Cystic echinococcosis (hydatidosis) is a world-wide zoonotic disease of mainly humans, livestock and dogs, caused by Echinococcus granulosus. The disease can negatively impact food production and animal welfare and causes socio-economic hardship. Here, we aimed to identify the local bovine hydatid cyst fluid (BHCF) antigen for developing a sero-diagnostic assay to be used for the pre-slaughter screening of food animals. In total, 264 bovines approved for slaughter in Pakistan were subjected to serum collection and post-mortem screening for hydatid cysts. These cysts were assessed microscopically to assess fertility and viability, and by PCR for molecular confirmation of species. A BHCF antigen was identified from positive sera via SDS-PAGE, confirmed by Western blot, and quantified via a bicinchoninic acid (BCA) assay. The quantified crude BHCF antigen (iEg67 kDa) was then used in ELISA screening to test all sera collected from known positive and negative animals based on hydatid cyst presence/absence. Of the 264 bovines examined, 38 (14.4%) showed hydatid cysts during post-mortem examination. All of these individuals, plus an additional 14 (total: 52; 19.6%) tested positive based on less time-consuming ELISA examination. Based on ELISA, occurrence in females (18.8%) was significantly higher than in males (9.2%) and was higher in cattle (19.5%) compared to buffalo (9.5%). The infection rate increased with age in both host species: cumulatively, 3.6% in animals aged 2–3 years, 14.6% in 4–5-year-olds and 25.6% in 6–7-year-olds. The occurrence of cysts in cattle was significantly higher in the lungs (14.1%) compared to their livers (5.5%), whereas the opposite was true in buffalo (6.6% livers, 2.9% lungs). For both host species, most cysts in the lungs were fertile (65%), while the majority in the liver were sterile (71.4%). We conclude that the identified iEg67 kDa antigen is a strong candidate for the development of a sero-diagnostic screening assay for the pre-slaughter diagnosis of hydatidosis.

Echinococcus granulosus is a zoonotic dog parasite, which causes cystic echinococcosis in humans and animals, adversely affecting health and food production. The resultant socio-economic impacts in lower–middle-income countries and the paucity of epidemiology data prompted the World Health Organization to classify cystic echinococcosis as a neglected tropical disease. Between November 2019 and December 2020, the prevalence of cystic echinococcosis was assessed from 3600 post-slaughter livestock (1142 sheep, 1258 goats and 1200 buffalo) from three districts, Narowal, Sheikhupura and Sialkot, in Northern Punjab, Pakistan. The collected hydatid cysts were subjected to microscopy and PCR followed by Sanger sequencing and phylogenetic analysis. A questionnaire targeted 3600 animal owners to assess disease risk factors. Of the 28 cyst samples sequenced for mtDNA COI, the species was confirmed as Echinococcus granulosus and phylogenetic analysis revealed the presence of both G1 and G3 lineages. Overall, we noted a regional difference in prevalence, where infections were significantly higher in Sheikhupura (9.8%) than Sialkot (6.1%) or Narowal (5.9%) districts. Prevalence was higher in buffalo (9.3%) than sheep (7%) or goats (5.5%), significantly higher (9.3%) in older (> 4 years) compared to younger (< 4 years) animals (5.7%) and higher in females (9.1%) compared to males (3.8%). In sheep and buffalo, more cysts were found in the liver (77.7% and 68.7%, respectively), whereas the lungs were more commonly infected in goats (67.1%). Epidemiological data revealed that combining dog deworming with effective disposal of viscera is needed for a significant reduction in infection prevalence. Similarly, animal deworming was only associated with significantly reduced infections when the animals were not kept near an abattoir, where dogs would normally be present and feeding on discarded viscera. This study adds to growing evidence that simple public health measures and awareness are needed to reduce the prevalence of cystic echinococcosis infection.