Peste des Petits Ruminants Virus Tissue Tropism and Pathogenesis in Sheep and Goats following Experimental Infection

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.     

Peste des petits ruminants (PPR): Disease appraisal with global and Pakistan perspective

Peste des petits ruminants (PPR), is a highly contagious viral disease affecting domestic and wild small ruminants. It is in the list of animal diseases to be notified to the World Organization for Animal Health (Office International des Epizooties, OIE). Because of the high magnitude of importance of sheep and goats for the poor and landless farmers, the control of disease, which has a negative impact on their productions, is aimed at poverty alleviation. A single injection of live attenuated monovalent vaccine (Nig/75/1) can induce protective immunity for at least the economic life of the animals, however vaccine efficacy only holds if the vaccination is done before exposure and when the animals at sub-clinical level of infection. In Pakistan, though published reports on existence of PPR in the country are few but findings on clinical signs and course of the disease are consistent with the internationally published reports on PPR elsewhere and the neighboring countries. The limited reports of incidence of PPR from few places/provinces do not exclude the possibility of presence of infection in other parts/province of the country. Therefore, the epidemiology, pathogenicity, host susceptibility/resistance and molecular nature of PPR virus have become multifaceted than considered formerly. It is anticipated that patent PPR vaccines and sophisticated diagnostic tests that differentiate infected and vaccinated animals might improve the diagnostic and epidemiological capabilities.     

Rapid quality control of a live attenuated Peste des petits ruminants (PPR) vaccine by monoclonal antibody based sandwich ELISA.

Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of goats and sheep. A homologous Vero cell-based attenuated PPR vaccine developed in our laboratory and used extensively throughout the country, is available for control of PPR. The presently used quality control test, titration in Vero cells for PPR virus titre in vaccine batches, takes at least 6-8days to determine the quality and dose of vaccine. In this study, 74 freeze-dried PPR vaccine batches were tested simultaneously by both virus titration and PPR sandwich ELISA (S-ELISA) to correlate the titre of the vaccine virus with reactivity in S-ELISA. It was found that the vaccine batches with titre more than 10(3)TCID(50)/ml gave positive results in S-ELISA and correlated well with the virus titre of the freeze-dried vaccines. The correlation coefficient between the virus titration and S-ELISA reactivity was estimated as 0.96, indicating a high correlation between the two parameters based on 74 batches of freeze-dried PPR vaccine. The vaccine batches with titres of 3.0, 4.3, 4.5, 5.0, 6.5 and 7.0 had shown a positive reaction when tested in two-fold dilutions in S-ELISA at 1, 5, 6, 7, 8 and 9log2 titres, respectively. The test vaccine batches were found to be negative in S-ELISA when the titre of the vaccine was less than 10(3)TCID50/ml, suggesting that the vaccine could not be passed for field use. It is concluded that S-ELISA could be a preliminary tool useful for the quality control of PPR vaccine as it is rapid and easy to perform when compared to virus titration.     

MVA recombinants expressing the fusion and hemagglutinin genes of PPRV protects goats against virulent challenge

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.     

Vaccination with Recombinant Adenoviruses Expressing the Peste des Petits Ruminants Virus F or H Proteins Overcomes Viral Immunosuppression and Induces Protective Immunity against PPRV Challenge in Sheep

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.     

Optimal time for vaccination against Peste des petits ruminants (PPR) disease in goats in humid tropical zone in southern Nigeria

Investigation of the incidence of peste des petits ruminants (PPR) disease in goats revealed seasonality in the natural occurrence of PPR disease in the environment. Tissue culture rinderpest vaccine was shown effective in protecting goats against the disease. The optimal time for vaccinating goats against PPR disease was when the least number of animals were incubating the disease. In the tropical humid zone, as in West Africa, the optimal time for vaccination against the PPR disease is from late November to middle of December.     

小反刍兽疫病毒结构与功能的研究进展

小反刍兽疫病毒属于副黏病毒科麻疹病毒属,主要感染山羊、绵羊和野生小反刍兽,临床症状以发热、肺炎、腹泻及呼吸道和消化道黏膜炎症为主要特征。迄今为止对于小反刍兽疫无特效药物进行治疗,该病可对家畜养殖业造成一定的经济损失,因此对小反刍兽疫病毒病原学、结构和功能的研究已成为迫切需求。主要综述了小反刍兽疫病毒的六种结构蛋白N、P、M、F、HN、L和两种非结构蛋白C、V的基因组结构及功能,探讨了新型疫苗的研发方向,以期为小反刍兽疫病毒的深入研究、小反刍兽疫的临床防控提供参考。     

Seroprevalence of Sheep and Goat Pox,Peste Des Petits Ruminants and Rift Valley Fever in Saudi Arabia

Sheep and goat pox, peste des petits ruminants and Rift Valley fever are important diseases of small ruminant livestock. Sheep and goat pox, along with peste des petits ruminants, are endemic throughout most of Africa, Asia and the Middle East. Whereas Rift Valley fever is endemic in Africa, outbreaks in the Middle East have been reported over the past decade, including the Arabian Peninsula. Saudi Arabia is a major importer of livestock, and understanding the prevalence of these viral infections would be useful for disease control. In this study, sera from sheep and goats were collected from 3 regions in Saudi Arabia. They were evaluated for antibodies specific to sheep and goat pox, peste des petits ruminants and Rift Valley fever by virus neutralization assays. To the best of our knowledge, this is the first study to evaluate the seroprevalence of these viruses in sheep and goats.     

Study on Passive Immunity:time of Vaccination in Kids Born to Goats Vaccinated Against Peste des petits ruminants

In this study,the decay of maternal peste des petits ruminants virus(PPRV) antibodies in kids born to goats vaccinated with Asian lineage IV PPR vaccine and the efficacy of passive immunity against PPRV was assessed to determine the appropriate period for vaccination in kids.Serum samples collected from kids born to vaccinated,unvaccinated and infected goats at different time intervals were tested by PPR competitive ELISA and serum neutralization test(SNT).Maternal antibodies in kids were detectable up to 6 months with a decline trend from the third month onwards and receded below the protective level by the fourth month.The kid with an SN titre of 1:8 at the time of immunization showed significant PPRV specific antibody response(percentage inhibition of 76;SN titers >1:16),when tested on 21 day post-vaccination and was completely protected from infection upon virulent PPRV challenge.Similarly,the kid with 1:8 SN titers was completely protected from PPR infection on active challenge.Therefore,PPR vaccination is recommended in kids,aged 4 months and born to immunized or exposed goats.This could be a suitable period to avoid window of susceptibility in kids to PPRV and the effort to eliminate PPR infection from susceptible populations.     

Study on passive immunity: Time of vaccination in kids born to goats vaccinated against Peste des petits ruminants

In this study, the decay of maternal peste des petits ruminants virus (PPRV) antibodies in kids born to goats vaccinated with Asian lineage IV PPR vaccine and the efficacy of passive immunity against PPRV was assessed to determine the appropriate period for vaccination in kids. Serum samples collected from kids born to vaccinated, unvaccinated and infected goats at different time intervals were tested by PPR competitive ELISA and serum neutralization test (SNT). Maternal antibodies in kids were detectable up to 6 months with a decline trend from the third month onwards and receded below the protective level by the fourth month. The kid with an SN titre of 1:8 at the time of immunization showed significant PPRV specific antibody response (percentage inhibition of 76; SN titers >1:16), when tested on 21 day post-vaccination and was completely protected from infection upon virulent PPRV challenge. Similarly, the kid with 1:8 SN titers was completely protected from PPR infection on active challenge. Therefore, PPR vaccination is recommended in kids, aged 4 months and born to immunized or exposed goats. This could be a suitable period to avoid window of susceptibility in kids to PPRV and the effort to eliminate PPR infection from susceptible populations.     

A Retrospective Study on the Epidemiology of Anthrax,Foot and Mouth Disease,Haemorrhagic Septicaemia,Peste des Petits Ruminants and Rabies in Bangladesh, 2010-2012

Anthrax, foot and mouth disease (FMD), haemorrhagic septicaemia (HS), peste des petits ruminants (PPR) and rabies are considered to be endemic in Bangladesh. This retrospective study was conducted to understand the geographic and seasonal distribution of these major infectious diseases in livestock based on data collected through passive surveillance from 1 January 2010 to 31 December 2012. Data analysis for this period revealed 5,937 cases of anthrax, 300,333 of FMD, 13,436 of HS, 247,783 of PPR and 14,085 cases of dog bite/rabies. While diseases were reported in almost every district of the country, the highest frequency of occurrence corresponded to the susceptible livestock population in the respective districts. There was no significant difference in the disease occurrences between districts bordering India/Myanmar and non-border districts (p>0.05). Significantly higher (p<0.01) numbers of anthrax (84.5%), FMD (88.3%), HS (84.9%) and dog bite/rabies (64.3%) cases were reported in cattle than any other species. PPR cases were reported mostly (94.8%) in goats with only isolated cases (5.2%) in sheep. The diseases occur throughout the year with peak numbers reported during June through September and lowest during December through April, with significant differences (p<0.01) between the months. The annual usages of vaccines for anthrax, FMD, HS and PPR were only 7.31%, 0.61%, 0.84% and 11.59% of the susceptible livestock population, respectively. Prophylactic vaccination against rabies was 21.16% of cases. There were significant differences (p<0.01) in the administration of anthrax, FMD and HS vaccines between border and non-border districts, but not PPR or rabies vaccines. We recommend that surveillance and reporting of these diseases need to be improved throughout the country. Furthermore, all suspected clinical cases should be confirmed by laboratory examination. The findings of this study can be used in the formulation of more effective disease management and control strategies, including appropriate vaccination policies in Bangladesh.     

Randomized Controlled Field Trial to Assess the Immunogenicity and Safety of Rift Valley Fever Clone 13 Vaccine in Livestock

BackgroundAlthough livestock vaccination is effective in preventing Rift Valley fever (RVF) epidemics, there are concerns about safety and effectiveness of the only commercially available RVF Smithburn vaccine. We conducted a randomized controlled field trial to evaluate the immunogenicity and safety of the new RVF Clone 13 vaccine, recently registered in South Africa.MethodsIn a blinded randomized controlled field trial, 404 animals (85 cattle, 168 sheep, and 151 goats) in three farms in Kenya were divided into three groups. Group A included males and non-pregnant females that were randomized and assigned to two groups; one vaccinated with RVF Clone 13 and the other given placebo. Groups B included animals in 1^st half of pregnancy, and group C animals in 2^nd half of pregnancy, which were also randomized and either vaccinated and given placebo. Animals were monitored for one year and virus antibodies titers assessed on days 14, 28, 56, 183 and 365.ResultsIn vaccinated goats (N = 72), 72% developed anti-RVF virus IgM antibodies and 97% neutralizing IgG antibodies. In vaccinated sheep (N = 77), 84% developed IgM and 91% neutralizing IgG antibodies. Vaccinated cattle (N = 42) did not develop IgM antibodies but 67% developed neutralizing IgG antibodies. At day 14 post-vaccination, the odds of being seropositive for IgG in the vaccine group was 3.6 (95% CI, 1.5 – 9.2) in cattle, 90.0 (95% CI, 25.1 – 579.2) in goats, and 40.0 (95% CI, 16.5 – 110.5) in sheep. Abortion was observed in one vaccinated goat but histopathologic analysis did not indicate RVF virus infection. There was no evidence of teratogenicity in vaccinated or placebo animals.ConclusionsThe results suggest RVF Clone 13 vaccine is safe to use and has high (>90%) immunogenicity in sheep and goats but moderate (> 65%) immunogenicity in cattle.     

Molecular characterization of Corynebacterium pseudotuberculosis isolated from goats using ERIC-PCR

Corynebacterium pseudotuberculosis, the infectious agent of caseous lymphadenitis (CLA), is responsible for substantial economic losses in goat and sheep production. Molecular characterization of C. pseudotuberculosis isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR has shown promising results in genotyping strains isolated from sheep with CLA. We evaluated the genetic diversity of C. pseudotuberculosis isolates collected from the Sert?o region of the Pernambuco (PE) State, Brazil, and investigated the potential of ERIC-PCR as a tool for the molecular typing of strains of C. pseudotuberculosis isolated from goats. Thirty-two C. pseudotuberculosis strains isolated from goats in the municipalities of Floresta and Ibimirim, PE, C. pseudotuberculosis type strain ATCC 19410, the 1002 vaccine strain, and a field isolate of Rhodococcus equi were fingerprinted using the primers ERIC-1R and ERIC-2 and the primer pair ERIC- 1R+ERIC-2. Using 100% similarity as the cutoff, 8, 10, and 7 genotypes were obtained with ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively. The Hunter-Gaston discriminatory index calculated for the ERIC-1-PCR was 0.75. The index for the ERIC-2-PCR was 0.88, and the index for the ERIC-1+2-PCR was 0.79. Among goat isolates of C. pseudotuberculosis, three, two and four genotypes (found by ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively) had been previously described among sheep isolates from Minas Gerais State, Brazil. These results showed that ERIC-PCR has good discriminatory power and typeability, making it a useful tool for discrimination among C. pseudotuberculosis isolates from goats.     

A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of <Emphasis Type="Italic">peste des petits ruminants</Emphasis> virus in the clinical samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T_m of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ～0.0001 cell culture infectious dose 50% units (TCID₅₀). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.     

Cytogenetic and blood group studies of sheep/goat chimaeras

Aggregation chimaeras were composed of quarter (or 1 cell) contributions from 4-cell blastocysts of sheep or goats, or of an 8-cell blastocyst of one species enveloped in three 8-cell blastocysts of the other. Gestation was in sheep or goat recipient females. Of the 10 living animals born, 3 were identified as interspecific chimaeras by body conformation and coat type among the 7 quarter/quarter aggregations and 1 among the 3 giant aggregates. Interspecific chimaerism was identified by cytogenetic study of umbilicus and blood lymphocytes respectively of 2 of these, one from each type of aggregate. Intraspecific sex chimaerism was found in 3 other animals; 2 were of giant aggregate origin, but the 1 of quarter/quarter origin must have acquired it by placental anastomosis with a twin conceptus. Tests using species-specific monoclonal antibodies and electrophoretic separation of haemoglobins and isoenzymes demonstrated sheep and goat erythrocytes in one giant aggregate chimaera; their relative proportions and those of the blood lymphocytes changed over a period of 31 months from approximately 60% goat and 40% sheep to more than 90% sheep. The plasma transferrins and amylases did not show similar relative changes from their predominantly goat-like character and, by implication, neither did their tissues of origin.     

Self-Assembly and Release of Peste des Petits Ruminants Virus-Like Particles in an Insect Cell-Baculovirus System and Their Immunogenicity in Mice and Goats

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential “differentiating infected from vaccinated animals” (DIVA) vaccine candidates for the surveillance and eradication of PPR.     

Presence of species-specific, monomorphic antigens on sheep and goat binucleate cells

Immunocytochemical assays for sheep and goat species-specific, monomorphic antigens were developed utilizing polyclonal antisera from sheep and goats immunized by interspecific pregnancy. The assays were applied to cell isolates from sheep and goat fetal cotyledons collected from allogeneic pregnancies at Days 35, 40 and 120 of gestation. The isolates contained 7 to 48% binucleate cells (BNC). Using these assays, the sheep-specific antigen was detected on sheep cotyledonary cell isolates on all days of gestation tested (P < 0.001); the assay also detected the antigen on the BNC subset of the cotyledonary cell isolate population (P < 0.001). The caprine-specific antigen was shown to be present on cotyledonary cell isolates (P < 0.05), although the presence of the antigen could not be demonstrated with statistical confidence on goat BNC due to insufficient numbers of discernible cells. Binucleate cells contribute to the formation of the syncytial layer of the placenta by fusing with maternal epithelial cells and with the syncytium. The species-specific antigen (or antigens) is present on BNC at the appropriate time of gestation at which it (they) could play a role in the humoral immune response to interspecific and hybrid pregnancies observed in ewes and does.     

A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and Taq Man RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID50). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.     

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.     

Sharing of Pasteurella spp. between free-ranging bighorn sheep and feral goats

Pasteurella spp. were isolated from feral goats and free-ranging bighorn sheep (Ovis canadensis canadensis) in the Hells Canyon National Recreation Area bordering Idaho, Oregon, and Washington (USA). Biovariant 1 Pasteurella haemolytica organisms were isolated from one goat and one of two bighorn sheep found in close association. Both isolates produced leukotoxin and had identical electrophoretic patterns of DNA fragments following cutting with restriction endonuclease HaeIII. Similarly Pasteurella multocida multocida a isolates cultured from the goat and one of the bighorn sheep had D type capsules, serotype 4 somatic antigens, produced dermonecrotoxin and had identical HaeIII electrophoretic profiles. A biovariant U(beta) P.haemolytica strain isolated from two other feral goats, not known to have been closely associated with bighorn sheep, did not produce leukotoxin but had biochemical utilization and HaeIII electrophoretic profiles identical to those of isolates from bighorn sheep. It was concluded that identical Pasteurella strains were shared by the goats and bighorn sheep. Although the direction of transmission could not be established, evidence suggests transmission of strains from goats to bighorn sheep. Goats may serve as a reservoir of Pasteurella strains that may be virulent in bighorn sheep; therefore, goats in bighorn sheep habitat should be managed to prevent contact with bighorn sheep. Bighorn sheep which have nose-to-nose contact with goats should be removed from the habitat.