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.

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.

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.

African swine fever: A permanent threat to Indian pigs.

India has 9 million pigs, of which 45% are in the North eastern (NE) states of India. Viral diseases affecting pigs are a major concern of mortality causing huge loss to the pig farmers. One such disease is African swine fever (ASF) that has already knocked the porous borders of NE states of India. ASF is a highly contagious devastating disease of pigs and wild boars causing 100% mortality. The causative agent African swine fever virus (ASFV) belongs to the genus Asfivirus, family Asfarviridae. Pig is the only species affected by this virus. Soft ticks (Ornithodoros genus) are shown to be reservoir and transmission vectors of ASFV. Transmission is very rapid and quickly engulfs the entire pig population. It is very difficult to differentiate classical swine fever from ASF since clinical symptoms overlap. Infected and in contact pigs should be culled immediately and buried deep, and sheds and premises be disinfected to control the disease. There is no vaccine available commercially. Since its first report in Kenya in 1921, the disease has been reported from the countries in Europe, Russian federation, China, and Myanmar. The disease is a threat to Indian pigs. OIE published the first report of ASF in India on May 21, 2020, wherein, a total of 3701 pigs died from 11 outbreaks (Morbidity - 38.45% and mortality - 33.89%) in Assam and Arunachal Pradesh states of India. ASF is non-zoonotic.

Prevalence and antibiotic susceptibility of Mannheimia haemolytica and Pasteurella multocida isolated from ovine respiratory infection: A study from Karnataka, Southern India.

BACKGROUND AND AIM: Respiratory infection due to Mannheimia haemolytica and Pasteurella multocida are responsible for huge economic losses in livestock sector globally and it is poorly understood in ovine population. The study aimed to investigate and characterize M. haemolytica and P. multocida from infected and healthy sheep to rule out the involvement of these bacteria in the disease. MATERIALS AND METHODS: A total of 374 healthy and infected sheep samples were processed for isolation, direct detection by multiplex PCR (mPCR), and antibiotic susceptibility testing by phenotypic and genotypic methods. RESULTS: Overall, 55 Pasteurella isolates (27 [7.2%] M. haemolytica and 28 [7.4%] P. multocida) were recovered and identified by bacteriological tests and species-specific PCR assays. Significant correlation between the detection of M. haemolytica (66.6%) with disease condition and P. multocida (19.1%) exclusively from infected sheep was recorded by mPCR. In vitro antibiotic susceptibility testing of 55 isolates revealed higher multidrug resistance in M. haemolytica (25.9%) than P. multocida (7.1%) isolates. Descending resistance towards penicillin (63.6%), oxytetracycline (23.6%), streptomycin (14.5%), and gentamicin (12.7%) and absolute sensitivity towards chloramphenicol were observed in both the pathogens. The antibiotic resistance genes such as strA (32.7%) and sul2 (32.7%) associated with streptomycin and sulfonamide resistance, respectively, were detected in the isolates. CONCLUSION: The study revealed the significant involvement of M. haemolytica together with P. multocida in ovine respiratory infection and is probably responsible for frequent disease outbreaks even after vaccination against hemorrhagic septicemia in sheep population of Karnataka, southern province of India.

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.

Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544.

The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage 'ϕLd' was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 10(8) CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ∼16, 19 and 23 kDa could be identified as immuno-dominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 µg protein and 60 µg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner.