Assessing and Prioritizing Zoonotic Diseases in Punjab, India: A One Health Approach.

Zoonotic diseases have a significant impact on both human and animal health globally. The present study was planned to prioritize the zoonoses in Punjab state of India. To develop a zoonotic disease prioritization scoring system, a comprehensive approach has been taken, including literature review, key person interviews with animal health experts (n = 12) and medical professionals (n = 7), and nine focus group discussions (FGDs) with veterinary academicians, medical professionals, and field veterinary doctors. The scoring system comprises of seven major criteria, each assigned a weightage score (ws): prevalence/incidence of the disease (ws = 0.20), severity of illnesses in humans (ws = 0.18), epidemic potential (ws = 0.16), socio-economic burden (ws = 0.16), availability of effective control and prevention measures (ws = 0.15), inter-sectoral collaborations (ws = 0.1), and bioterrorism potential (ws = 0.05). The finalized scoring system, accompanied by a list of 15 selected zoonotic diseases, was implemented among a group of 23 professionals engaged in zoonoses research (n = 7), animal health (n = 10), and medical health (n = 6) to determine their prioritization. The zoonotic diseases prioritized for the Punjab (India) included, Brucellosis (0.70) > Rabies (0.69) > Anthrax (0.64) > Leptospirosis (0.62) = Toxoplasmosis (0.62) = Highly Pathogenic Avian Influenza (HPAI) (0.62) > Bovine tuberculosis (0.61) > Q fever (0.60) > Cysticercosis (0.59) > Listeriosis (0.58) > Crimean-Congo haemorrhagic fever (CCHF) (0.57) > Japanese encephalitis (0.56) = Echinococcosis (0.56) > Dermatophytosis (0.53) > and Scrub typhus (0.48), respectively. Higher priority is suggested for endemic zoonoses (e.g., brucellosis and rabies) as compared to those with epidemic potential (e.g., CCHF, HPAI etc.) in Punjab. Results of the current study will help in the development of targeted control and prevention strategies for zoonotic diseases in Punjab and other geographical regions facing similar challenges.

Evaluation of the sensitivity and specificity of three diagnostic tests for Coxiella burnetii infection in cattle and buffaloes in Punjab (India) using Bayesian latent class analysis.

Q Fever is a zoonotic disease of significant animal and public health concern, caused by Coxiella burnetii (C. burnetii), an obligate intracellular bacterium. This study was done to evaluate the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of three diagnostic methods to diagnose C. burnetii infection in cattle and buffaloes in Punjab, India: an indirect ELISA method applied in serum samples and a trans-Polymerase Chain Reaction (trans-PCR) technique applied in milk samples and genital swabs, using a Bayesian latent class analysis. Conditional independence was assumed between the tests, given (i) the different biological principle of ELISA and trans-PCR and (ii) the fact that the trans-PCR was performed on different tissues. The ELISA method in the serum samples showed the highest DSe of 0.97 (95% Probability Intervals (PIs): 0.93; 0.99) compared to the trans-PCR method applied in milk samples 0.76 (0.63; 0.87) and genital swabs 0.73 (0.58; 0.85). The DSps of all tests were high, with trans-PCR in genital swabs recording the highest DSp of 0.99 (0.98; 1), while the DSp of trans-PCR in milk samples and ELISA in serum samples were 0.97 (0.95; 0.99) and 0.95 (0.93; 0.97) respectively. The study results show that none of the applied tests are perfect, therefore, a testing regimen based on the diagnostic characteristic of the tests may be considered for diagnosis of C. burnetii.

Assessment of aflatoxin contamination in dairy animal concentrate feed from Punjab, India.

Aflatoxins are one of the major environmental contaminants in animal feed and pose a potential threat to human health due to their secretion in the milk of lactating animals. The present study was conducted with the objectives to determine the occurrence of aflatoxins (B1, B2, G1, and G2) in dairy animal concentrate feed and to evaluate the effect of season, spatial variation, and dairy farm size on the levels of aflatoxins contamination. A total of 189 dairy animal concentrate feed samples were tested for aflatoxins with enzyme-linked immunosorbent assay (ELISA) as screening and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) as confirmatory techniques. Of the total, 59% feed samples were found positive for aflatoxins, while 44% samples were detected with total aflatoxins levels higher than the tolerance limit established by the Food and Drug Administration (FDA) and 58% samples were found with aflatoxins B1 (AFB1) levels above the European Commission (EC) legal limit. AFB1 levels in dairy animal concentrate feed were found significantly higher during rainy (41.6 µg kg-1) and winter (35.9 µg kg-1) seasons as compared to the summer season (25.5 µg kg-1). The theoretical extrapolation of the AFB1 carry-over from animal feed to milk (aflatoxins M1) in different seasons may lead to 50-100% contamination of milk at levels above the EC tolerance limit. The incidence and levels of aflatoxins especially AFB1 in animal feed, not only pose a direct effect on animals but may also pose a concern for food safety in relation to the occurrence of aflatoxins M1 in milk. Therefore, continuous surveillance of aflatoxins in dairy animal feeds is required to reduce animal and consequently human exposure.

Toxoplasma gondii in Chickens (Gallus domesticus) from North India.

PURPOSE: Toxoplasma gondii, an important food-borne parasite has been reported in a wide range of food animals globally. Poultry meat production is on rise in several developing nations including India. Several studies on status of T. gondii conducted globally had shown substantial risk of this parasite through poultry meat. Such information is lacking in the north India, where poultry meat is a preferred non-vegetarian food over other meats; therefore, this study determined the seroprevalence (based on detection of antibodies) and tissue prevalence (based on detection of DNA) of T. gondii in poultry meat intended for human consumption in north India. METHODS: Samples (skeletal muscle and/heart as well as blood) were collected from slaughter poultry intended for human consumption reared in Punjab state (n = 366), Himachal Pradesh (n = 44) state, and Chandigarh union territory (n = 112) in North India. Serum samples were tested using commercially available Chicken T. gondii IgG ELISA kit to determine the serostatus. The DNA of T. gondii was detected by nested PCR. To determine the agreement between ELISA and PCR, Kappa value was estimated. RESULTS: Overall, study reports 2.3% (95% CI 1.3%-4.1%) tissue prevalence and 6.5% (95% CI 4.6%-9.1%) seroprevalence from chickens reared in north India. Backyard poultry had significantly higher proportion of T. gondii (24.4%, 10/41; p < 0.001) than caged (0%, 0/284) and deep litter poultry (16.2%, 32/197). The sequence alignment score of three sequences obtained in this study showed 97-100% nucleotide identity with Type I RH strain and VEG type III strain. The agreement between ELISA and PCR was poor (Kappa = 0.145; 95% CI 0.07-0.22). Thus, a combination of serological and molecular tests can improve the diagnosis of T. gondii. CONCLUSION: A low prevalence indicates that T. gondii in poultry presents a lower food safety risk for public health in north India; however, this study recommend cooking of poultry meat before consumption. Further studies are required to check the viability of T. gondii in poultry meat intended for human consumption, and to determine the risk factors associated and the genotypes circulating in poultry in north India.

Using Dairy Value Chains to Identify Production Constraints and Biosecurity Risks.

The dairy industry plays an important role in the economy and food security of India. A study of the dairy value chains was conducted in Punjab, India, to identify production constraints and biosecurity risks. Focus group discussions and key informant interviews were conducted during 2018-2019 with a total of 119 participants comprising veterinarians (41), paraveterinarians (15), veterinary academics (12), dairy farmers (46) and key informants (5). Input and output value chains were created, and potential risk nodes were identified that could facilitate the transmission of pathogens between animals, farms and villages. The majority of the participants were male (93%), middle-aged (68%) or worked in rural areas (75%). Most of the farmers self-cultivated their green fodder (82%), used the wheat straw from their own fields (60%) but purchased commercial feed (63%). Artificial insemination was used by 85% of farmers for cattle, but only 68% for buffaloes. Most of the farmers (76%) reported getting their animals vaccinated against foot-and-mouth disease and hemorrhagic septicemia. Animals were sold and purchased without any health certification and testing in most cases. Adoption of biosecurity measures by farmers and the use of personal protective equipment by veterinary personnel were very low. We recommend conducting epidemiological studies to further characterize the identified risk nodes, training of veterinary practitioners and farmers to ensure adequate biosecurity practices and the appropriate use of personal protective equipment.

Potential risk factors associated with the occurrence of aflatoxin M1 in raw milk produced under different farm conditions.

The present study was carried out with the objectives to determine the occurrence of aflatoxin M1 (AFM1) in raw milk and identification of associated risk factors from three different types of animal farming systems. A total of 189 pooled raw milk samples were tested for AFM1 with enzyme linked immunosorbant assay (ELISA) as screening and high performance liquid chromatography with fluorescence detection (HPLC-FLD) as confirmatory techniques. AFM1 was found positive in 58% pooled milk samples with the mean level of 0.917 µg/L, while 50.8% and 36.5% samples were found above the legal limits set by European Commission (EC) and Food safety and standard authority of India (FSSAI), respectively for AFM1 in milk. Among all investigated risk factors, feeding of readymade concentrate feed and leftover household cereals, longer feed storage duration and feed storage quality were found significantly associated with presence of AFM1 in farm milk. High prevalence as well as levels of AFM1 in farm milk poses a health risk to the consumers especially children. Therefore, efforts are urgently required to create awareness among dairy farmers about aflatoxins contamination of feed and milk, to improve farm management practices and to control AFM1 level in milk below the legal limits.

Antibiotic residues and mycotoxins in raw milk in Punjab (India): a rising concern for food safety.

The present study was envisaged with an aim to determine the occurrence of antibiotic residues; enrofloxacin, oxytetracycline, penicillin G, sulphamethoxazole and chloramphenicol as well as mycotoxins; aflatoxin M1 and ochratoxin A in raw milk samples collected from individual animals from dairy farms located in 9 districts of Punjab, India. A total of 168 raw milk samples were collected and analysed using commercially available competitive Enzyme linked immunosorbent assay kits. Out of these, 19 (11.3%) and 9 (5.4%) samples were found positive for antibiotic residues and mycotoxins, respectively. The milk samples were positive for enrofloxacin (4.8%), oxytetracycline (3.0%), penicillin G (3.0%) and sulphamethoxazole (0.6%) residues. The percentage of samples found above maximum residue limit established by Europen Union (EU)/Codex Alimentarius Commission (CAC) were 1.7%, 1.2% and 0.6% for enrofloxacin, oxytetracycline and penicillin G residues, respectively. Aflatoxin M1 was detected in 5.4% of monitored milk samples with 1.2% samples exceeded the established maximum levels of EU but were below the maximum levels established by CAC. The occurrence of antibiotic residues and mycotoxins in raw milk samples above maximum limits is a rising concern for food safety due to possibility of health risks to the consumers.

Prevalence and distribution of Taenia solium cysticercosis in naturally infected pigs in Punjab, India.

BACKGROUND: Taenia solium (T. solium) cysticercosis remains a neglected zoonotic disease in India. The current study was planned to estimate the prevalence of T. solium porcine cysticercosis in the Punjab state of India, to compare this prevalence with the disease prevalence in pigs reared outside Punjab and to assess the distribution of the parasite in pig carcasses. METHODS: Two slaughter shops were selected in each of the 22 districts of Punjab. Pigs slaughtered on the day/s of inspection were post-mortem inspected to identify the presence of T. solium cysts. Estimated true prevalence was estimated by taking into account the diagnostic sensitivity (38%) and specificity (100%) of post-mortem inspection using the Rogan-Gladen estimator. Positive carcasses were purchased and brought to the laboratory to assess the tissue distribution of T. solium cysts and to conduct PCR targeting large subunit rRNA gene, internal transcribed spacer 1 gene, ITS1 gene and Cytochrome oxidase I gene. The selected PCR products were submitted for sequencing and phylogenetic analyses were performed. FINDINGS: We contacted 71 shop owners to achieve a sample of 44 shops for the study. We inspected 642 pigs reared in Punjab and 450 imported from other states at these slaughter shops. In addition, we sampled 40 pigs from an abattoir located in the state capital. Of the 642 pigs reared in Punjab, 9 had T. solium cysts with an apparent prevalence of 1·40% (95% CI: 0·74%, 2·64%) and the estimated true prevalence of 3.69% (95% CI: 1·95%, 6·95%). Pigs imported from outside the state had a significantly higher prevalence (odds ratio: 2·58; 95% CI: 1·12, 5·98; p-value: 0·026) as 15 of the 450 imported pigs were positive (apparent prevalence: 3.33%; 95% CI: 2.03%, 5.43%; estimated true prevalence: 8.77%; 95% CI: 5.34%, 14.28%). None of samples was positive from the pigs sampled at the abattoir in the state capital. The PCR confirmed T. solium cysts from all the 24 positive samples. We counted a median of 897 (range 526-1964) cysts per infected pig from the 19 infected pig carcasses inspected. The phylogenetic tree based on the alignment of partial cytochrome oxidase 1 sequences indicated all positive samples to be clustered with the T. solium Asian genotype. The analysis did not indicate the presence of T. asiatica in the slaughter pigs. CONCLUSIONS: Despite the underestimation of the prevalence due to missing mildly-infected carcasses, low participation and lack of representative sampling, the presence of heavily infected carcasses containing viable cysts, particularly those imported from outside the state, indicates that T. solium cysticercosis is an important food safety concern for pork consumers in Punjab, India. Measures should be taken to reduce the disease prevalence in pigs to reduce the disease burden in the public.

The Clean India Mission: Public and animal health benefits.

The Clean India Mission is a national campaign that aims for complete elimination of open defecation from the country. In India, 564 million people do not have access to toilets and defecate in the open environment. The 'Millennium development goals' have given increased weightage to elimination of open defecation for improving health, nutrition and productivity of developing country populations. The Indian economy bears an estimated annual total loss (in terms of health, education, access time and tourism) of US$ 54 billion due to lack of toilets, poor hygiene and over US$ 38.5 billion in treatment costs for diseases occurring due to poor hygiene. Out of 1415 human pathogens, at least more than 10% of pathogens are transmitted through the faecal-oral route. The practice of open defecation helps pathogens persist in the environment and cause diseases. This review focuses on the current status and harms of open defecation, as well as the public and animal health benefits of implementing 'The Clean India Mission' in India.

Prevalence and first molecular identification of Sarcocystis species in cattle and water buffaloes in India.

The importance of Sarcocystis hominis in causing zoonotic infections is well known. Recently, S. hominis like cysts have been reported from water buffalo in China. Previous studies indicate prevalence of Sarcocystis species in bovine populations in India but molecular evidence is required for proper species differentiation. We examined two hundred and ninety six cardiac tissue samples of Indian water buffaloes and cattle from northern and western parts of the country. Tissues were examined for Sarcocystis using intact cyst isolation method, pepsin acid digestion method and Sarcocystis 18S rRNA PCR. The combination of primers was used for 18S rRNA PCR amplification followed by sequencing. Twenty five representative samples were sent for sequencing and 19 readable sequences were obtained for phylogenetic analysis. Overall, the Sarcocystis cysts/zoites were recorded in 44% (95% CI 38-49%), 58% (95% CI 53-64%) and 68% (95% CI 63-73%) from both cattle and buffalo samples using intact cyst isolation, pepsin-HCl digestion method and conventional PCR, respectively. The results indicate that pepsin-HCl digestion method and conventional PCR are more sensitive than intact cyst isolation for detection of Sarcocystis species in tissue samples. The prevalence of Sarcocystis species was high in buffalo as compared to cattle intermediate hosts. Phylogenetic analysis indicated that more than one Sarcocystis species are circulating in cattle and water buffaloes in India. The results further indicate that experimental transmission studies are required to re-confirm the identities and host ranges of the Sarcocystis species in cattle and water buffaloes in India.

Prevalence and morphological characterisation of Echinococcus granulosus from north India.

The purpose of this study was to document the prevalence and to analyze morphological characteristics from hydatid cysts to test their suitability for strain identification. In the present study, 4,130 animals, including 278 cattle, 298 buffaloes, 760 sheep, 2,439 goat and 355 pigs were examined for the presence of hydatid cysts on post-mortem inspection at different slaughter houses/shops in northern India. Morphological characteristics from hydatid cysts were analyzed to test their suitability for strain identification. For statistical analysis, five variables were considered: number of hooks per rostellum, blade length of large and small hooks, and total length of large and small hooks. Principal component analysis was applied for analysis of morphological parameters. Out of a total of 4,130 animals examined, 66 were positive for hydatid cysts (prevalence 1.598 %). The prevalence of hydatid cysts was highest in cattle (5.39 %) followed by buffaloes (4.36 %), pigs (3.09 %), sheep (2.23 %) and goat (.41 %). The results indicate significant prevalence of hydatidosis in all the food producing animals and further that morphological analysis can also be used as a valid criterion for differentiation of different strains of E. granulosus particularly in developing countries where molecular studies could not be performed due to lack of infrastructure or financial constraints.

Detection and sequencing of plasmid encoded tetracycline resistance determinants (tetA and tetB) from food-borne Bacillus cereus isolates.

OBJECTIVE: To investigate the detection and sequencing of plasmid encoded tetracycline resistance genes (tetA and tetB) from food-borne and standard strains of Bacillus cereus (B. cereus). METHODS: A PCR was carried out to detect the tetracycline resistance genes (tetA and tetB) in food-borne B. cereus strains and the amplified products were sequenced. RESULTS: The phenotypic resistance against tetracycline was observed in 39 of the 118 food-borne isolates and two reference strains (MTCC 430 and MTCC 1307) of B. cereus. Among the phenotypically resistant isolates, tetA was detected in 36 food-borne isolates and two reference strains (MTCC 430 and MTCC 1307), whereas, tetB was detected in 12 food-borne isolates and MTCC 1307 strain. CONCLUSIONS: A close association was therefore found between phenotypic resistance against tetracycline and presence of tetracycline resistance genes. The tetA and tetB gene fragments were amplified, purified and sequenced. The gene sequences of the isolates studied herein were found similar to tetA and tetB gene sequences of other bacteria available in NCBI. The occurrence of tetA and tetB genes in B. cereus indicate the horizontal transfer of antibiotic resistance determinants from other bacteria into B. cereus. The transfer of these resistant determinants to other potentially pathogenic bacteria may be a matter of great concern.

Determination of organochlorine pesticide residues in freshwater fish species in Punjab, India.

The levels of organochlorine pesticides residues were determined in five freshwater fish species in Punjab State, India. These species were selected in view of their importance to local human fish consumer. DDTs were the predominant organochlorine contaminants in all species with pp DDT and pp DDE as the main pollutants. Other organochlorines, such as HCH isomers and dieldrin were also found at lower levels in fish species. The alpha-HCH was the dominant isomer of HCH in all fish species followed by gamma-, beta- and delta-HCH. The residues of aldrin, chlordane, heptachlor and endosulphan were not detected in any fish sample. The levels of organochlorines detected in present study were below the tolerance limits recommended at National and International standards.

Prevalence of Cryptosporidium parvum infection in Punjab (India) and its association with diarrhea in neonatal dairy calves.

A prevalence study was contemplated to determine the prevalence of Cryptosporidium spp. in dairy farms in Punjab, India. The cryptosporidium oocysts were detected from 50 and 25.68% from 80 diarrheic and 74 non-diarrheic animals, respectively. Both shedding and intensity of shedding were significant in calves with diarrhea. The Cryptosporidium spp. appears to be common in dairy calves and an important contributor of calf diarrhea in the Punjab province. The prevalence of the infection peaked in young calves between 0 and 30 days in both the diarrheic and non-diarrheic groups (86.4 and 66.6%, respectively). The percentage distribution of positive samples, with reference to age groups of diarrheic and non-diarrheic animals was negatively correlated with increase in age. High mortality rate and case fatality rate of 35.2 and 44.4% were observed in young calves between 0 and 30 days of age.