First evidence of lumpy skin disease in mithun (Bos frontalis) in India.

Lumpy skin disease (LSD) is a disease of cattle that is also known to cause mild infection in buffaloes. To date, there have been no reports of LSD in mithun (Bos frontalis), a bovine species distributed in Northeast India, Bangladesh, Myanmar, and parts of China. In the present study, the presence of typical clinical signs, virus isolation, PCR amplification, sequence analysis, and the demonstration of antibodies in serum by indirect enzyme-linked immunosorbent assay and serum neutralization test, confirmed the occurrence of LSD in mithun for the first time in India. Phylogenetic analysis based on the full-length RPO30 and P32 genes of LSD virus from mithun and cattle revealed 100% sequence identity, indicating circulation of the same strain in both species in India and the possibility of spillover between species.

Post-Vaccination Sero-Monitoring of Peste des Petits Ruminants in Sheep and Goats in Karnataka: Progress towards PPR Eradication in India.

Peste des petits ruminants (PPR) presents economic challenges in enzootic countries impacting small ruminant productivity. The state of Karnataka, India, implemented a mass vaccination campaign in alignment with the PPR-Global Eradication Programme (GEP) and the National Strategic Plan for PPR eradication. This study was conducted from January to March 2023 to assess seroconversion in post-vaccinated goats and sheep at the epidemiological unit (epi-unit) level, aligning with the World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) guidelines in the PPR Global Control and Eradication Strategy (GCES). Before vaccination, 3466 random serum samples were collected from small ruminants of three age groups (6-12 months, 1-2 years, and >2 years) across 116 epi-units, spanning 82 taluks in 28 districts. Post-vaccination sero-monitoring included 1102 serum samples collected from small ruminants of the 6-12-month age group only, across 111 epi-units covering 64 taluks in 23 districts. The PPRV antibody status was determined using an indigenous hemagglutinin (H) protein monoclonal antibody-based competitive ELISA kit. Pre-vaccination, the PPR seropositivity rates were 55%, 62%, and 66% in the age groups of 6-12 months, 1-2 years, and >2 years, respectively, with a 61% PPRV antibody prevalence across all the age groups. Notably, 41% of the epi-units exhibited antibody prevalence rates of ≥70%, indicating a substantial population immunity, possibly attributed to the previous vaccination program in the state since 2011. In contrast, only 17% of the epi-units had below 30% seroprevalence rates, emphasizing the need for intensified vaccination. Statistical analysis of the data revealed significant correlations (p < 0.05) between the presence of PPRV antibodies and host factors such as species, breed, and sex. Post-vaccination seroprevalence in the 6-12 months age group was found to be 73.4%, indicating the use of an efficacious vaccine. On the evaluation of vaccination immunity in the 6-12 months age group, it was revealed that over 69% of the epi-units achieved a response surpassing ≥70%, indicating a significant improvement from 42% of the epi-units in pre-vaccination. For active PPR eradication, a mass vaccination campaign (>95% coverage) targeting small ruminant populations aged >4 months is advocated, aiming to achieve the desired herd immunity of >80%. This study offers crucial insights into PPR baseline seroprevalence/immunity status and vaccine efficacy, guiding national strategies towards a PPR-free India and further supporting the global eradication initiative.

Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Epidemiological investigation of ASF outbreaks in Kerala (India): detection, source tracing and economic implications.

This study investigates suspected African swine fever (ASF) outbreaks in two villages of Kannur district in Kerala, India, with the aim of identifying the causative agent and its genotype, the source of infection, and estimating the economic losses due to the outbreaks. Clinically, the disease was acute with high mortality, while gross pathology was characterized by widespread haemorrhages in various organs, especially the spleen, which was dark, enlarged and had friable cut surfaces with diffuse haemorrhages. Notably, histopathological examination revealed multifocal, diffuse haemorrhages in the splenic parenchyma and lymphoid depletion accompanied by lymphoid cell necrosis. The clinico-pathological observations were suggestive of ASF, which was confirmed by PCR. The source of outbreak was identified as swill and it was a likely point source infection as revealed by epidemic curve analysis. The phylogenetic analysis of p72 gene identified the ASFV in the current outbreak as genotype-II and IGR II variant consistent with ASFVs detected in India thus far. However, the sequence analysis of the Central Variable Region (CVR) of the B602L gene showed that the ASFVs circulating in Kerala (South India) formed a separate clade along with those found in Mizoram (North East India), while ASFVs circulating in Arunachal Pradesh and Assam states of India grouped in to different clade. This study represents the first investigation of ASF outbreak in South India, establishing the genetic relatedness of the ASFV circulating in this region with that in other parts of the country. The study also underscores the utility of the CVR of the B602L gene in genetically characterizing highly similar Genotype II ASFVs to understand the spread of ASF within the country.

Lumpy Skin Disease (LSD) in Yak (Bos grunniens): An Evidence of Species Spillover from Cattle in India.

Lumpy skin disease (LSD), caused by the lumpy skin disease virus (LSDV), is a global concern that affects cattle and buffalo. Recently, the disease has been reported in new species such as the Indian Gazelle, Camel, Banteng, Gaur, and Giraffe from various parts of the world. This report provides an insight into the occurrence of LSD in Yak from Sikkim, a North-Eastern state of India. During the investigation, both cattle and yak exhibited typical clinical signs of LSD, including skin nodular lesions. The morbidity, mortality, and case fatality rates for cattle were 9.08%, 1.84%, and 20.24%, respectively. Similarly, the morbidity, mortality, and case fatality rates in yak were 7.57%, 1.24%, and 16.33%, respectively. The virus isolation and amplification of LSDV-specific genes confirmed the presence of LSDV in cattle, yak, and vectors. Further, demonstrated antibodies in randomly collected sera from naïve and unvaccinated cattle and yak using indirect Enzyme Linked Immuno-sorbent Assay (iELISA) and Serum Neutralisation test (SNT) from this region. Sequencing and phylogenetic analysis of P32, GPCR, and RPO30 genes revealed that the virus isolated from both species was 100% identical to each other and also closely related to the field LSDV isolates circulating in the Indian subcontinent. The study highlighted the emergence of LSDV in unconventional hosts and underscored the need to include other bovine species in national disease control programs, encompassing disease surveillance initiatives.

Diagnosis of rabies using reverse-transcription polymerase chain reaction on post-mortem skin tissue specimens of the nasolabial plate in a rabies suspected cow: a case study.

In India, rabies in cattle is under-reported. Religious sentiments hamper its diagnosis, discouraging post-mortem examination, particularly opening the cranium. Specimens of peripheral tissue innervated by the cranial nerves could potentially be used as alternative diagnostic specimens to the brain. Herein, we present a case study of a novel approach for diagnosing rabies in a cow suspected of having rabies, using skin tissue specimens of the nasolabial plate obtained post-mortem. Brain and nasolabial tissue specimens tested positive for rabies using conventional reverse-transcription polymerase chain reaction. This approach has been previously shown to have a high diagnostic sensitivity in animals. We encourage further studies with more nasolabial plate skin specimens for both post- and antemortem diagnosis of rabies in cattle.

Sero-positivity of Japanese Encephalitis Virus in Equine Population of India Using IgG ELISA: Unraveling the Need for Vaccination.

Japanese encephalitis (JE) is a mosquito borne flaviviral zoonoses, causing fatal disease in equines and humans. JE is endemic in most of the states of India with occurrence of human cases every year. The horses are not vaccinated against JE in India and thus they are at more risk of acquiring the disease. Due to nonavailability of indigenously developed ELISA and high cost of imported kits, regular sero-surveillance is not being carried out to assess the true picture of JE virus in equine population of India. Therefore, a recombinant NS1 protein based indirect IgG ELISA was developed with the objective to assess the sero-positivity of JE virus in equine population of India. The diagnostic sensitivity and specificity of developed ELISA was 84.73% and 86.70%, respectively. The validation studies revealed good reproducibility of ELISA with kappa value ranging from 0.75 to 1 between the results of different laboratories. A total of 2,069 horse serum samples were screened using the developed ELISA and 401 samples were positive for IgG against JEV with an overall sero-positivity of 19.38% in equine population of India. A sero-positivity of 25.90% and 12.22% was recorded in Himachal Pradesh and Jammu-Kashmir, both hill states of North zone of India for the first time, revealing the spread of virus to the nonendemic parts of the country. The high sero-positivity of JE virus recorded in equine population warrants the need for initiation of vaccination of horses in India to prevent the morbidity and mortality.

Serological surveillance and clinical investigation of glanders among indigenous equines in India from 2015 to 2018.

Equine glanders is an infectious and notifiable bacterial disease caused by Burkholderia mallei. The disease has been reported in South American, African and Asian countries including India. Here, we present the outcome of glanders serosurveillance carried out between January 2015 and December 2018 to know the status of equine glanders among different states in India. A total of 102,071 equid sera from 299 districts of twenty-one states and one union territory were tested for glanders. Samples were screened with Hcp1 indirect ELISA followed by confirmatory diagnosis by CFT. During this four-year surveillance, a total of 932 glanders-positive cases were detected from 120 districts of 12 states. The study also revealed increasing trend of glanders from 2016 onwards with maximum occurrence in northern India. Overall seroprevalence ranged between 0.62% (95% CI, 0.52-0.72) and 1.145% (95% CI, 1.03-1.25). Seasonal shifting from winter to summer (March to June) coincided with highest number glanders incidence with corresponding seroprevalences of 1.2% (95% CI, 1.09-1.30). The present surveillance unveils territorial ingression of glanders to six states like Jammu & Kashmir, Gujarat, Rajasthan, Madhya Pradesh, Delhi and Tamil Nadu. In addition, re-emerging cases have been reported in Maharashtra, Haryana and Punjab after a gap of 10 years. Lack of awareness, little veterinary care and unrestricted movement of equids across state borders might have led to the introduction and establishment of the infection to these states. We believe that information from this study will provide a baseline data on glanders for devising surveillance and control strategies in India. Being a zoonotic disease, the persistence of glanders poses a potential threat to occupationally exposed humans especially equine handlers and veterinarians. Therefore, targeted surveillance of human population from each glanders outbreak is also recommended.

Latex agglutination test for rapid on-site serodiagnosis of Japanese encephalitis in pigs using recombinant NS1 antigen.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) is a mosquitoe-borne viral zoonotic disease and globally around three billion people are at the risk of disease. The occurrence of JE cases has shown a rising trend during last decade in India. Pig is the amplifying host for JE virus and serves as a suitable sentinel model for the prediction of disease outbreak in humans. The development of a diagnostic test that is suitable for surveillance of JE in pigs is the need of the hour. The existing tests require elaborate laboratory facilities which make their application in rural settings difficult. Therefore, realizing the need for a rapid test, efforts were made to standardize a latex agglutination test (LAT) for serodiagnosis of JE in pigs. METHODS: Standardization of LAT by physical adsorption of recombinant NS1 (non-structural) protein of JE virus onto latex beads was done by altering six different variables, namely the antigen concentration, sensitization condition, surface blocking agent, blocking condition, particle concentration and reaction time. The standardized latex-protein complex was used for screening 246 pig serum samples under optimal conditions. RESULTS: The test was standardized with a diagnostic sensitivity and specificity of 82.24 and 87.83%, respectively. Screening of 246 field pig serum samples using standardized LAT showed a seropositivity of 50.4%. The results were available within 5 min after addition of test serum sample to the sensitized beads. INTERPRETATION & CONCLUSION: The findings of the study highlight the potential of LAT as a rapid on-site assay for JE diagnosis in pigs which would aid in predicting JE outbreaks in humans.

Genetic characterization of equine herpesvirus 1 isolates from abortion outbreaks in India.

Equine herpesvirus 1 (EHV1) is a common pathogen of horses that causes upper respiratory tract disease, abortion, neonatal death and neurological disease. The neurological form of disease is called equine herpesvirus myeloencephalopathy (EHM). During the past decade, the incidence of EHM has been on the rise in Europe, North America, Australia and Asia. Some EHV1 isolates causing EHM exhibit a single-nucleotide polymorphism (SNP) in the DNA polymerase gene (ORF30) at position 2254 (A2254 to G2254). Further, based on polymorphism in the ORF68, EHV1 isolates have been classified into different groups. The aim of the present study was to estimate the genetic diversity of EHV1 and to determine the prevalence of the neuropathogenic genotype of EHV1 in India. Out of 133 clinical specimens from abortion cases in northern India, 56 were positive for EHV1 infection. Analysis of the A/G SNP by real-time PCR and sequence analysis revealed that 54 of 56 samples (96.43 %) were of the non-neuropathogenic genotype (A2254), while two (3.57 %) had the neuropathogenic marker (G2254). Sequence analysis of the polymorphic region of ORF68 of EHV1 isolates (n = 9) from India indicated that the Delhi/1998, Tohana-2/2013, Hisar-2/2014 and Hisar-15/1990 isolates belonged to group 4, while the Jind/1996, Rajasthan/1998, Delhi-3/2007 and Tohana-5/1996 isolates clustered within group 5. One isolate (Hisar-7/1990) exhibited SNPs at positions C710 and C713, forming a separate group. Here, we report for the first time the detection of neuropathogenic genotypes of EHV1 in India and show that Indian EHV1 isolates cluster within groups 4 and 5.

Pathology of Equine Influenza virus (H3N8) in Murine Model.

Equine influenza viruses (EIV)-H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×106.24 EID50 with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.