Clostridium perfringens from fresh water fish of Kashmir Himalaya and their aquatic environment: Toxinotyping and phylogenetic analysis.

Clostridium perfringens is a common anaerobic foodborne pathogen known to produce >20 toxins. In nature, this bacterium has 7 different toxinotypes (A-G) based on the presence of its 6 main toxins. The present study examined the occurrence of different toxinotypes of this bacterium in the ichthyofauna and aquatic environments of Kashmir Himalayan lakes, India. A total of 510 samples (210 water; 150 each of common carp and snow trout) were collected from 3 different lacustrine habitats (Dal, Anchar and Nigeen Lakes) of the region. By performing 16S rRNA PCR test, it was observed that all 210 water samples and 80 (26.66%) of 300 fish samples tested for this specific bacterial species were positive. Then by using multiplex-PCR targeting six virulence genes of C. perfringens, it was confirmed that all the 290 isolates from water and fish samples were positive for Toxinotype A, as only cpa toxin gene was amplified. Phylogenetic analysis of the amplified gene and its amino-acid sequences revealed 95%-98% homology with analogous sequences of this bacterial strain reported from China, Egypt and India. The study documents the existence of C. perfringens toxinotype A in the ichthyofauna of Kashmiri Himalayan lakes, entailing that fish can likely act as transmission medium for C. perfringens food poisoning to humans via food.

Genome sequence of Dichelobacter nodosus JKS-07B isolate from J&K, India associated with virulent footrot of sheep.

INTRODUCTION: Virulent footrot of sheep caused by Dichelobacter nodosus is associated with tremendous economic losses due to recurrent treatment costs and increased culling rates. This organism being a fastidious anaerobe is difficult to isolate on ordinary media that does not support its growth. The D. nodosus serogroup B isolate described in the present study has been used in the preparation of the whole-cell killed vaccine against footrot in India. D. nodosus serogroup B is the predominant serogroup involved in virulent footrot (lesion score 4) in India as well as in many sheep-rearing countries of the globe. METHODS: Genomic DNA was extracted using wizard Genomic DNA purification kit. The whole genome of the D. nodosus strain B was sequenced using an Illumina HiSeq 2500 platform and annotated according to functional gene categories. Annotations were performed using in-house developed Perl scripts using Nr/Nt database, uniprot, Pfam, KEGG, Panther DB, and GO database. RESULT: The assembled genome size is 1.311,533 Mb and GC content is 44.38. A total of 1215 protein-coding genes, 44tRNA and 7 rRNA were identified. The genome shows 98.63% sequence homology with the reference genome. However, 21 new genes have been identified in this genome. The information will provide insights into the various genes and regulators necessary for D. nodosus growth and survival. DISCUSSION: The genome information of this serogroup B of D. nodosus isolate involved in 85-90% cases of virulent footrot of sheep in India provides further insights for improvement of the killed vaccine (B serogroup) developed recently in India. For the development of an efficacious vaccine against virulent footrot, it is essential to know the serological diversity as well as the virulent status of the strains of the D. nodosus. This serogroup isolate is a potential vaccine candidate to mitigate ovine footrot in India as the majority of virulent footrot cases belong to serogroup B of D. nodosus.

Methicillin resistance genes and in vitro biofilm formation among Staphylococcus aureus isolates from bovine mastitis in India.

INTRODUCTION: Biofilms, an assemblage of microbial cells irreversibly associated with a surface and enclosed in a matrix of polysaccharide material pose serious health challenges, resulting in high economic losses. The emergence of methicillin-resistant S. aureus (MRSA) infections and ability to form biofilms in dairy animals is of emerging concern for livestock and public health owing to their association with serious infections. The present study was undertaken to examine the presence of methicillin resistance genes among the biofilm forming Staphylococcus aureus strains isolated from cases of acute and subacute bovine mastitis. A total of 150 mastitic milk samples referred to Veterinary Clinical Complex, Shuhama (Aulesteng) SKUAST-K were screened in present study. The methicillin resistant Staphylococcus aureus isolates were also screened for in vitro biofilm forming ability. RESULTS: A total of 80 (53.33%) S. aureus isolates were recovered from cases of bovine mastitis of which 20 (25%) were methicillin (mecA) gene positive. Of the 20 mecA positive isolates, 20% were positive for SCCmec I, 35% for SCCmec IV and 45% for SCCmec V subtypes. In vitro antibiotic sensitivity testing of MRSA revealed complete resistance towards methicillin and other pencillin group of antibiotics. CONCLUSION: A significant correlation was observed between in vitro biofilm formation and presence of methicillin resistance gene in S aureus isolates recovered from acute and subacute mastitis. The Staphylococcus aureus isolates positive for methicillin resistance gene (mecA) were either strong or moderate biofilm formers.

Comparative evaluation of different therapeutic protocols for contagious caprine pleuropneumonia in Himalayan Pashmina goats.

Therapeutic management of contagious caprine pleuroneumonia (CCPP) involves mostly the use of oxytetracycline followed by enrofloxacin and rarely tylosin. In many parts of the world including India, the former antibiotics are commonly available than the latter. Therefore, prolonged use of the same leads to the development of antibiotic resistance and decreased efficacy of drug. Besides, inflammatory and allergic pathogenesis of CCPP envisages combination therapy. In this study, we evaluated the effectiveness of the combination therapy using different antibiotics (oxytetracycyline @ 10: group I, enrofloxacin @ 5 group II, and tylosin: group III, @ 10 mg/kg body weight), along with anti-inflammatory (meloxicam @ 0.5 mg/kg) and anti-allergic (pheneramine maleate @ 1.0 mg/kg) drugs. These drugs were given intramuscularly at the interval of 48 h for four times in three test groups (n = 10) of Pashmina goats, viz. groups I, II, and III, respectively, affected with CCPP. Group IV (n = 10) was kept as healthy control when group V (n = 10) treated with oxytetracycline @ 10 mg/kg alone was used as positive control. Clinical signs, clinical parameters, pro-inflammatory cytokine (tumor necrosis factor alpha (TNF-α)), and oxidative stress indices (total oxidant status (TOS), total antioxidant status (TAS)) were evaluated at hours 0, 48, 96, and 144 of experimental trial. Tylosin-based combination therapy resulted in a rapid and favorable recovery resulting in restoration of normal body temperature (102.46 ± 0.31 °F), respiration rate (16.30 ± 0.79 per minute), and heart rate (89.50 ± 2.63 per minute) compared to the oxytetracycline (102.95 ± 0.13, 21.30 ± 1.12, 86.00 ± 2.33, respectively) and enrofloxacin (102.97 ± 0.19, 21.00 ± 1.25, 90.00 ± 2.58, respectively) treated groups. By hour 144, all the groups showed restoration of clinical parameters of normal health and diminishing signs of CCPP, viz. fever, dyspnea, coughing, nasal discharge, weakness, and pleurodynia. Significant (P ≤ 0.05) decrease in levels of TNF-α and non-significant (P > 0.05) decrease in levels of TOS and an increase in levels of TAS were noted from hour 0 to 144 in all the test groups. Within the groups, no significant (P > 0.05) change was noted in TNF-α, TOS, and TAS levels; however, TNF-α levels were comparatively lower in group III. Hematological parameters did not differ significantly (P > 0.05). From these findings, it can be inferred that tylosin-based combination therapy is relatively better for early, rapid, and safe recovery besides minimizing inflammatory and oxidative cascade in CCPP affected Pashmina goats compared to oxytetracycline- and enrofloxacin-based therapies.

Efficacy of bacteriophage Lysed Pasteurella marker vaccine in laboratory animal models with a novel DIVA for haemorrhagic septicaemia.

OBJECTIVE: The present study aimed at evaluating the efficacy of an improved phage lysate marker vaccine for haemorrhagic septicaemia in mice and rabbit model and development of a DIVA ELISA based on iron restricted outer membrane protein (IROMP). METHOD: The experimental vaccine was prepared by lysing P. multocida B:2 grown under iron restricted conditions with a Pasteurella bacteriophage and addition of an alum adjuvant to enhance the immunogenicity. The vaccine was administered in mice and rabbits divided into two group each. Phage lysate vaccine (PL-VacI) was administered to group I mice and rabbits whereas group II mice and rabbits received alum precipitated HS vaccine (HS-VacII). Antibody titres were monitored 0, 30, 60, 90, 210 and 240 dpv. An IROMP (130 kDa) based indirect ELISA was also developed to differentiate between infected and vaccinated animals. The Pasteurella phage isolated in present study was sequenced at Georgia Genomic Facilty, Georgia. RESULT: The sequence of PMP-GAD-IND (Pasteurella bacteriophage) was deposited in GenBank under no KY203335. The group I mice and rabbits vaccinated with Phage lysate vaccine (PL-VacI) group revealed significantly higher antibody titres than group II mice and rabbits receiving alum-precipitated bacterin (HS-VacII) by MAT, IHA and ELISA (P < 0.05 and P < 0.001). The peak log 10 values (3.46) in case of group I mice by ELISA were attained at 90DPI whereas in group II mice the peak values at 90DPI were 2.82. Mean log10 titres by ELISA in group I and II rabbits were 2.43 and 2.35 respectively at 30DPI whereas at 120DPI the titres were 3.29 and 2.75, respectively. The DIVA ELISA detected presence of a novel 137 kDa IROMP/siderophore antibody in sera of group I mice and rabbits (PL-VacI) absent in sera of mice and rabbits given HS-VacII. CONCLUSION: The bacteriophage based marker vaccine (PL-VacI) had a more effective and longer immune response against HS in mice and rabbit in comparison to the widely used alum precipitated HS vaccine (HS-VacII). Moreover, the development of a recombinant IROMP based indirect ELISA could serve as an excellent tool to differentiate between infected and vaccinated cattle and buffaloes for effective control of HS.

Draft genome sequence of Dichelobacter nodosus JKS-07 serogroup E from India.

OBJECTIVES: Dichelobacter nodosus is an anaerobic bacterium with fastidious growth requirements that is the principal cause of footrot associated with lameness in sheep and goats. In India, D. nodosus serogroups B and E have been recorded as major causes of footrot. Here we report the draft genome sequence of a D. nodosus serogroup E strain (JKS-07) from a case of virulent footrot in India. METHODS: The whole genome of the D. nodosus JKS-07 serogroup E was sequenced using an Illumina HiSeq 2500 platform and was annotated according to functional gene categories. De novo genome assembly and annotation were performed using Perl scripts developed in-house using the Nr/Nt and UniProt databases. RESULTS: The assembled genome is 1389350bp and contains 1301 genes. The genome has 45 tRNAs and 9 rRNAs. The draft genome sequence will provide insight into the various genes and regulators involved in D. nodosus growth and survival. CONCLUSION: Information on the genome of the D. nodosus serogroup E strain is important bearing in mind the fact that both serogroups B and E are associated with virulent footrot, either alone or frequently together. In order to develop an efficacious vaccine against virulent footrot, it is essential to know the serological diversity as well as the virulence status of the D. nodosus strains. Serogroups B and E are potential vaccine candidates to mitigate ovine footrot in India.