Search

Mannheimia haemolytica is recognized as principal pathogen associated with pneumonic pasteurellosis leading to huge economic losses to small ruminant farmers. Even though the disease causes huge economic losses, epidemiology of M. haemolytica is less studied, hindering the formulation of effective control strategies. Current study aimed to highlight molecular characterisation of M. haemolytica strains isolated from ovine pneumonic infection. M. haemolytica 27 isolates with two reference strains were characterised using capsular and virulence gene typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. M. haemolytica serotype A2 recognized as predominant serotype (74%) followed by A6 (11%) and A1 (5%) serotypes. Virulence gene profiling by PCRs showed dominance of all five virulent genes [such as adh and gcp (100% each)] followed by gs60 (88.8%), lktC (85.2%), tbpB (51.9%) and least nmaA gene (14.8%). MLST profiling delineated M. haemolytic isolates into 11 sequence types (STs) with most prevalent being ST37 (27.9%) and ST16 (23%) and nine new STs (ST37, 38, 39, 40, 41, 42, 47, 48, and 49). These new STs did not belong to any of the three clonal complexes (CC4, CC8 and CC28). ST16 was exclusively noted in A1 and A6 serotypes. Amongst 25 isolates, 22 pulsotypes (GD 0.88) recorded indicated variability of the M. haemolytica isolates in PFGE analysis. In conclusion, the study suggested dominance of M. haemolytica serotype A2 harbouring different virulent genes, diverse STs and pulsotypes responsible for pneumonic pasteurellosis frequently encountered in sheep. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Haemorrhagic septicaemia (HS) is a highly contagious and fatal disease of cattle and buffaloes and causes major economic losses to farmers. Though indirect hemagglutination (IHA) test has been used to detect a specific antibody against P. multocida, it has low specificity for sero-diagnosis of HS. Therefore, development of a rapid, highly sensitive and specific serological test is a prerequisite for detection of antibodies against HS. In this context, we explored an in-house ELISA method using recombinant antigens for detection of antibodies against P. multocida in bovines. nanH gene from P. multocida B:2 strain P52 was cloned and the recombinant mature protein with a C- and N-terminal truncation was produced as a fusion protein (~63 kDa) in Escherichia coli. The immunogenic potential of purified rNanH-Tr was assessed by the Western blot method using specific anti-rNanH-Tr antibody responses in sera collected from immunized rabbits. An indirect-ELISA based on rNanH-Tr was developed and optimized. Furthermore, the rNanH-Tr ELISA was applied to screen bovine serum samples (n=250). The receiver operating characteristic curve analysis for the detection of anti P. multocida specific antibodies indicated a diagnostic sensitivity of 86.2 (CI 73.26-96.80%) and specificity of 80.0 (63.06-91.56%). No cross reactivity was noted with antibodies against other bovine diseases (e.g., foot-and-mouth disease and brucellosis). Screening of random bovine serum samples showed a 22% sero-positivity for anti P. multocida specific antibodies. [ABSTRACT FROM AUTHOR]

Flagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. Upon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. rFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti- Clostridium chauvoei- specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). The study indicated the production of conformational recombinant flagellin—rFliA(C)—antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei- specific antibodies in BQ-recovered and/or vaccinated animals. [Display omitted] • Construct design based on structural features of flagellin of Clostridium chauvoei. • Native purification of highly soluble recombinant FliA(C) by a single-step process. • Elicitation of rFliA(C) specific conformational antibodies in rabbit & guinea pig. • In ELISA, rFliA(C) detected antibodies against Clostridium chauvoei in bovine sera. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Elevation, land surface temperature, host variables (Cattle, Buffalo and Pigs) determine the spatial risk of Anthrax in Karnataka state of India • Response curves for selected predictor variables developed using Bayesian Penalised spline method • Risk map for Anthrax developed using remote sensed variables • Risk map will be helpful in planning future surveillance, prevention and control 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. Risk map for Anthrax in Karnataka using Fourier processed MODIS variables. The probability of suitability is on a scale from zero to one. [Display omitted] [ABSTRACT FROM AUTHOR]

• Whole genome sequencing of Indian virulent Pasteurella multocida B:2 strains. • Comparative genome analysis of Haemorrhagic septicaemia (HS) causing P. multocida B:2 strains of bovines. • A high divergence of SNPs among circulating P. multocida strains. • Virulent field strains differ from existing HS vaccine strain in India. 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. [ABSTRACT FROM AUTHOR]

Background: Pasteurellosis is an economically important disease of livestock worldwide and vaccination is effective mean to control the disease outbreaks. One of alternative method for vaccine preparation could be the use of lytic phages as bacterial inactivating agents and employing "phage lysate immunogen" as an immunizing agent. Therefore, the study was undertaken to explore the protective immune response of novel phage lysate vaccine against Pasteurella multocida. Methods: A phage lysate vaccine of Pasteurella multocida was prepared using isolated lytic phage and subjected to vaccine response study in rabbits. In this experiment, two mouse fixed-dose of lysate was used and the prophylactic efficacies of lysates were evaluated in rabbits by passive mouse protection test (PMPT), western blotting and direct virulent challenge. Result: Passive transfer of antibodies to mice using post-immunization sera of rabbits protected the animals against the challenge with A: 1 strain (75%) and B: 2 strain (50%). In western blotting, a total number of 4 bands were observed in the region between 130 kDa to 25 kDa in the protein of the phage lysate. The study suggested the significance increase in humoral immunity by phage lysate in murine Pasteurellosis. [ABSTRACT FROM AUTHOR]

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. • Virulence and repetitive elements based typing of Pasteurella multocida isolates. • No significant influence of host or place on prevalence of virulence genes (VAGs). • Significant correlation (positive & negative) between broad function groups of VAGs. • Repetitive elements of P. multocida were independent of host or place or clinical condition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • Isolation and whole genome sequencing of P. multocida type A NIVEDI/PMS-1 from respiratory infection of sheep. • Phylogenetic analysis of P. multocida type A NIVEDI/PMS-1 revealed as closest neighbor to bovine strain P. multocida P1933. • Various virulence factors, antibiotic and metal resistance were identified in the ovine strain of P. multocida serotype A. • In P. multocida type A NIVEDI/PMS-1 genome, 19 genomic islands (GIs) and 10 phage regions were identified. • Among these, 3 GIs and 7 phage regions were found novel to P. multocida type A NIVEDI/PMS-1. Abstract Pasteurella multocida is a causative agent of pneumonic pasteurellosis in ruminants. Bovine and porcine strains of P. multocida have been sequenced and analyzed worldwide. However limited genomic information is available on ovine strains involved in causation of pneumonic pasteurellosis. Therefore, the present study focused on genome sequencing and analysis of a P. multocida type A isolate recovered from respiratory infection of sheep to unravel the genetic contents and its involvement in the disease. P. multocida type A strain NIVEDI/PMS-1 genome was 2,543,931bp in size, assembled into 67 contigs, consisted of 1879 protein-coding regions, 19 genomic islands (GIs) and 10 phage regions. The identified GIs and phage regions showed high sequence identity mostly with other Pasteurellaceae family respiratory pathogens indicating horizontal gene transfer in ovine respiratory niche. The phylogenetic analysis revealed strain NIVEDI/PMS-1 as closest neighbor to P. multocida P1933 strain reported from bovine. Comparative genomic analysis of strain NIVEDI/PMS-1 with virulent GX-Pm and avirulent Pm70 strains revealed presence of 45 virulence genes common with virulent strain. Further, various virulence factors and accessory genes needed for survival and pathogenesis were also identified. Dermonecrotic toxin (ToxA) like protein presence in the strain appears interesting and further study is needed to understand its role in ovine pasteurellosis. [ABSTRACT FROM AUTHOR]

Aim: To use partially characterized phages for generation of phage lysates having potential to be developed as vaccines against Salmonella Abortusequi. Material and Methods: Phage/s isolation from several sewage samples was attempted against Salmonella Abortusequi strains procured from the National Salmonella Reference Centre, IVRI using enrichment and agar overlay technique. A concentrated stock of the phage was prepared after its purification. The lytic range, morphology and DNA size were determined. Results: Two lytic phages could be isolated and purified. The phage with wider lytic range was found to possess a hexagonal head with a very short tail. The DNAsize was found to be 21kb. Conclusion: The investigation reports first isolation of a lytic phage against Salmonella Abortusequi. The phage can be used in future for development of phage lysate based immune therapeutic agent against equine abortions. [ABSTRACT FROM AUTHOR]