Understanding the role of pigs in the transmission of zoonotic Echinococcus ortleppi in Haryana, India.

The etiological agents of zoonotic cystic echinococcosis comprise the Echinococcus granulosus sensu lato (s.l.) species complex. The present study was aimed at investigating the zoonotic genotypes of Echinococcus granulosus s.l. circulating in the pig population of Haryana, India. Out of 253 slaughtered pigs screened, 5 showed the presence of hydatid cysts. The amplification of the partial mitochondrial NADH dehydrogenase subunit 1 (nad1) gene for the molecular confirmation and phylogenetics of the retrieved metacestodes (n = 2) revealed the presence of E. ortleppi. The sequences generated herein exhibited 99.80% homology to the GenBank archived E. ortleppi sequences. Cladistics targeting genetic diversity and haplotype network analysis involved 37 E. granulosus s.l. GenBank archived sequences from India corresponding to different hosts (large and small ruminants and humans) along with the sequences (n = 2) generated in the present study. Overall, 14 haplotypes with high haplotype (0.780 ± 0.059) and low nucleotide (0.033 ± 0.010) diversities were recorded for the overall data set, which evinced a population expansion. The median-joining haplotype network revealed a stellate shape of E. granulosus sensu stricto (s.s.) sequences, which was indicative of rapid population expansion. High genetic differentiation (FST = 0.840 - 0.983) and low gene flow (Nm = 0.003 - 0.047) were recorded between the pig intermediate hosts infected with E. ortleppi and other hosts infected with E. granulosus s.s. The findings are of paramount significance for the formulation of effective control strategies considering the public health and economic impact of cystic echinococcosis.

Seroprevalence and risk factors associated with Toxoplasma gondii in pigs in Haryana, India.

AIMS: Toxoplasmosis is one of the most common food-borne parasitic zoonosis, caused by Toxoplasma gondii, an obligate intracellular protozoan parasite. A cross-sectional study was carried out to determine the seroprevalence of T. gondii and associated risk factors in pigs in Haryana, India. METHODS AND RESULTS: Serum samples were collected from 429 pigs from three agroclimatic zones (I-III) of Haryana and analysed for the presence of antibodies against T. gondii using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Anti-T. gondii antibodies were detected in 106 animals (24.7%), with the highest seropositivity in zone II (31.3%) followed by zone III (24.4%) and zone I (18.3%). Risk factors associated with higher seropositivity in pigs were farm size (higher in large-sized farms), age (higher in pigs >1 year of age), sex (higher in males), type of feeding (higher in combination of homemade and hotel waste) and housing (higher in free-ranging pigs). CONCLUSIONS: The findings of the study testify to the exposure of pigs (of all agro-climatic zones) to T. gondii. Hence, the observations are of significant medical and veterinary importance for devising and implementing control measures to check the dissemination of toxoplasmosis to pigs and eventually to humans.

Cladistics of Echinococcus granulosus Sensu Stricto Genotypes Infecting the Slaughtered Pigs.

PURPOSE: Echinococcus granulosus sensu stricto (s. s.) consists of the most widespread genotypes (G1, G3) implicated in human cystic echinococcosis worldwide. The present study aimed to evaluate the role of pigs in the transmission dynamics of E. granulosus s. s. genotypes, including the phylogenetics, evolutionary divergence and haplotype network analyses of north Indian pig isolates along with GenBank™ archived sequences. METHODS: In totality, 223 slaughtered pigs were thoroughly screened for the presence of hydatid cysts. The amplification of the partial mitochondrial cytochrome C oxidase subunit 1 gene was performed for the molecular confirmation and phylogenetics of the retrieved metacestodes. RESULTS: The molecularly confirmed and sequenced present study isolates (n = 2) clustered with the E. granulosus genotype G3. The very low evolutionary divergence values (< 0.0027) of the present study isolates with E. ganulosus genotype G3 indicated the absence of diverged lineages. The significantly negative values of Tajima's D (- 2.598) and Fu and Li's D (- 4.054) of the overall dataset and for the Asian sequences signified an expansion of population size. The overall dataset exhibited low nucleotide (0.067 ± 0.055) and high haplotype (0.574 ± 0.015) diversities, also alluding to demographic expansion. The haplotype network showed that the pig isolates from South America and Europe constituted the predominant haplotype, Hap_2 along with Hap_3 and Hap_6, primarily associated to E. granulosus genotype G1; whereas, the Indian isolates formed different haplotypes (Hap_1 and Hap_5) belonging to genotype G3. CONCLUSIONS: The present study highlighted the important role of pigs in the transmission of E. granulosus s. s., which is of paramount significance given the public health and economic impact of cystic echinococcosis.

First Molecular Confirmed Outbreak of Malignant Ovine Theileriosis in Sheep from North India.

CONTEXT: Malignant ovine theileriosis is a tick-borne disease of sheep and goats, caused by protozoan Theileria lestoquardi. The disease has serious economic implications for small ruminant production around the world. METHODS: An outbreak of malignant ovine theileriosis in a sheep flock was investigated from Hisar district of Haryana, India, in March 2022. The etiological agent was identified using polymerase chain reaction assay with genus specific primers targeting 18S rRNA gene and subsequently confirmed by sequencing. RESULTS: The morbidity, mortality and case fatality rate reported in the outbreak were 22.2, 18.8 and 85%, respectively. The phylogenetic analysis clustered the present study T. lestoquardi isolate in the same clade with T. lestoquardi from Iraq, Iran and Pakistan with maximum nucleotide identity of 99.37% with strains from Iraq. The tick vector Hyalomma anatolicum recovered from dead animals was implicated in the disease's transmission. CONCLUSIONS: Malignant ovine theileriosis resulted in high case fatality rate. This study presents the first molecularly confirmed outbreak of malignant ovine theileriosis outbreak in the North Indian region, with characteristic post-mortem findings.

Molecular investigations on outbreaks of ovine theileriosis among sheep and goats in Haryana, India.

During February 2020 to October 2020, four outbreaks of theileriosis in small ruminants were recorded with overall morbidity, mortality and case fatality rates of 27.95%, 17.46% and 62.5%, respectively. The disease was characterized by high fever (up to 106°F), superficial lymphadenopathy, anaemia, anorexia, lethargy, respiratory distress and death. The presence of pleomorphic intra-erythrocytic piroplasms of Theileria species in Giemsa's stained blood smears was a common finding in all the episodes. Significant haematological alterations including high total leucocyte count and low haemoglobin and packed cell volume were characteristic. Necropsy findings of the icteric liver, enlarged spleen, pulmonary oedema and abomasal ulcerations were observed in three flocks. Smear-positive blood samples from all the episodes were screened by PCR using 18S rRNA gene-specific primer sets for T. lestoquardi, T. luwenshuni, T. uilenbergi and T. ovis. T. lestoquardi which was detected in all four flocks, while there was co-infection of T. ovis in two flocks. Phylogenetic analysis revealed that T. ovis and T. lestoquardi identified in this study had 100% and ~ 99.86% homology, respectively, with the published sequences used for comparison. This is the first confirmed report of outbreaks of malignant ovine theileriosis in the Haryana state of India which caused high morbidity, mortality and case fatality among sheep and goats. Further studies on theilerioses in small ruminants are required to understand epidemiology better.

Epidemiology, clinical features, and molecular detection of orf virus in Haryana (India) and its adjoining areas.

Orf is an acute, highly contagious, and economically important viral disease of small ruminants. In this study, six orf suspected outbreaks among goats and sheep were investigated from Haryana state and adjoining areas of Rajasthan state during the year 2021. The disease was diagnosed on the basis of clinical signs and molecular identification. The causative agent of the disease, orf virus (ORFV), was confirmed using polymerase chain reaction (PCR) targeting immunodominant envelope antigen (B2L) gene and confirmed by sequencing. The morbidity in goats ranged from 8.75 to 100%, whereas in sheep, it ranged from 0 to 8%. The higher mortality was observed among flocks with mixed infections of orf and peste des petits (PPR) or orf and haemonchosis as compared to other outbreaks. The phylogenetic analysis of sequenced PCR products clustered the current study strains in the same clad with Indian as well as strains from other countries with nucleotide identity more than 99%, signifying a close genetic relationship. The study highlighted the circulation of strains of a single cluster among sheep and goats in Haryana and adjoining areas. Prompt diagnosis of the disease is highly important for facilitating the implementation of control measures to minimize the losses suffered by small and marginal farmers in this region. Further detailed studies are required to delineate the molecular details of ORFV for better understanding the dynamics and molecular epidemiology of strains circulating in the country and for designing the effective vaccines against the disease which are currently lacking in the country.

Epidemiology, risk factors and molecular characterization of small ruminant morbillivirus in Haryana, India.

Peste des petitis ruminants is an economically important transboundary and notifiable viral disease of sheep and goats. In this study, 14 PPR suspected outbreaks among sheep and goats were investigated in four districts of Haryana, India, during July 2020 to October, 2021. The causative agent of the disease; small ruminant morbillivirus was detected by Reverse Transcription Polymerase Chain Reaction targeting full gene sequences of fusion protein gene and confirmed by sequencing. The overall morbidity and cumulative mortality in these outbreaks were 37.56% and 12.09%, respectively. Risk factor analysis identified significant difference in mortality based on age with higher mortality in young ones; 21% as compared to adults; 7.55%. Analysis of the vaccination status revealed significant difference in morbidity and mortality with higher morbidity and mortality in un-vaccinated animals as compared to vaccinated ones. The sequencing and phylogenetic analysis of representative samples revealed that the strains of the present study fall in lineage IV (96.6-99.1%) along with other Indian isolates but made a separate cluster (sub-lineage). The comparison of deduced amino acid (aa) sequence analysis of fusion protein of circulating field strains with reference vaccine strain and other lineage IV strains revealed four N-linked glycosylation sites instead of three. The findings of the present study revealed changes in fusion protein of some of the circulating field strains of SRMV in Haryana, India. Further detailed studies are warranted to delineate the molecular details of these circulating field strains and to evaluate the effectiveness of currently used vaccine against these mutated strains.

Sub-lineages of Taenia solium Asian Genotype Recorded in North India.

PURPOSE: Porcine cysticercosis is a neglected zoonotic disease of significant veterinary and medical importance owing to its economic impact and public health significance. The present study aimed at genetic characterization of Taenia solium metacestodes in slaughtered pigs of Haryana (North India). METHODS: A total of 213 (160 and 53 from Chandigarh and Hisar, respectively) slaughtered pigs intended for human consumption were screened for the presence of T. solium metacestodes. The retrieved metacestodes were confirmed molecularly based on the partial amplification of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Evolutionary divergence, haplotype and nucleotide diversities and neutrality indices of the retrieved isolates were also assessed. RESULTS: Out of the 213 pigs, 2 (0.94%) revealed the presence of metacestodes involving 1 pig each from Chandigarh (0.62%) and Hisar (1.9%). The sequences obtained after custom sequencing were submitted to GenBank under the accession numbers LC661682-83. The present study haplotype clustered with haplotypes of Asian origin and showed variation from other haplotypes by 1-23 mutational steps. However, the present study isolates also showed nucleotide polymorphisms (A198T, A199G, A201T, G204A, T206A, C210T, T212G, T213A, T216G/A, T217C, T221C, C524T, G994A) at different positions, which indicated the presence of sub-lineages. Low nucleotide diversity (π = 0.020) and negative value of Tajima's D (- 1.304) observed for the haplotypes under consideration was indicative of purifying selection and recent population expansion. CONCLUSIONS: Our study confirms the circulation of T. solium Asian genotype (with distinct sub-lineages) in study area and recommends strict control measures to contain the zoonotic disease.

Phylogenetics and Pathology of Hydatid Disease in Slaughtered Buffaloes of North India.

PURPOSE: Hydatid disease is one of the neglected and challenging (for diagnosis as well as for treatment) parasitic diseases. Along with adverse effect on animal's health leading to production losses, hydatidosis is also associated with huge economic losses. The present study was envisaged with an aim to assess the phylogeny and pathological changes due to natural hydatid cysts in lungs and liver of slaughtered buffaloes in north India. METHODS: A total of 137 slaughtered buffaloes intended for human consumption were screened for the presence of cysts. The retrieved cysts were confirmed molecularly based on the amplification of mitochondrial cytochrome oxidase 1 gene (mtCO1), exhibiting a product size of approximately 446 bp. The samples collected from infected lungs and liver were subjected to histopathological examination. RESULTS: The hydatid cysts were recorded in 25 (18.2%) animals. Phylogenetic analysis revealed the isolated strain to be closely related to Echinococcus granulosus sensu stricto (G1) genotype. The nucleotide diversity (π) obtained was 0.014685, whereas, Tajima's D was negative (- 2.796053), which indicated purifying selection or recent population expansion. Histopathologically, in the infected lungs, fibrosis and inflammatory reaction comprising of mononuclear cells and fibroblasts around the thick coat of granulation tissue were observed. Marked calcified masses and necrosis were also observed in the calcified cysts. However, in case of infected livers, Kupffer cell hyperplasia, degeneration of hepatocytes, fibrosis and inflammatory cells were most commonly observed around the hydatid cysts. CONCLUSION: The findings of the present study are of significant veterinary and medical importance owing to economic and public health impact of G1 genotype of E. granulosus.

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.

Gastrointestinal parasitic infections in Indian Gaddi (goat) breed bucks: clinical, hemato-biochemical, parasitological and chemotherapeutic studies.

The present study was envisaged with an aim to evaluate gastrointestinal parasitic infections in a herd of conserved Gaddi (goat) breed bucks (6-9 months of age) kept on semi-intensive feeding management. The individuals (n = 20) representing the herd were selected and divided into two groups; group I (n = 10), clinically ill and group II (n = 10), sub clinically infected individuals. The clinical examination revealed anemia, emaciation and rise in body temperature of the individuals of group I as compared to group II. The detailed copro-parasitological examination and copro-culture revealed the presence of eggs of Moniezia expansa and larvae of Haemonchus species, respectively in the fecal samples of both clinically and sub clinically infected individuals. The hemato-biochemical parameters proved vital indicators of the health of group I individuals and exhibited decline in the values of hemoglobin, packed cell volume and total erythrocyte count as compared to group II. Significant (P < 0.05) hypoproteinemia, hypoalbuminemia, hypoglycemia and increased levels of alanine aminotransferase and aspartate aminotransferase were observed in infected individuals as compared to treated ones. The detailed parasitological, hemato-biochemical observations and clinical findings elucidated and supported the presence of concurrent gastrointestinal parasitism in the herd. The significant improvement was observed in the health status of the herd after 1 month of the therapeutic management, which was carried out using a combination of fenbendazole and praziquantel in both clinically and sub clinically infected individuals.

Cyathostomum catinatum infection in a mule of Palam Valley, India: a case report.

The small strongyles also called as cyathostomes, cyathostomins or trichonemes, affect the health status of equines, leading to morbidity and mortality in heavily infested individuals. The present study was carried out with the aim to identify the cause of colic in a mule of Palam valley, Himachal Pradesh, India. The detailed faecal sample examination revealed heavy intensity of strongyle eggs with an egg per gram value of 2300. The collected faecal sample also exhibited presence of adult worms. The morphological observations after clearing the retrieved parasites revealed the presence of adults of Cyathostomum catinatum species. The haematological and serum biochemical studies revealed alterations related to parasitic invasion. Fenbendazole used for the treatment proved completely effective. To the best of our knowledge and thorough literature search this is the first report of C. catinatum infestation in equines of India.