Novel fluconazole-resistant zoonotic yeast isolated from mastitis.

A hospital-based cross-sectional study was conducted during 2018-2019 to decipher the prevalence of yeast mastitis. The results indicated a 19.68% prevalence of clinical mastitis in bovines. Among them, 5.51% of samples revealed yeasts constituting 1.09% overall prevalence. Candida albicans was recorded as a significant fungal agent involved in clinical bovine mastitis. We record the association of Kodamaea ohmeri in clinical bovine mastitis. On proteomic and molecular confirmation, K. ohmeri isolates were re-identified from phenotypically identified Candida isolates associated with bovine mastitis. After conventional identification, the yeast isolates were re-identified by MALDI-TOF MS-based proteomic approaches. The D1/D2 domains of 26S-rRNA gene and 5.8S-internal transcribed spacer (ITS) rDNA regions based molecular phylogenetic analysis identified the isolates as K. ohmeri. The isolates were resistant to fluconazole. This study reports the first systemic study of K. ohmeri isolates recovered from bovine clinical mastitis, utilizing conventional, automated, proteomic, and genomic approaches followed by antifungal susceptibility. The findings suggest K. ohmeri as a potent opportunistic emerging pathogen of veterinary and public health concern, need for accurate identification of fungal agents from mycotic mastitis, and use of validated antifungal susceptibility assay because of developing resistance to antimycotic agents. Our findings suggest judicious use of fluconazole and alternative antifungal agents may be considered in case of an outbreak.

The investigation on allelic, genotypic frequencies and gene expression study in coding region of tyrosinase gene in Deoni cattle breed of western India.

An investigation was carried out on Deoni animals of western India to study the allelic and genotypic frequencies in coding region of TYR gene as well as gene expression profile. The animals were grouped according to age, gender, strain and intensity of partial albinism (low, medium and high). The present study revealed that the genotypic frequency of TYR gene across different strains, gender, age group and level of partial albinism was found to be non-significant for both exon-I and exon-II. The AB genotype in Balankya (0.70) was observed highest genotypic frequency followed by Wanera (0.55) and Shewara (0.55) strains. The genotypic frequency of AB and BB genotypes were observed highest in male and female, respectively. In exon-I, genotype frequency of AA genotype was found highest (0.55) in low level of partial albinism. The allelic frequencies in Shewara strain, male and low level of partial albinism were 0.75, 0.63 and 0.73, respectively. However, in exon-II genotype frequency of AB and BB was observed highest (0.70) in Wanera and Balankya strains followed by AA genotype in Shewara (0.50). The highest genotypic frequency of AA (0.87) and BB (0.50) were in male and female, respectively. The genotype frequency of AB genotype was found highest in all level of partial albinism. The allelic frequency was highest (0.85 for B allele) in Wanera strain, male (0.80 for A allele) and high level (0.60 for A allele) of particle albinism. The highly significant (p = 0.002) expression of tyrosinase gene was observed in young animals as compared to adult animals. The TYR gene expression was significantly (p = 0.047) higher in animals with low intensity of partial albinism followed by in the animals with medium and high intensity. Therefore, it is inferred that the TYR gene expression in young animals were high and as compared to the old animals of Deoni cattle breed.

Molecular phylodynamics of fowl adenovirus serotype 11 and 8b from inclusion body hepatitis outbreaks.

Fowl adenovirus (FAdV) serotypes are involved in a variety of clinical manifestations in poultry and has resulted in substantial economic loss to the poultry farmers. Despite the endemicity of Inclusion body hepatitis (IBH) in South Asian countries, including India, its etiology is not well studied. In western India, the rural poultry flocks obtained from the vaccinated parents were experiencing disease outbreaks with substantial economic losses due to heavy outbreaks and mortality. Therefore, the study was conducted to decipher the molecular epidemiology of the FAdV from field outbreaks in western India. A total of 37 commercial broiler poultry flocks and 29 village poultry flocks of western India were visited during 2019 to 2021. Out of these, 19.14% flocks showed incidence of IBH during the age of 15 to 35 days. The mortality ranged from 3.3 percent to 55.28 percent. The samples were subjected for amplification of partial hexon gene covering loop 1 and loop 2. The results revealed 48.28% positivity by PCR. The sequence analysis identified 14 isolates as species D serotype 11 with 0.97 to 0.99% divergence and two as species E serotype 8b with 0.99% divergence. The FAdV-11 isolates showed amino acid substitutions D195N, T399A, N417S, and N496H. The amino acids I188 and N195 were conserved in FAdV-11. The molecular clock in Bayesian methods was used to determine most common ancestor. The isolates MH379249 and MH379248 were determined the most recent common ancestor for FAdV-11 and FAdV-8b isolates. The analysis suggested evolution of 10 FAdV-11 strains in 2012, and four FAdV-11 strains and two FAdV-8b strains in 2018.

Molecular phylodynamics of infectious bursal disease viruses.

The present study was conducted to study the molecular phylodynamics of the Indian field IBDVs. A total of 13 organized commercial poultry farms and 29 village poultry flocks were recruited in the study. The broiler flocks showed 15.25-60.18% mortality, followed by 12.4% in improved native poultry varieties and 5% in indigenous birds. The 664 bp hypervariable VP2 gene fragment of Western and Central Indian vvIBDVs showed 97.14-98.79 and 94.49-96.69% identity to Pakistani and South Indian vvIBDVs, respectively. An isolate was 99.54% identical to the Ventri-Plus vaccine strain, while three IBDVs showed maximum identity with the Georgia strain. Out of 22, 19 strains showed typical vvIBDV amino acid signature, while three strains showed substitutions specific to classical IBDVs. Central Indian vvIBDVs showed conserved substitutions at N212D and E300A, which can be used as a regional marker. Phylogenetic genogrouping placed global IBDVs into seven genogroups based upon virulence and geographical distribution. Nineteen field vvIBDVs were placed in the G3 genogroup, and the other three were grouped with classical IBDVs in G1 genogroup. A nucleotide span from 584 to 1248 covering VP2 hypervariable fragment was found suitable for correct genogrouping of field IBDVs. The Bayesian evolutionary analysis showed tMRCA of the year 2009 for 8 Western Indian vvIBDVs with vvIBDV from Pakistan. Central Indian vvIBDVs were evolved in the year 1991 from BD-3 and PY12 strains of vvIBDVs from Bangladesh and Pondicherry, respectively. An isolate showed evolution in year 2010 from the Nigerian ABIC strain, while three classical strains showed tMRCA of the year 2009 with the Georgia strain as a recent common ancestor.

Bacteria from bovine clinical mastitis showed multiple drug resistance.

Mastitis, which often manifests as udder infection in dairy animals, is of great concern as it affects public health and results in heavy economic losses to the dairy industry. A hospital-based cross-sectional study was conducted to determine the cultivable bacterial species associated with bovine clinical mastitis and their resistance patterns towards different antimicrobials. The milk samples from cows suffering from clinical mastitis during monsoon season were investigated. The prevalence of clinical mastitis was significantly high in Holstein-Friesian crossbred cows, followed by in Jersey crossbred, Red Kandhari and Deoni. Significantly high prevalence was observed during 4th to 6th months of lactation. A total of 110 bacterial isolates belonging to 14 different genera were isolated and identified. Aminoglycosides and quinolones were found to be the most effective antibiotics. Vancomycin resistant penicillinase producing Gram positive bacteria were demonstrated. Gram negative bacteria resistant to extended spectrum ß lactamases, cephalosporins, tetracyclines, vancomycin and chloramphenicol as well as vancomycin resistant enterococci, multiple drug resistant (MDR) gram negative rods, MDR Pseudomonas and MDR Acinetobacter were found. Widespread resistance of Streptococcus uberis towards cephalosporins was documented. Variable MDR patterns were recorded within a single species. MDR transfer from non-pathogens to emerging foodborne and established mastitis pathogens could be a potential problem to the dairy industry as well as to public health.

Journey towards National Institute of One Health in India.

BACKGROUND & OBJECTIVES: Issues such as emerging and re-emerging infectious diseases, antimicrobial resistance, food security, biosafety and biosecurity are associated with changes in land use, population growth, urbanization, global travel and trade and climate change. As a result, a trans-disciplinary approach among human, animal and environmental health disciplines gained support. The Indian Council of Medical Research (ICMR) and Indian Council of Agricultural Research (ICAR) decided to establish a National Institute of One Health at Nagpur, Maharashtra, India. In this context, two collaborative research projects, funded by the ICAR and ICMR were initiated to conduct the epidemiological surveillance of selected zoonotic diseases in Central India. METHODS: Disease surveillance and molecular detection employing standard techniques like enzyme linked immunosorbent assay (ELISA), immuno-fluroscent assay (IFA), standard tube agglutination test (STAT) , Rose Bengal plate test (RBPT) and polymerase chain reaction (PCR) were undertaken based on the disease to be screened. RESULTS: In animals, the seropositivities for listeriosis (7.66%) and brucellosis (11.69%) were recorded. The occurrence of tuberculosis (3.8%) and leptospirosis (6.33%) was detected by PCR. Through cross-sectional studies from suspected human population with associated risk factors for zoonotic diseases, the seropositivity of brucellosis (1.83-11%), listeriosis (1.01-10.18 %), leptospirosis (8.14-12.67%) and scrub typhus (1.78-20.34%) was recorded. The investigations on scrub typhus indicated bimodal pattern during the months of pre-monsoon and post-monsoon season with a peak in post-monsoon in human cases. Ornithonyssus bacoti mites were identified from the rodents as a vector harbouring Orientia tsutsugamushi. The bovine tuberculosis was detected in 1.43 per cent human cases employing molecular assay. INTERPRETATION & CONCLUSIONS: The data indicated the occurrence of important zoonotic diseases adversely affecting the livestock health and human wellbeing. The scientific collaboration between veterinary and medical faculties has set an example for effective implementation of One Health (OH) programme for the establishment of National Institute of OH.

Molecular epidemiology of Canine parvovirus shows CPV-2a genotype circulating in dogs from western India.

Canine parvovirus (CPV) has emerged as an acute pathogen of young canine causing haemorrhagic enteritis and myocarditis. It is widely distributed and underreported in India. Therefore the study was conducted to type the CPV circulating in western Maharashtra. The faecal samples (n = 150) from clinically ill dogs showing diarrhoea and vomition were collected and subjected to haemagglutination (HA) with porcine RBC's. The DNA was extracted from the samples showing HA titres above 64 and subjected for amplification of VP2 gene fragment by PCR. The amplicons were subjected for restriction fragment length polymorphism (RFLP), sequencing and BEAST phylogenetic analysis. The results revealed 6% positivity by PCR. The RFLP results indicated single cleavage site for ApaLI and HinfI with an exception of two sites for HinfI. The nucleotide sequences showed nonfunctional nucleotide changes at different locations. The sequence analysis indicated that the nucleotide divergence within isolates under study was 0.00-0.42%, while the nucleotide homology was 99.58-100%. The most recent common ancestor was determined by molecular clock analysis using Bayesian methods. The sequence and phylogenetic analysis suggested the isolates as CPV-2a and KATN1 (KU866391, 2014) isolate from Tamilnadu, India as time to most recent common ancestor (TMRCA). The results revealed the circulating CPV in canines from western India as CPV2a genotype.

Identification and characterization of a novel infectious bursal disease virus from outbreaks in Maharashtra Province of India.

AIM: The study was undertaken to isolate infectious bursal disease virus (IBDV) from clinical cases in broiler and cockerel flocks of Maharashtra state, India, and its molecular epidemiological investigation. MATERIALS AND METHODS: The morbid bursal tissues were collected from flocks suspected for IBD. The samples were subjected for virus adaptation in primary chicken embryo fibroblast (CEF) cells followed by confirmation by reverse transcription polymerase chain reaction (RT-PCR) for partial VP2 sequence and phylogenetic analysis. RESULTS: The isolation of IBDV from field samples took seven blind passages for adaptation in CEF. The cytopathic effects included rounding, aggregation, vacuolation, and detachment of the cells. The RT-PCR showed amplification of 627 bp amplicon specific to the primers for VP2 gene fragment which confirmed successful adaptation and isolation of IBDV using CEF. The nucleotide and deduced amino acids based on phylogeny clustered the current isolate in a distinct clade with classical virulent and antigenic variants. It showed divergence from very virulent (vv) and vaccine strains of Indian origin. The isolate showed unique amino acid substitution at A329V as compared to all other IBDVs. The variation in key amino acids was reported at A222, I242, Q249, Q253, A256, T270, N279, T284, I286, L294, N299, and V329. It shared conserved amino acids at position A222, I242, and Q253 as reported in vvIBDV isolates. However, the amino acids reported at position T270, N279, T284, L294, and N299 are conserved in classic, antigenic variant and attenuated strains of IBDV. The amino acids at positions N279 and T284 indicated that the isolate has key amino acids for cell culture replication. CONCLUSION: The IBDV field isolate does not reveal the full nucleotide sequence signature of vvIBDV as well as vaccine strains. Hence, we can conclude that it might not belong to vvIBDVs of Indian origin and the vaccine strain used in the region. This may be suggestive of the evolution of the IBDV in the field due to the coexistence of circulating field strains and live attenuated hot strains, resulting into morbidity and mortality, warranting the need for safer protective vaccines, and implementation of stringent biosecurity measures to minimize loss to farmers.