Pathomolecular epidemiology, antimicrobial resistance, and virulence genes of Streptococcus dysgalactiae subsp. equisimilis isolates from slaughtered pigs in India.

AIMS: We aimed to investigate the prevalence, pathology, and characterization of Streptococcus dysgalactiae subsp. equisimilis (SDSE) in slaughtered pigs of India. METHODS AND RESULTS: We collected 1254 morbid tissues (lungs-627 and spleen-627) and 627 heart-blood from 627 slaughtered pigs. The bacterial isolation, antibiogram, virulence gene profiling, and mouse pathogenicity testing were performed for the detection and characterization of SDSE. A total of 177 isolates (heart-blood-160 and tissues-17) were recovered from 627 slaughtered pigs with higher isolation rate in heart-blood (25.51%). The prevalence of SDSE was 11% in morbid tissues by polymerase chain reaction. Majority of isolates showed higher detection of streptolysin O, followed by streptokinase and extracellular phospholipase A virulence genes with higher degree of resistance to azithromycin, clindamycin, erythromycin, and penicillin antibiotics. Mouse pathogenicity testing confirmed virulence based on histopathological lesions and re-isolation of SDSE. CONCLUSIONS: Our findings highlight the high prevalence of SDSE in slaughtered pigs. The presence of virulence genes and mouse pathogenicity testing confirm their pathogenic potential.

A reverse transcription-multiplex PCR strategy devised for concomitant detection and differentiation of foot and mouth disease virus serotypes O, A and Asia 1 in India.

Serotype identification occupies the central part of foot and mouth disease (FMD) diagnosis workflow and vaccination decision tree. In this study, a reverse transcription-multiplex PCR (RT-mPCR) strategy wherein three assays with unique combinations of serotype specific primers targeting the VP1 region was developed to differentiate FMD virus serotypes O, A and Asia 1 based on differential size of the PCR amplicons on agarose gel. Their diagnostic performance relative to the mPCR assay in use in India was evaluated on 169 clinical samples and 210 cell culture grown virus isolates. The relative diagnostic sensitivity was found to be 99.69%, 98.78% and 99.08% for primer combinations 1, 2 and 3, respectively. These assays proved their worth by detecting serotype in three FMD suspected specimens that went undiagnosed in the existing mPCR and also by identifying multiple serotypes in the same sample. Their detection limits varied from log10 2 to log10 4 viral RNA dilution and from 100 to 0.1 TCID50 virus depending on the serotype. The validated novel mPCR assays show promise to be included in the routine diagnostic tool-box to augment the efficiency of diagnosis of FMD virus serotypes that display extreme genetic diversity and a tendency of transboundary dispersal.

Foot-and-Mouth Disease Virus Serotype O Exhibits Phenomenal Genetic Lineage Diversity in India during 2018-2022.

In India, widespread foot-and-mouth disease (FMD) outbreaks occurred in 2021. The objective of this study was to identify genetic lineages and evaluate the antigenic relationships of FMD virus (FMDV) isolates gathered from outbreaks reported between 2019 and 2022. Our study shows that the lineages O/ME-SA/Ind2001e and the O/ME-SA/Cluster-2018 were both responsible for the FMD outbreaks on an epidemic scale during 2021. This observation is in contrast to earlier findings that suggested epidemic-scale FMD outbreaks in India are often connected to a single genetic lineage. Additionally, we report here the identification of the O/ME-SA/PanAsia-2/ANT10 sub-lineage in India for the first time, which was connected to two intermittent outbreaks in Jammu and Kashmir. The current study demonstrates that the O/ME-SA/ind2001e lineage has a strong presence outside of the Indian subcontinent. Furthermore, the O/ME-SA/Cluster-2018 was observed to have a wider geographic distribution than previously, and like the O/ME-SA/Ind2001d and O/ME-SA/Ind2001e lineages in the past, it may eventually spread outside of its geographic niche. For O/ME-SA/Ind2001e and O/ME-SA/Cluster-2018, the predicted substitution rate for the VP1 region was 6.737 × 10-3 and 8.257 × 10-3 nt substitutions per site per year, respectively. The time of the most recent common ancestor of the O/ME-SA/Ind2001e and O/ME-SA/Cluster-2018 strains suggests that the viruses possibly emerged during 2003-2011 and 2009-2017, respectively. Recent sightings of the O/ME-SA/PanAsia2/ANT10 virus in India and the O/ME-SA/Ind2001e virus in Pakistan point to possible cross-border transit of the viruses. The results of a two-dimensional viral neutralization test revealed that all of the field isolates were antigenically matched to the currently used Indian vaccine strain O INDR2/1975. These results suggest that the serotype O vaccine strain can protect against outbreaks brought on by all three circulating lineages.

Emergence of a novel genetic lineage 'A/ASIA/G-18/2019' of foot and mouth disease virus serotype A in India: A challenge to reckon with.

Foot and mouth disease (FMD) has engendered large scale socioeconomic crises on numerous occasions owing to its extreme contagiousness, transboundary nature, complicated epidemiology, negative impact on productivity, trade embargo, and need for intensive surveillance and expensive control measures. Emerging FMD virus variants have been predicted to have originated and spread from endemic Pool 2, native to South Asia, to other parts of the globe. In this study, 26 Indian serotype A isolates sampled between the year 2015 and 2022 were sequenced for the VP1 region. BLAST and maximum likelihood phylogeny suggest emergence of a novel genetic group within genotype 18, named here as 'A/ASIA/G-18/2019' lineage, that is restricted so far only to India and its eastern neighbour, Bangladesh. The lineage subsequent to its first appearance in 2019 seems to have displaced all other prevalent strains, in support of the phenomenon of 'genotype/lineage turnover'. It has diversified into two distinct sub-clusters, reflecting a phase of active evolution. The rate of evolution of the VP1 region for the Indian serotype A dataset was estimated to be 6.747 × 10-3 substitutions/site/year. India is implementing a vaccination centric FMD control programme. The novel lineage showed good antigenic match with the proposed vaccine candidate A IND 27/2011 when tested in virus neutralization test, while the existing vaccine strain A IND 40/2000 showed homology with only 31% of the isolates. Therefore, in order to combat this challenge of antigenic divergence, A IND 27/2011 could be the preferred strain in the Indian vaccine formulations.

Novel pan-lineage VP1 specific degenerate primers for precise genetic characterization of serotype O foot and mouth disease virus circulating in India.

Analysis of the VP1 gene sequence of the foot and mouth disease virus (FMDV) is critical to understanding viral evolution and disease epidemiology. A standard set of primers have been used for the detection and sequence analysis of the VP1 gene of FMDV directly from suspected clinical samples with limited success. The study validated VP1-specific degenerate primer-based reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and sequencing of serotype O FMDV lineages circulating in India. The novel degenerate primer-based RT-PCR amplifying the VP1 gene can circumvent the genetic heterogeneity observed in viruses after cell culture adaptation and facilitate precise viral gene sequence analysis from clinical samples.

Diagnostic application of formalin fixed archived tissues for detection of foot-and-mouth disease.

Early and definitive disease diagnosis is critical for effective disease control. 50% buffered glycerine is commonly used viral transport medium, which is not always available and required cold chain. Tissues samples archived in 10% neutral buffered formalin (NBF) can preserve nucleic acid that can be used in molecular studies and disease diagnosis. The present study's goal was to detect the foot-and-mouth disease (FMD) viral genome in formalin-fixed archived tissue which may avoid cold chain during transportation. This study used FMD suspected samples preserved in 10% neutral buffered formalin from 0 to 730 days post fixation (DPF). All archived tissues were positive for FMD viral genome by multiplex RT-PCR and RT-qPCR up to 30 DPF, whereas archived epithelium tissues and thigh muscle were positive for FMD vial genome up to 120 DPF. FMD viral genome was detected in cardiac muscle up to 60 DPF and 120 DPF, respectively. The findings suggest that 10% neutral buffered formalin could be used for sample preservation and transportation for timely and accurate FMD diagnosis. More samples need to be tested before implementing the use of 10% neutral buffered formalin as a preservative and transportation medium. The technique may add value in ensuring biosafety measures for creation during disease free zone as well.

Production and characterization of monoclonal antibodies against foot-and-mouth disease virus serotype O and development of a sandwich ELISA for virus antigen detection.

Foot-and-mouth disease (FMD) is endemic in India with a majority of outbreaks caused by FMD virus (FMDV) serotype O. In the present study a panel of eight (2F9, 2G10, 3B9, 3H5, 4C8, 4D6, 4G10 and 5B6) mouse monoclonal antibodies (MAbs) were developed against FMDV serotype O Indian vaccine strain, O/IND/R2/75 via hybridoma systems. The MAbs generated were FMDV/O specific without cross-reactivity against FMDV type A and Asia 1. All the MAbs were identified as IgG1/kappa type. Out of eight, three MAbs (3B9, 3H5 and 4G10) demonstrated virus neutralizing activity. The reactivity of all MAbs increased with heat treated (@560C) serotype O antigen compared to untreated antigen in sandwich ELISA indicating that their binding epitopes are linear. Six MAbs (except 2F9 and 4D6) reacted with recombinant P1 protein of homologous virus in an indirect ELISA among which only MAb 3B9 bound to VP1. MAb profiling of 37 serotype O field viruses isolated between the years 1962 and 2021 demonstrated antigenic similarity between field isolates and reference vaccine strain. MAbs 5B6 and 4C8 consistently reacted with all 37 isolates. In indirect immunofluorescence assay MAb 5B6 bound well with FMDV/O antigen. Finally, a sandwich ELISA was successfully developed using rabbit polyclonal anti-FMDV/O serum and MAb 5B6 for detection of FMDV/O antigen in clinical samples (n = 649). The new assay exhibited 100% and 98.89% diagnostic sensitivity and specificity respectively compared to traditional polyclonal antibody-based sandwich ELISA suggesting that the MAb-based ELISA developed here could be an effective method for detection of FMDV serotype O.

Thermostable negative-marker foot-and-mouth disease virus serotype O induces protective immunity in guinea pigs.

Foot-and-mouth disease (FMD) is a contagious viral disease of high economic importance, caused by FMD virus (FMDV), a positive-sense single-stranded RNA virus, affecting cloven-hoofed animals. Preventive vaccination using inactivated virus is in practice to control the disease in many endemic countries. While the vaccination induces antibodies mainly to structural proteins, the presence of antibodies to the non-structural proteins (NSP) is suggestive of infection, a criterion for differentiation of infected from vaccinated animals (DIVA). Also, there is a growing demand for enhancing the stability of the FMD vaccine virus capsid antigen as the strength of the immune response is proportional to the amount of intact 146S particles in the vaccine. Considering the need for a DIVA compliant stable vaccine, here we report generation and rescue of a thermostable and negative marker virus FMDV serotype O (IND/R2/1975) containing a partial deletion in non-structural protein 3A, generated by reverse genetics approach. Immunization of guinea pigs with the inactivated thermostable-negative marker virus antigen induced 91% protective immune response. Additionally, a companion competitive ELISA (cELISA) targeting the deleted 3A region was developed, which showed 92.3% sensitivity and 97% specificity, at cut-off value of 36% percent inhibition. The novel thermostable-negative marker FMDV serotype O vaccine strain and the companion cELISA could be useful in FMDV serotype O enzootic countries to benefit the FMD control program. KEY POINTS: • Thermostable foot-and-mouth disease virus serotype O with partial deletion in 3A. • Inactivated thermostable marker vaccine induced 91% protection in guinea pigs. • Companion cELISA based on deleted region in 3A could potentially facilitate DIVA.

Foot-and-mouth disease status in India during the second decade of the twenty-first century (2011-2020).

Foot-and-mouth disease (FMD) is a major disease of livestock in India and causes huge economic losses. The formal FMD control program started in 2003-04 in selected districts and was gradually expanded. The present study provides a descriptive review of the FMD outbreaks, prevalent serotypes, and genetic and antigenic features of the FMD virus (FMDV) that circulated in the country between 2011 and 2020. FMD outbreaks were regularly reported in cloven-hoofed domestic livestock and wildlife, with three serotypes including O, A, and Asia1. During the study period, a total of 2226 FMD outbreaks were documented and serotypes confirmed. FMDV serotype O dominated the outbreak scenario, accounting for about 92% of all outbreaks, followed by Asia1 (5% of all outbreaks) and A (3% of all outbreaks). Two major epidemics of FMD on an unprecedented scale during the years 2013 and 2018 by serotype O were recorded. The spatial distribution of FMD was characterized by a larger number of outbreaks in the southern region of the country. In an annual-scale analysis, 2020 was the year with the lowest outbreaks, and 2013 was the year with the highest. The month-scale analysis showed that outbreaks were reported throughout the year, with the highest numbers between October and March. The emergence of three major lineages (O/ME-SA/Ind2001d, O/ME-SA/Ind2001e, and O/ME-SA/Ind2018) of serotype O was observed during the period. In the cases of serotype A and Asia1, the appearance of at least one novel lineage/genetic group, including A/G-18/non-deletion/2019 and Asia1/Group-IX, was documented. While serotype A showed the advent of antigenic variants, serotypes O and Asia1 did not show any antigenic diversity. It was noticed during the course of an outbreak that animal movement contributes significantly to disease transmission. Except for 2018, when numerous FMD outbreaks were recorded, the number of annual outbreaks reported after 2016 has been lower than in the first half of the decade, probably due to mass vaccination and COVID-19 pandemic-linked movement restrictions. Even during outbreaks, disease symptoms in ruminant populations, including cattle, were found to be less severe. Regular six-monthly immunization certainly has a positive impact on the reduction of disease burden and should be followed without fail and delay, along with intensive disease surveillance.

Impact of mass vaccination on the spatiotemporal dynamics of FMD outbreaks in India, 2008-2016.

Foot-and-mouth disease (FMD) is endemic in India, where circulation of serotypes O, A and Asia1 is frequent. Here, we provide an epidemiological assessment of the ongoing mass vaccination programs in regard to post-vaccination monitoring and outbreak occurrence. The objective of this study was assessing the contribution of mass vaccination campaigns in reducing the risk of FMD in India from 2008 to 2016 by evaluating sero-monitoring data and modelling the spatiotemporal dynamics of reported outbreaks. Through analyzing antibody titre data from >1 million animals sampled as part of pre- and post-vaccination monitoring, we show that the percent of animals with inferred immunological protection (based on ELISA) was highly variable across states but generally increased through time. In addition, the number of outbreaks in a state was negatively correlated with the percent of animals with inferred protection. We then analyzed the distribution of reported FMD outbreaks across states using a Bayesian space-time model. This approach provides better acuity to disentangle the effect of mass vaccination programs on outbreak occurrence, while accounting for other factors that contribute to spatiotemporal variability in outbreak counts, notably proximity to international borders and inherent spatiotemporal correlations in incidence. This model demonstrated a ∼50% reduction in the risk of outbreaks in states that were part of the vaccination program. In addition, after controlling for spatial autocorrelation in the data, states that had international borders experienced heightened risk of FMD outbreaks. These findings help inform risk-based control strategies for India as the country progresses towards reducing reported clinical disease.

Selective isolation of foot-and-mouth disease virus from coinfected samples containing more than one serotype.

The foot-and-mouth disease virus (FMDV) causes a highly infectious disease of all cloven-footed animals. The RNA genome of the virus continuously evolves, leading to the generation of new strains; this necessitates the selection of new vaccine strains to ensure complete protection. Infection with one FMDV serotype does not provide cross-protection against the other FMDV serotypes. Many of the recovered animals may become carriers of the FMDV, but they still remain susceptible to the other serotypes. Coinfection with multiple FMDV serotypes has been reported and studied to understand the virus evolution. Isolation and characterization of all the involved serotypes in the mixed infection case is essential to understand the molecular evolution of the virus. In this study, two cases of coinfection were studied by selective isolation of each of the FMDV serotypes under the cross-serotype-specific immune pressure. It was estimated that the virus present in a minimum of 10-0.92 TCID50 could be isolated from the mixed population containing other serotypes in infective doses of 100.25 TCID50 or less. All involved serotypes present in the mixed infection cases were isolated, without any cross-contamination. Virus characterization revealed that genotype 2 was of serotype A virus from a sample collected in 1995, which was last reported in 1986, indicating a possible subdued prevalence of the genetic group even after vanishing from the field.

Short communication: preliminary observations on the serum levels of HSP70 and its correlation with serum cortisol, thyroid hormones, and acute-phase protein concentration in cattle naturally infected with foot-and-mouth disease virus.

The study aimed to explore the serum levels of HSP70 and identify its possible association with serum cortisol, thyroid hormones, and acute-phase protein concentrations in cattle naturally infected with foot-and-mouth disease (FMD) virus. After the FMD outbreak in an organized dairy cattle farm in India, blood samples were obtained from clinically infected (n = 40) and apparently healthy (n = 30) animals. Samples were processed and tested by an in-house DIVA assay for confirmation of FMD infection. Serum was analyzed for HSP70, cortisol, T4, T3, haptoglobin, and serum amyloid A by enzyme-linked immunosorbent assay (ELISA). HSP70 concentrations were significantly higher in the serum of clinically infected cattle (p < 0.01) than the healthy group. To the best of our knowledge, this is the first report describing the elevated serum levels of HSP70 under infectious diseases of bovines. Cortisol (p < 0.05), haptoglobin (p < 0.001), and serum amyloid A (p < 0.05) concentrations also markedly increased in the diseased animals; however, no differences (p > 0.05) were found in T4 and T3 levels between healthy and infected cattle. Elevated HSP70 concentration correlated positively with high cortisol (p < 0.05) and haptoglobin (p < 0.001) levels suggesting an essential link between these acute events during clinical infectious phase of FMD.

Genetic characterization of foot-and-mouth disease virus serotype O isolates collected during 2014-2018 revealed dominance of O/ME-SA/Ind2001e and the emergence of a novel lineage in India.

Foot-and-mouth disease (FMD) is endemic in India with a preponderance of outbreaks caused by FMD virus (FMDV) serotype O. Out of the 11 global topotypes of serotype O, only ME-SA topotype has been reported in the country so far. Lineage O/ME-SA/Ind2001 and O/ME-SA/PanAsia are documented as the most dominant ones in terms of the number of outbreaks caused by them. To understand the distribution of topotype/lineages in India and their antigenic behaviour during the year 2014-2018, a total of 286 FMDV serotype O viral isolates were sequence determined at the VP1 region, and 109 isolates were characterized antigenically. All the isolates grouped in the ME-SA topotype, being distributed in lineage O/ME-SA/Ind2001 (within sub-lineages O/ME-SA/Ind2001d and O/ME-SA/Ind2001e), and a new group designated here as O/ME-SA/2018 cluster. The sub-lineage O/ME-SA/Ind2001e reported for the first time in India during the year 2015, replaced sub-lineage O/ME-SA/Ind2001d gradually, which was dominating since 2008. During the years 2014-2018, the sub-lineage O/ME-SA/Ind2001e was found to be the most predominant one whose mean evolutionary rate was observed to be faster than that of the sub-lineage O/ME-SA/Ind2001d. The codon sites 45 and 85 of VP1 were found to be under diversifying selection in a large proportion of trees. The common ancestor predicted for sub-lineages O/ME-SA/Ind2001e and O/ME-SA/2018 dates back to 2012 and 2016, respectively. The sustenance and spread of the new O/ME-SA/2018 cluster need to be assessed by continued surveillance. The Indian vaccine strain O/INDR2/1975 was found to provide adequate antigenic coverage to the emerging and prevalent serotype O lineages. The trait association tests showed frequent virus exchange among different states, which could be an important confounder in the region-specific assessment of effectiveness of FMD control programme.