immunity, have broad spectrum of activity against all the variants of the virus circulating in the region, induce rapid immune response, potent, e cacious, safe and effective in all age groups, no adverse reactions, thermostable, protection even with a single dose of immunization and economical [5]. Duration of immunity following single shot immunization is an important attribute of a vaccine as it is economical and more effective in reducing the transmission rates particularly in the enzootic settings. A number of vaccines have been shown to induce long duration of immunity like smallpox and hepatitis in humans, while, rinderpest and PPR vaccines have been tested for long term immunity in their homologous hosts.
Goatpox is enzootic in India and can be controlled by homologous vaccine. There are several protective studies on capripoxviruses in general and goatpox virus in particular which report protection following vaccination for a period up to few days to years [1,4,6,[10][11][12]21] and some claim possible long-time protection. However, there is no systematic study to prove the protection beyond two years post vaccination [5]. In the current study, we studied the protection of goats up to 52 months on single shot vaccination with goatpox vaccine following intradermal route of inoculation. Animals were monitored for local and systemic reactions like thermal and immune responses. Immune response was evaluated by serum neutralization and periodic protection studies for a period of 52 months following single immunization.
In this controlledtrial, vaccine was found safe and induced signi cant antibody and protection response in the vaccinated animals. The important objective of this study was to study the duration of immunity following single immunization. Each animal was immunized with a recommended dose of goatpox vaccine and in contact control animals maintained along with the vaccinated ones to rule out shedding of the virus from the vaccinates. The animals were monitored daily for 14 days for thermal and local reactions. Following vaccination, all the vaccinated animals had slight increase in body temperature and small takes [erythema] at the site of inoculation implying that the replication of the virus at low levels. These reactions were transient and subsided gradually without affecting the health of the animal. These signs are not uncommon following vaccination with a pox vaccine [4,6,7,12].
At 12, 24, 48 and 52 months post vaccination, goats were challenged with virulent goatpox virus. Vaccine could confer protection up to 52 months post vaccination on single dose vaccination as evident from humoral antibody response and protection on challenge. Both humoral and cell mediated immunity play a major role in protection. Nevertheless, the cell mediated immunity is predominant. However, the challenge study is the indicator of protection. In all the instances, vaccinated and challenged animals were protected indicating 100% protection even after 52 months of single immunization. Even then, serum collected periodically from vaccinated and vaccinated challenged goats indicated a rise in antibody levels which is signi cantly different from the control animals. On 21/28 th day post vaccination with goatpox vaccine in goats, a range of 0.5 to 1.75 neutralization index [NI] has been reported [2,20] with 100% protection [12]. However, some studies have reported the protection for a year post vaccination based on the neutralizing index, which is ≥ 1.5 [1][2]. There are studies which used ELISA titres as the indicators of protection [12,15]. A very few studies have demonstrated protection following vaccination and challenge after a year of immunization [14].
Long term immunity trial based on the challenge studies on vaccination with live attenuated goatpox vaccine in goats demonstrated protective immunity up to 52 months. Protection was substantiated by the presence of neutralizing antibodies also resistance to live virus challenge. Vaccine that provides long duration of immunity is highly useful in resource poor countries as it obviates the cost of repeat vaccination in mass immunization program, where cost of vaccination is signi cant as compared to the cost of vaccine, besides avoiding stress to the animal. Therefore, live attenuated goatpox vaccine reported here has an abundant potential impact towards achieving control with vaccination and subsequent eradication of goatpox. Clinical reaction of vaccinated (12,24, 42 and 52 months) and control goats following challenge. Vaccinated goats have shown only mild local skin reaction whereas the control animals were showing severe local and generalized skin lesions along with varying degree of clinical disease.

Figure 3
Post vaccination and challenge sero-monitoring of goats by serum neutralization test indicating the trend of neutralizing antibody prevalence over the period of long term study