Spatial and temporal analysis of haemorrhagic septicaemia outbreaks in India over three decades (1987-2016).

Haemorrhagic septicaemia (HS) is an economically important disease affecting cattle and buffaloes and the livelihoods of small-holder farmers that depend upon them. The disease is caused by Gram-negative bacterium, Pasteurella multocida, and is considered to be endemic in many states of India with more than 25,000 outbreaks in the past three decades. Currently, there is no national policy for control of HS in India. In this study, we analysed thirty year (1987-2016) monthly data on HS outbreaks using different statistical and mathematical methods to identify spatial variability and temporal patterns (seasonality, periodicity). There was zonal variation in the trend and seasonality of HS outbreaks. Overall, South zone reported maximum proportion of the outbreaks (70.2%), followed by East zone (7.2%), Central zone (6.4%), North zone (5.6%), West zone (5.5%) and North-East zone (4.9%). Annual state level analysis indicated that the reporting of HS outbreaks started at different years independently and there was no apparent transmission between the states. The results of the current study are useful for the policy makers to design national control programme on HS in India and implement state specific strategies. Further, our study and strategies could aid in implementation of similar approaches in HS endemic tropical countries around the world.

Comparative genome analysis of Pasteurella multocida strains of porcine origin.

Pasteurella multocida causes acute/chronic pasteurellosis in porcine, resulting in considerable economic losses globally. The draft genomes of two Indian strains NIVEDIPm17 (serogroup D) and NIVEDIPm36 (serogroup A) were sequenced. A total of 2182-2284 coding sequences (CDSs) were predicted along with 5-6 rRNA and 45-46 tRNA genes in the genomes. Multilocus sequence analysis and LPS genotyping showed the presence of ST50: genotype 07 and ST74: genotype 06 in NIVEDIPm17 and NIVEDIPm36, respectively. Pangenome analysis of 61 strains showed the presence of 1653 core genes, 167 soft core genes, 750 shell genes, and 1820 cloud genes. Analysis of virulence-associated genes in 61 genomes indicated the presence of nanB, exbB, exbD, ptfA, ompA, ompH, fur, plpB, fimA, sodA, sodC, tonB, and omp87 in all strains. The 61 genomes contained genes encoding tetracycline (54%), streptomycin (48%), sulphonamide (28%), tigecycline (25%), chloramphenicol (21%), amikacin (7%), cephalosporin (5%), and trimethoprim (5%) resistance. Multilocus sequence type revealed that ST50 was the most common (34%), followed by ST74 (26%), ST13 (24%), ST287 (5%), ST09 (5%), ST122 (3%), and ST07 (2%). Single-nucleotide polymorphism and core genome-based phylogenetic analysis clustered the strains into three major clusters. In conclusion, we described the various virulence factors, mobile genetic elements, and antimicrobial resistance genes in the pangenome of P. multocida of porcine origin, besides the rare presence of LPS genotype 7 in serogroup D.

Quantifying the influence of climate, host and change in land-use patterns on occurrence of Crimean Congo Hemorrhagic Fever (CCHF) and development of spatial risk map for India.

Crimean Congo Hemorrhagic Fever (CCHF), is an emerging zoonosis globally and in India. The present study focused on identifying the risk factors for occurrence of CCHF in the Indian state of Gujarat and development of risk map for India. The past CCHF outbreaks in India were collated for the analyses. Influence of land use change and climatic factors in determining the occurrence of CCHF in Gujarat was assessed using Bayesian spatial models. Change in maximum temperature in affected districts was analysed to identify the significant change points over 110 years. Risk map was developed for Gujarat using Bayesian Additive Regression Trees (BART) model with remotely sensed environmental variables and host (livestock and human) factors. We found the change in land use patterns and maximum temperature in affected districts to be contributing to the occurrence of CCHF in Gujarat. Spatial risk map developed using CCHF occurrence data for Gujarat identified density of buffalo, minimum land surface temperature and elevation as risk determinants. Further, spatial risk map for the occurrence of CCHF in India was developed using selected variables. Overall, we found that combination of factors such as change in land-use patterns, maximum temperature, buffalo density, day time minimum land surface temperature and elevation led to the emergence and further spread of the disease in India. Mitigation measures for CCHF in India could be designed considering disease epidemiology and initiation of surveillance strategies based on the risk map developed in this study.

Human anthrax in India in recent times: A systematic review & risk mapping.

The disease anthrax occurs generally in herbivores and the causative organism (Bacillus anthracis) infects humans who come in contact with infected animals or their products. The persistence of anthrax spores for decades and its lethality contribute to its biowarfare potential. We conducted this systematic review along with risk mapping to investigate the spatio-temporal distribution, clinico-epidemiological, socio-behavioural and programmatic issues pertaining to anthrax in India over the last two decades. Peer reviewed quantitative and qualitative studies and grey literature comprising weekly reports of the 'Integrated Disease Surveillance Program' (IDSP), were accessed for extracting data. IDSP data were used for geo-referencing of the villages of anthrax cases; Pseudo-absence was generated to fit a Bayesian Additive Regression Trees (BART) model to develop anthrax risk map. The case fatality rate of cutaneous anthrax ranged from 2% to 38%, while the gastrointestinal and inhalational types were 100% fatal. Our synthesis revealed that human anthrax outbreaks in India were clustered around the eastern coastal regions. The states of Odisha, West Bengal, Andhra Pradesh and Jharkhand reported maximum number of outbreaks. Odisha reported a maximum number of 439 human anthrax cases since 2009, of which Koraput district contributed to 200 cases (46%). While handling or consumption of infected animal product were proximal drivers of these events, poverty, lack of awareness, traditional beliefs and local practices served as facilitatory factors. Other structural determinants were wild life-livestock interface, historical forest loss, soil pH, soil-water balance, organic carbon content, temperature, rainfall and humidity. The programmatic issues identified through this review were lack of active surveillance, non-availability of diagnostic facility at the periphery, delayed reporting, absence of routine livestock vaccination and lack of adequate veterinary services. Interventions based on One-health approach in the country merit immediate policy and program attention; high risk zones for anthrax identified during present investigation, should be prioritized.

Elevation determines the spatial risk of Anthrax outbreaks in Karnataka, India.

Anthrax is an economically important livestock disease affecting subsistence farmers and it is of zoonotic importance. Anthrax is endemic in many states of India including Karnataka. Identification of spatial risk factors for occurrence of anthrax and development of predictive risk maps are required for planning adequate vaccination in high-risk areas as well as targeted surveillance activities in animals, humans and environment. In this study, village level anthrax outbreak locations from Karnataka (1997-2016) were geo-referenced and predictive risk map was developed using temporally Fourier Processed remotely sensed variables. A non-linear discriminant analysis approach was used to develop the risk map for Karnataka. Elevation was identified as top predictor variable in the 10 variables selected. The predicted risk map showed good accuracy and validation statistics when evaluated using different metrics (Kappa, sensitivity, specificity, AUC). The predicted risk map also showed good correspondence with past outbreaks. Further, we used Bayesian Penalised spline method to estimate species response curves for top 10 variables selected. The validated risk map can be used in planning vaccination strategy and surveillance in high-risk areas.

Temporal pattern and risk factors for occurrence of Canine Rabies in Chennai.

Rabies is one of the most important zoonoses resulting in a high case fatality rate in humans. Most of the human Rabies cases are due to dog bites which can be prevented by effective vaccination in dogs. Globally, epidemiological studies on understanding the seasonality and risk factors for occurrence in canines are limited. The present study aimed to understand the temporal pattern of Rabies occurrence in Chennai city of Tamil Nadu, India, and address the suggestive clinical signs for better clinical ante-mortem rabies diagnosis. Data of 598 suspected canine hippocampus brain smear samples with Seller's staining and/or FAT percent positivity of 71.57% (428/598) from March 2010 to February 2019 were included in this study. Cross-correlation between rabies cases and meteorological factors showed that maximum temperature (lag 15), morning relative humidity (lag 0 and lag 5) and evening relative humidity (lag 4) were significantly associated with rabies cases. Auto-Regressive Integrated Moving Average (ARIMA) model with exogenous variables (significant lags of meteorological variables) was used to fit the time series of canine Rabies in Chennai. In logistic regression analysis, the following risk factors were found to be playing a significant role in Rabies positivity viz., behavioural changes in dogs (P < 0.001), free-roaming, unprovoked biting, hyper salivation (P < 0.05), dog bite history and drop jaw (P < 0.01). Hence, the study results highlight the need for continuous surveillance of canine Rabies for devising and implementing future preventive strategies and is helpful to establish the above-identified risk factors as a criterion to help in clinical rabies diagnosis.

Whole-genome sequence analysis of Clostridium chauvoei isolated from clinical case of black quarter (BQ) from India.

Black quarter (BQ) is an infectious disease affecting cattle and small ruminants worldwide caused by Gram-positive anaerobic bacterium Clostridium chauvoei. In this study, a draft genome sequence of C. chauvoei NIVEDIBQ1 strain isolated from clinical case of black quarter was analyzed. Sequence analysis indicated that genome had 2653 predicted coding DNA sequences, harbored numerous genes, mobile genetic elements for pathogenesis, and virulence factors. Computational analysis revealed that strain contained 30 virulence-associated genes. An intact genomic region highly similar to the Clostridium phage was present in the genome. Presence of CRISPR systems and the transposon components likely contribute to the genome plasticity. Strain encode diverse spectrum of degradative carbohydrate-active enzymes (CAZymes). Comparative SNP analysis revealed that the genomes of the C. chauvoei strains analyzed were highly conserved. Phylogenetic analysis of strains and available genome (n = 21) based on whole-genome multi-locus sequence typing (wgMLST) and core orthologous genes showed the clustering of strains into two different clusters suggesting geographical links.

Comparative genome analysis of Pasteurella multocida serogroup B:2 strains causing haemorrhagic septicaemia (HS) in bovines.

Pasteurella multocida, a Gram-negative bacterium with ubiquitous nature, is known to affect wide range of host species worldwide with varied clinical manifestations including haemorrhagic septicaemia (HS) in bovines. Although, HS causing P. multocida strains were identified and characterized by conventional tools and PCR assays, diverse strains are indistinguishable by these tools in the face of disease outbreaks. In this study, draft genomes of three virulent P. multocida serotype B:2 strains (NIVEDIPm32, NIVEDIPm34 and NIVEDIPm35) were analyzed following whole genome sequencing, assembly, annotation and compared them with existing global genomes (n = 43) of bovine origin in the database. Three draft genomes of NIVEDIPm strains consisted of 40-52 contigs with GC content of ∼40.4%. The genome size and predicted genes content was ∼2.3 Mb and 2181-2189, respectively. Besides, the presence of various mobile genetic elements, antimicrobial resistance genes and biofilm related genes suggested their vital roles in virulence; further, adaptation to the host immune system as well as host pathogen interaction. Multi locus sequence analysis based on RIRDC scheme showed the presence of ST122 in all the three strains. wgMLST based phylogenic analysis suggested that HS causing Indian virulent field strains differed geographically and showed diversity from existing HS vaccine strain P52. The phylogenetic tree revealed that North Indian strains share high similarity with strains of Pakistan than South Indian Strain. Notably, a high divergence of SNPs between the HS causing circulating virulent strains of India and current HS vaccine strain P52 suggested an imminent need for relook in to HS vaccination strategy for livestock in India.

Countrywide cross-sectional study of swine brucellosis sero-prevalence in Indian subcontinent during 2018-2019.

Brucellosis in swine is a contagious disease with greater zoonotic potential caused by Brucella suis. The study describes PAN India swine brucellosis sero-prevalence in 5431 stratified random serum samples collected during 2018-2019 from 26 out of 29 states and two out of seven union territories. The serum samples were tested for anti-Brucella antibodies by indirect ELISA and overall, 4.33% apparent prevalence (AP) was recorded. The AP is ≥ 10% in five states among 26 states, P ≥ 50% in four districts out of 117 districts screened and cent percent prevalence in two epi units out of 264 sampled. Significantly high seropositivity (p < 0.05) in male (6.08%) than female pigs (3.46%) and in ≥ 24-month-old pigs indicated older and male pigs as potential carriers of the disease. The study recorded endemicity of the swine brucellosis in few regions of India requiring periodical surveillance for control of the disease. Brucella testing of boars before breeding and awareness among farmers and veterinarians will aid in reduction of disease burden in the absence of vaccination policy.

Development of recombinant NS1-NS3 antigen based indirect ELISA for detection of bluetongue antibodies in sheep.

Bluetongue is an insect borne (Culicoides) viral disease of small ruminants. The virus blankets the globe with a wide serotypic variation, numbered from 1 to 28. In India 21 different serotypes have been reported to be circulating across the various agro-climatic zones of the country. Non-structural proteins (NSPs) of bluetongue virus have always remained ideal target for differentiation of infected from vaccinated animals. The current study is an extrapolation of our previous work where a novel fusion construct comprising of bluetongue viral segment NS1 and NS3 was successfully cloned, expressed, purified with an efficient strategy for its suitable implementation as a diagnostic antigen. In this study, the applicability of the fusion construct has been further evaluated and optimised for field applicability. The fusion construct used in an ELISA platform projected a relative diagnostic sensitivity and specificity of 98.1% and 95.5% respectively against a pre-established test panel. The rNS1-NS3 ELISA showed substantially good agreement with the commercial BTV antibody detection kit. Finally, the study brings together the diagnostic capability of two NSPs, which can be a handy tool for sero-surveillance of bluetongue.

Variability in in vitro biofilm production and antimicrobial sensitivity pattern among Pasteurella multocida strains.

Biofilm production, hitherto an uncharacterized feature among circulating Pasteurella multocida strains, was studied along with the antibiotic susceptibility pattern. On the basis of biofilm formation ability, all the strains were categorized into four groups under six different culture conditions: strong biofilm-forming (22%), moderate (19%), weak (51%), and non-adherent (7%). Strains from serogroups A and B formed significant biofilms in at least one culture condition whereas strains from serogroup D were unable to form biofilms. All strains were found to be susceptible to tetracycline. In addition, the correlation between diverse factors (host, capsule type, clinical condition and the tadD gene) as well as antimicrobial susceptibility in biofilm production were analyzed by Joint distribution models, and showed that enrofloxacin and azithromycin resistant strains were positively correlated with strong biofilm production.

Comparative genetic diversity analysis based on virulence and repetitive genes profiling of circulating Pasteurella multocida isolates from animal hosts.

Virulence associated and/or housekeeping/repetitive genes either in single or multiple copies are being extensively targeted for bacterial pathogen detection and differentiation in epidemiological studies. In the present study, isolation of Pasteurella multocida from different animals and their genetic profiling based on the capsular types, virulence and repetitive elements (ERIC/REP) were carried out. A total 345 clinical samples from apparently healthy and diseased (pneumonic, septicaemia) animals (sheep, goat, pig, cattle, buffalo and rabbits) from different geographical regions of Karnataka, Uttar Pradesh, Mizoram and Assam states of India were screened. A total of 32% of the samples were found positive, of which 41 P. multocida isolates recovered. Virulence profiling of isolates indicated that omp87, ompA, ptfA, sodA, sodC, nanB, fur and exbB were present in 100% of isolates. Whereas, prevalence of other genes were; nanH (90%), ompH (71%), pfhA (63%), plpB (80%), hsf-1 (12%), hsf-2 (37%), pmHAS (78%), toxA (73%), hgbA (37%), hgbB (81%), tbpA (78%) and fimA (98%), among isolates. There was no influence of host or place on prevalence of virulence genes when assessed by fitting a Hierarchial Bayesian ordinal regression model. There was correlation (positive and negative) between broad groups of virulence genes. Both repetitive gene profiles (ERIC and REP) generated multiple amplicons (~200 to ~4000 bp). Cluster analysis with ERIC profiles revealed 5 clusters and 3 non- typable isolates with higher discriminatory power (D = 0.7991) than the REP-PCR profiles (D = 00.734) which revealed 4 clusters and 6 non- typable isolates. The results showed that a considerable level of genetic diversity exists among circulating P. multocida isolates despite belonging to the same geographical origin. The genetic diversity or clustering based on either virulence or repetitive elements among isolates could be largely driven by multiple factors acting together which lead to manifestations of particular disease symptoms.