Isolation, molecular characterization and first report of Dothiorella gregaria associated with fruit rot of walnuts of Jammu and Kashmir, India.

Walnuts are known for their high levels of antioxidants, which are linked to various health benefits. However, challenges related to distribution and storage, as well as the risk of fungal infections, can affect the quality of walnut kernels. Fungal pathogens from the Botryosphaeriaceae family, including Dothiorella species and Diplodia species, can damage fruit and reduce its antioxidant content. To comprehend the cause of fruit rot in walnuts, Dothiorella gregaria isolates were studied using polyphasic methods, including multiple gene sequences and morphological identification, as well as analysis of polyphenol content and pathogenicity. The walnuts kernels purchased from market places of Jammu and Kashmir (J&K), India were observed to be affected by Dothiorella gregaria species causing the quality detoriation and decrease in polyphenol content thus undeniably with decreased antioxidant properties. D. gregaria Infected walnut kernels were having some brown and black spots and some were having white mycelial growth and however, most samples were asymptomatic. Pathogenicity testing revealed that the pathogen was able to develop all the symptoms under experimental conditions and the reisolated pathogen was morphologically similar to D. gregaria. The samples infected with this pathogen showed considerable decrease in polyphenol content, 10.9 ± 2.66 mgGAE/g (mean ± standard deviation) thus decreased antioxidant quality as compared to the samples which showed zero incidence of this pathogen, 52.50 ± 4.27 mgGAE/g (mean ± standard deviation). Furthermore, the pathogen was studied using polyphasic approach involving morphological, molecular and phylogenetic analysis. Combined nucleotide dataset of nuclear ribosomal ITS and tef1-α revealed that Dothiorella gregaria (NY6) formed a clade with Dothiorlla iberica (MAEC33), Dothiorella sarmentorium (MAEC28) and Dothiorella iberica (CAA905) strains with 83% bootstrap support. Besides, we observed six nucleotide changes, four were insertions or deletions and two were substitutions in the 502-bp region of the ITS rRNA gene when we compared our isolate to the most equivalent sequences submitted to NCBI GenBank. This is the first report of Dothiorella gregaria affecting walnuts purchased from various markets in J&K, India, causing fruit rot in walnuts after harvest. Given that local farmers store and export walnuts, it could pose an emerging threat to their livelihood. Thus, creating post-harvesting interventions for D. gregaria and knowing more about the fruit rot in walnuts can be benefited from morphological and molecular identification using several gene loci, genetic variability in the ITS rRNA gene, and total phenol analysis.

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.

Determination of prevalence, serological diversity, and virulence of Dichelobacter nodosus in ovine footrot with identification of its predominant serotype as a potential vaccine candidate in J&K, India.

The aim of this study was to determine the prevalence, serological diversity, and virulence of Dichelobacter nodosus in footrot lesions of sheep and identification of its predominant serotype as a potential vaccine candidate. The overall prevalence of footrot in sheep was 16.19%, and ranged from 13.69 to 19.71%, respectively. A total of 759 flocks with 22,698 sheep were investigated for footrot and 2374 clinical samples were collected from naturally infected sheep exhibiting footrot lesions. Of the 2374 samples collected, 1446 (60.90%) were positive for D. nodosus by polymerase chain reaction (PCR). These positive samples when subjected to serogroup-specific multiplex PCR, 1337 (92.46%) samples carried serogroup B, 247 (17.08%) possessed serogroup E, 86 (5.94%) serogroup I, and one (0.069%) serogroup G of D. nodosus. While mixed infection of serogroups B and E was detected in 127 (8.78%), B and I in 46 (3.18%) and B, E, and I in 26 (1.79%) samples, respectively. The serogroup B of D. nodosus was the predominant (92.47%) serogroup affecting sheep population with footrot followed by serogroup E (19.91%) and serogroup I (4.57%), respectively. Virulent status of D. nodosus strains were confirmed by presence of virulence-specific integrase A (intA) gene and the production of thermostable proteases. The intA gene was detected in 709 (72.79%) samples while gelatin gel test carried out on 246 representative isolates all positive for intA gene produced thermostable proteases, confirming their virulence nature. The PCR-restriction fragment length polymorphism (PCR-RFLP) of whole fimA gene of serogroup B revealed the predominance of serotype B5 (82.97%) of serogroup B. This information suggests that serotype B5 is the predominant serotype of D. nodosus associated with severe footrot lesions in sheep in Jammu & Kashmir (J&K), India. Hence, this serotype can be a potential vaccine candidate for the effective control and treatment of ovine footrot.

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.

Isolation, molecular characterization and prevalence of Clostridium perfringens in sheep and goats of Kashmir Himalayas, India.

AIM: The study was conducted to report the occurrence of the Clostridium perfringens in sheep and goats of the Kashmir valley for the 1st time and to characterize them molecularly with respect to toxin genes to determine the prevalence of the various toxinotypes. MATERIALS AND METHODS: A total of 177 samples (152 from sheep and 25 from goats) collected from healthy, diarrheic animals, and morbid material of animals suspected to have died of enterotoxaemia were screened for C. perfringens toxinotypes. The presumptive positive isolates were confirmed using 16S rRNA gene-based polymerase chain reaction (PCR). All the confirmed isolates were screened for six toxin genes, namely; cpa, cpb, etx, cpi, cpb2, and cpe using a multiplex PCR. RESULTS: The PCR amplification of 16S rRNA gene revealed that out of 177 samples collected, 125 (70.62%) were found positive for C. perfringens, of which 110 (72.36%) were from sheep and 15 (60%) were from goats. The highest prevalence of C. perfringens toxinotype D was observed in lambs (56.16%) and kids (46.16%) followed by 3.84% in adult sheep while it was absent in samples obtained from adult goats. The multiplex PCR revealed that 67 (60.90%) isolates from sheep and 8 (53.33%) isolates from goats belonged to toxinotype A, while 43 (39.09%) isolates from sheep and 7 (46.66%) isolates from goats were detected as toxinotype D. None of the isolates was found to be toxinotype B, C, or E. All the C. perfringens toxinotype A isolates from sheep were negative for both cpb2 and cpe genes, however, 27.90% toxinotype D isolates from sheep carried cpb2 gene, and 6.97% possessed cpe gene. In contrast, 12.50% C. perfringens toxinotype A isolates from goats harbored cpb2 and cpe genes while 14.28% isolates belonging to toxinotype D carried cpb2 and cpe genes, respectively. CONCLUSION: The high prevalence of C. perfringens was observed, even in day-old lambs. The toxinotypes A and D are prevalent in both sheep and goats. The severity of disease and mortality may be associated with the presence of minor toxins in both the detected toxinotypes.

The detection of Dichelobacter nodosus and Fusobacterium necrophorum from ovine footrot in Kashmir, India.

In a study conducted, a total of 450 swab samples from footrot lesions of naturally infected sheep were collected in all the ten districts of the Kashmir valley and were examined for the presence of Dichelobacter nodosus (D. nodosus) and Fusobacterium necrophorum (F. necrophorum), in order to determine if F. necrophorum was associated with ovine footrot. The detection of F. necrophorum and D. nodosus was carried out by polymerase chain reaction targeting the leukotoxin (lktA) and 16S rRNA genes, respectively. In this study, only less than 50% of positive samples contained both the bacteria, so it is not possible to conclude with certainty that both bacteria are together required for the disease manifestation.

Investigation of diarrhoeic faecal samples for enterotoxigenic, Shiga toxin-producing and typical or atypical enteropathogenic Escherichia coli in Kashmir, India.

Three hundred and twenty-six Escherichia coli isolates recovered from 326 human faecal specimens from sporadic cases of diarrhoea in Kashmir valley, India, were investigated for the presence of stx(1), stx(2), eaeA, hlyA and lt virulence genes. None of the samples was positive for stx genes or Shiga toxins by PCR or enzyme-linked immunosorbent assay. Twenty-three E. coli isolates showed the presence of the eaeA gene, whereas three isolates harboured the lt gene. Enteropathogenic E. coli (EPEC) belonged to 10 different serogroups. Out of 23 EPEC isolates, the majority (78.26%) were atypical while five (21.73%) were typical. Only one of the typical EPEC harboured the EAF plasmid. Subtyping of the eaeA gene showed the presence of eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta in one, two, four and two isolates, respectively. None of the E. coli isolates possessed eaeA-delta, eaeA-epsilon and eaeA-zeta. This study further upholds the opinion that Shiga toxin-producing E. coli do not pose a major threat to human health in India and eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta could be common EPEC subtypes prevalent in humans with diarrhoea in India. The present study appears to be the first report of subtype analysis of the eaeA gene from India and also records the isolation of EPEC with the eaeA-xi gene from humans.