Occurrence and exposure assessment of Aflatoxin M1 in milk and milk products in India.

Milk containing Aflatoxin M1 (AFM1) poses a serious health risk to consumers. Present study was undertaken to determine levels of AFM1 in 146 milk and value added dairy products sold in retail markets of Chhattisgarh, India using HPLC coupled with fluorescence detector. A total of 52 samples (35.6%) were found to contain AFM1 with overall concentrations ranging from nd - 2.608 µg/L. The contamination levels were higher in non-fermented milk products than fermented milk products samples, although this difference was statistically non-significant (p > 0.05). AFM1 concentrations above maximum permissible limits established by the European Commission were found in 94.2% of positive samples. Health risk assessments ascertained that the estimated daily intakes for AFM1 is higher than the established tolerable daily intakes for both adults and children (Hazard Index > 1), there by implying a potentially high risk to consumer's health. Current investigation provides valuable information regarding contamination of raw as well as value added milk products sold in Indian markets. Therefore, to protect consumer's health and promote dairy trade; there is an urgent need to increase farmer's knowledge on good storage practices of feed and fodder. Further, stringent enforcement of food safety regulations is imperative to safeguard and promote human health.

Detection of aflatoxin M1 in bovine milk from different agro-climatic zones of Chhattisgarh, India, using HPLC-FLD and assessment of human health risks.

Concerns regarding food safety and 'One Health' are increasing globally. Aflatoxin M1 (AFM1), a human carcinogenic toxin, is excreted by lactating animals in their milk after consumption of feed contaminated with aflatoxin B1. The present cross-sectional study aimed to determine the occurrence of AFM1 in cattle and buffalo milk produced in rural and peri-urban areas under different agro-climatic conditions of Chhattisgarh, India, and assesses human health risks. Analyses of 545 milk samples by validated high-performance liquid chromatography revealed high level of AFM1 contamination in 224 (41.1%) samples with mean concentration of 0.137 ± 0.029 µg/L. Statistically significant differences (p< 0.05) were observed in the levels and frequency of AFM1 occurrence among different agro-climatic zones. AFM1 was more frequently detected in milk samples from Northern hills (64%) followed by Bastar plateau (40.7%) and Chhattisgarh plain (27.3%), with mean concentration levels of 0.396 ± 0.099 µg/L, 0.081 ± 0.025 µg/L and 0.013 ± 0.002 µg/L, respectively. Species wise no significant difference was observed in the detection frequency and concentration of AFM1 in milk from cattle and buffalo. AFM1 contamination above maximum permissible limits established by European commission and Food Safety and Standard Authority of India was detected in 21.3% and 4.4% of samples, respectively. The estimated daily intakes for AFM1 were found to be higher than tolerable daily intakes for both adults and children, especially of Northern hills implying a potentially high risk to consumer's health. This study provides valuable information on the contamination status of milk in one of the fastest developing state of India. It also highlights the importance and need for continuous farmers' awareness on good animal husbandry practices, routine surveillance of mycotoxins in animal feeds and food commodities to safeguard human health.

Myxovirus resistance 1 (Mx1) gene: Molecular characterization of complete coding sequence and expression profile in the endometrium of goat (Capra hircus).

Myxovirus resistance 1 (Mx1) gene plays an important role in uterine receptivity and conceptus development by creating a strong defense mechanism in the uterine environment. However, the specific role of Mx1 gene is not yet documented in the goat. Therefore, in the present study, full-length coding sequence (CDS) of the Mx1 gene was amplified, sequenced and characterized through various Bioinformatic tools. Temporal expression profile of Mx1 mRNA and protein was also examined by quantitative real-time PCR (qPCR) and western blot, respectively, in the endometrium of cyclic stage (non-pregnant), pregnancy stage I (16-24 days of gestation) and pregnancy stage II (25-40 days of gestation) of caprine (cp). A fragment of the cpMx1 gene, 2144 bp in length, was amplified from complementary DNA (cDNA) with a 1965 bp open reading frame. Coding and deduced amino acid sequences of the cpMx1 were aligned with other species and it exhibited 98.8-81.5 % identities with different species. On phylogenetic analysis, sheep and goat were found belonging to the same clade but differing from large ruminants. The cpMx1 protein possess the conserved signature motif (LPRGTGIVTR) of dynamin superfamily and the tripartite guanosine-5'-triphosphate (GTP) binding motif (GDQSSGKS, DLPG, TKPD) at the N-terminal end, and the leucine zipper motifs at the C-terminal end. Both cpMx1 mRNA and protein were found to be expressed maximally (P < 0.05) in the pregnancy stage I as compared to cyclic stage. It was concluded that the cpMx1 gene shares major structural and probably functional similarities with other species.

Characterization of Salmonella Gallinarum from an outbreak in Raigarh, Chhattisgarh.

AIM: The present investigation was conducted to isolate and characterize Salmonella Gallinarum from an outbreak of fowl typhoid in layer birds. MATERIALS AND METHODS: Clinically ill and dead layer birds from an outbreak were investigated. History, clinical signs, and postmortem lesions were suggestive of fowl typhoid. Postmortem samples including heart blood, intestinal contents, pieces of ovary, and liver were collected and processed immediately for bacterial culture, serotyping and antibiotic sensitivity tests. Isolates were further screened for the presence of extended spectrum beta lactamase (ESBL) (blaTEM) gene by polymerase chain reaction. RESULTS: On the basis of cultural, staining and biochemical characteristics; three bacterial isolates were confirmed as S. Gallinarum. On serotyping, somatic antigen O: 9 and 12 with nonflagellated antigen were detected in all three isolates. Isolates were intermediate sensitive to amoxycillin, amoxyclav, gentamicin and ciprofloxacin and resistant to most of the antibiotics including chloramphenicol, ampicillin, ceftazidime, cefexime, cefepime, azithromycin, nalidixin, tetracycline, oxytetracycline, and streptomycin. Two isolates were found to harbor ESBL (blaTEM) gene. CONCLUSION: Beta lactamase producer S. Gallinarum was confirmed as cause of increased mortality in layer birds during present investigation. Existence of multi drug resistant Salmonella poses serious threat to poultry industry in Chhattisgarh.

Occurrence and characteristics of extended-spectrum ß-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India.

AIM: To assess the prevalence of antimicrobial resistance producing extended-spectrum ß-lactamases (ESBL) (blaTEM, blaSHV, and blaCTX-M) genes in Escherichia coli isolated from chicken meat, chevon meat, raw milk, and human urine and stool samples collected from tribal districts of Chhattisgarh, viz., Jagdalpur, Dantewada, Kondagaon, and Kanker. MATERIALS AND METHODS: A total of 330 samples, comprising 98 chicken meat, 82 chevon meat, 90 raw milk, and 60 human urine and stool samples, were processed for isolation of E. coli. Isolates were confirmed biochemically and further tested against commonly used antibiotics to know their resistant pattern. The resistant isolates were tested for ESBL production by phenotypic method followed by characterization with molecular method using multiplex-polymerase chain reaction technique. RESULTS: Overall 57.87% (191/330) samples were found positive for E. coli, which include 66.32% (65/98) chicken meat, 46.34% (38/82) chevon meat, 81.11% (73/90) raw milk, and 25% (15/60) human urine and stool samples. Isolates showed the highest resistance against cefotaxime (41.36%) followed by oxytetracycline (34.03%), ampicillin (29.31%), cephalexin (24.60%), cefixime (16.75%), and ceftazidime (13.08%). Phenotypic method detected 10.99% (21/191) isolates as presumptive ESBL producers, however, molecular method detected 3.66% (7/191), 2.09% (4/191), and 0.00% (0/191) prevalence of blaTEM, blaCTX-M, and blaSHV, respectively. CONCLUSION: The present study indicates a high prevalence of E. coli in raw chicken meat, chevon meat, and milk due to poor hygienic practices. The antibiotic susceptibility test detected the presence of the resistance pattern against ESBL in E. coli isolated from raw chicken meat, chevon meat, milk, and also in human clinical samples is of great concern. The appearance of E. coli in the human food chain is alarming and requires adaptation of hygienic practices and stipulate use of antibiotics.

Isolation and adaptation of bovine herpes virus Type 1 in embryonated chicken eggs and in Madin-Darby bovine kidney cell line.

AIM: Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin-Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test. MATERIALS AND METHODS: Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test. RESULTS: Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as "bunch of grapes" at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test. CONCLUSION: BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test.