Quantitative risk assessment of human Taenia solium exposure from consuming pork produced in Punjab, India.

Taenia solium cysticercosis is a neglected zoonosis that affects people throughout much of the developing world. The disease is endemic in Punjab state of India and controlling it is a public health challenge. No studies have been conducted to quantify the risk of T. solium exposure from consuming pork produced in the state. A stochastic quantitative risk assessment (QRA) model was constructed to understand the risk of human T. solium infection from consuming pork produced in Punjab. Input data were collected from official records, published literature, active surveillance and specifically for this study. Sensitivity analyses were conducted to assess the influence of the input parameters on the main output probability that any one pork meal is infective (contains at least one viable cyst) at consumption. The probability of any pork meal in Punjab containing at least one viable T. solium cyst post-storage and post-cooking was median 5.57 × 10-4 (95% PI 1.06 × 10-4 -1.95 × 10-3 ). Sobol' sensitivity analysis demonstrated that the most influential input parameters on consumption of a pork meal with at least one viable T. solium were the probability that a meal is insufficiently cooked to render cysts unviable, and the proportion of infected carcasses following informal slaughter. Whilst improved sanitation and hygiene can prevent cysticercosis in people, efforts to reduce the prevalence of T. solium cysts in pig carcases by preventing pigs' access to human waste, increasing meat inspection and promoting effective cooking practices are also important to reduce this source of taeniasis.

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.

Molecular detection of Toxoplasma gondii in the slaughter sheep and goats from North India.

Toxoplasma gondii is an obligate intracellular parasite that infects almost all the warm blooded animals, including human beings. The disease usually remains asymptomatic but is a serious concern for pregnant women, developing foetus and immuno-compromised individuals. We collected 400 cardiac/skeletal muscle tissue samples from slaughter sheep (177) and goat (223) intended for human consumption from Punjab, Uttar Pradesh and Chandigarh states/union territory in North India. The samples were pepsin-HCl digested and DNA was extracted from all the digested samples. Nested-PCR was carried out to amplify 580bp and 531bp bands with external and internal sets of primers specific for B1 gene of T. gondii. Molecularly, six (1.5%) isolates of T. gondii were detected. In PCR, T. gondii DNA were detected from 1.69% and 1.34% of the sheep and goat samples, respectively. Three PCR amplified products were sequenced in both the directions and readable sequences were obtained. Due to a low level of polymorphism in the targeted B1 gene, the clonal lineages of different isolates could not be determined. The results indicate that T. gondii in slaughter sheep and goat presents a low food safety risk for public health in North India.

Prevalence of gastro intestinal parasites in pigs in Punjab, India.

Gastrointestinal parasites are a common problem in pigs in India. The important risk factors include coprophagic behaviour of pigs and their free access to garbage. To investigate the gastrohelmenthic spectrum in pigs of Punjab, we examined 265 faecal samples from farm (n = 47) and scavenging pigs (n = 218) using faecal floatation method. Ascaris suum, unsporulated oocysts, Trichuris spp. and Strongyloides were recorded in 27.5, 15.4, 1.8 and 4.5 % of the pig faecal samples, respectively. Overall prevalence was significantly higher in pigs >1 year (56.5 %) than pigs ≤1 year (39.6; p = 0.01) Parasite positivity was neither significantly related with location (p = 0.309) nor with management practices (p = 0.69). High prevalence of gastro intestinal parasites in pigs in Punjab warrants intervention policies to control this problem.

Epidemiological studies on trichinellosis in pigs (Sus scofa) in India.

The present study was conducted to determine the prevalence of trichinellosis in domestic pigs (Sus scrofa) in India. A total of 319 tissue and 279 serum samples were collected from 279 slaughter pigs. The samples were collected from small slaughter shops located in Assam, Chandigarh (Union territory), Punjab and Uttarakhand states of the country. The presence of Trichinella spp. antibodies in serum was determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit and tissue samples were digested using the OIE recommended pepsin-HCl digestion method. The overall seroprevalence of trichinellosis was found to be 2.87 %. The age wise seroprevalence was found to be 2.83 % in pigs aged more than 6 months and 3.12 % in pigs <6 months of age. The seroprevalence was more in male (3.38 %) than female (1.96 %) pigs. None of the samples were found positive using pepsin-HCl digestion method. Since none of the samples was found positive for Trichinella, PCR assay could not be employed in the current study. The age, sex and management practices of the animals were not significantly (p > 0.05) associated with seroprevalence. The current research confirms that trichinellosis remains a rare zoonosis in India. However, extensive studies must be carried out in wildlife to identify reservoir hosts in the country.

Comparison of radiography and ultrasonography in the detection of lung and liver cysts in cattle and buffaloes.

AIM: Echinococcosis is the major cause of lung and liver cysts in ruminants. This study compared usefulness of radiography and ultrasonography (USG) in the detection of lung and/or liver cysts in sick bovine animals. The study also worked out cooccurrence of lung and liver cysts, and whether these cysts were primary cause of sickness or not. MATERIALS AND METHODS: This study was conducted on 45 sick bovine (37 buffaloes and 8 cattle) suffering from lung and liver cysts. A complete history of illness and clinical examination was carried out. Lateral radiographs of chest and reticular region were taken. In radiographically positive or suspected cases of cysts, USG of the lung and liver region was done. Depending on the location of cyst and clinical manifestations of the animal, the cysts were categorized as primary or secondary causes of sickness. RESULTS: Using either imaging technique, it was observed that 46.7% of the animals had both lung and liver cysts, whereas 33.3% had only lung and 20% had only liver cyst. Cysts were identified as primary cause of sickness in 31.1% animals only. For diagnosing lung cysts, radiography (71.1%) and USG (62.2%) had similar diagnostic utility. However, for detecting liver cysts, USG was the only imaging tool. CONCLUSION: The lung and liver cysts, depending on their number and size may be a primary cause of sickness in bovine. Radiography and USG are recommended, in combination, as screening tools to rule out echinococcosis.

Morphological characterization of Cysticercus cellulosae in naturally infected pigs in Punjab (India).

Porcine cysticercosis is an important medical and veterinary concern in the developing world. The present study was carried out to determine the morphological characteristics of Cysticercus cellulosae, so as to differentiate the C. viscerotropica (larval form of T. asiatica) which is having only rudimentary hooks on their rostellum. Morphological analysis was conducted on 22 Cysticercus positive samples. Measurements for number of hooks (large and small), the total length and blade length of large and small hooks per rostellum were carried out as per previous studies. Microscopic examination of all the cysts showed typical characteristic of T. solium i.e. presence of hooks in all the cysts. The results indicated absence of T. asiatica from naturally infected pigs in Punjab (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.

Genetic diversity and population genetic structure analysis of Echinococcus granulosus sensu stricto complex based on mitochondrial DNA signature.

The genetic diversity and population genetics of the Echinococcus granulosus sensu stricto complex were investigated based on sequencing of mitochondrial DNA (mtDNA). Total 81 isolates of hydatid cyst collected from ungulate animals from different geographical areas of North India were identified by sequencing of cytochrome c oxidase subunit1 (coxi) gene. Three genotypes belonging to E. granulosus sensu stricto complex were identified (G1, G2 and G3 genotypes). Further the nucleotide sequences (retrieved from GenBank) for the coxi gene from seven populations of E. granulosus sensu stricto complex covering 6 continents, were compared with sequences of isolates analysed in this study. Molecular diversity indices represent overall high mitochondrial DNA diversity for these populations, but low nucleotide diversity between haplotypes. The neutrality tests were used to analyze signatures of historical demographic events. The Tajima's D test and Fu's FS test showed negative value, indicating deviations from neutrality and both suggested recent population expansion for the populations. Pairwise fixation index was significant for pairwise comparison of different populations (except between South America and East Asia, Middle East and Europe, South America and Europe, Africa and Australia), indicating genetic differentiation among populations. Based on the findings of the present study and those from earlier studies, we hypothesize that demographic expansion occurred in E. granulosus after the introduction of founder haplotype particular by anthropogenic movements.

Molecular epidemiology of Echinococcosis from food producing animals in north India.

Echinococcosis is an important medical, veterinary and economic concern in India. Ten cysts were randomly selected from each intermediate host species (cattle, buffalo, sheep, goat and pigs). Either the germinal layer (sterile cysts) or protoscoleces (fertile cysts) were collected for molecular characterization. A 434 base pair fragment of the mitochondrial cytochrome oxidase-1 gene was amplified using PCR from each isolate. Ten representative samples (2 from each intermediate host species) were sequenced in both the directions from which readable sequences were obtained from nine for phylogenetic analysis (NCBI, Blast). Phylogenetic analysis of cytochrome oxidase I gene revealed that seven (77.7%) isolates, from cattle (2), pigs (2), buffaloes (1) and goat (2) were clustered with the Indian Buffalo (G3) strain of Echinococcus granulosus, while two (22.2%) isolates from sheep were clustered with the sheep strain (G1) of E. granulosus. Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the buffalo strain (G3) and common sheep strain (G1) are cycling among livestock in north India and that these strains are highly adapted to cattle, buffalo, sheep, goats and pigs.

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.