Prevalence and molecular genotyping of cystic echinococcosis among Buffalo isolates from South-Punjab, Pakistan.

Echinococcus granulosus sensu lato (s.l.), the causative agent of cystic echinococcosis (CE) cause enormous health, production, and economic impacts on livestock and human population. Buffaloes play a central role in Pakistan's livestock economy, and often serve as key intermediate hosts in the transmission of E. granulosus s.l. However, little is known about CE in buffaloes and associated productive and financial risks in southern Punjab, Pakistan. Out of 1689 buffaloes (male-678, female-1011), 256 were found infected (95%CI = 13.45-16.87), with a 15.16% prevalence (X2 = 30.30). Samples primarily the liver, lungs, heart, spleen, and kidneys were collected from three cities of Bahawalpur division demonstrated maximum prevalence (18.1%) in Bahawalpur (95%CI = 15.35-20.85). The prevalence was higher in females (19.09%) (95%CI = 16.67-21.33) than males (9.29%), (95%CI = 7.10-11.48). The highest age-wise prevalence (21.72%) was found in older buffaloes >7 years (95%CI = 19.03-24.41), followed by 11.34% in >5-≤7 years (95%CI = 8.35-14.33) and 3.09% in >3-≤5 years (95%CI = 1.30-4.90). The most favorable organ for cyst development was the liver (11.78%), followed by the lungs (3.37%, X2 = 85.22). The partial mitochondrial cytochrome oxidase 1 (COX1) sequence was used for molecular characterization of randomly selected five buffalo isolates of E. granulosus sensu stricto (s.s). Sequence analysis showed that five E. granulosuss.l. isolates obtained from buffaloes were 96.4-99.15% similar to E. granulosus s.s. but differed from other reference sequences by significant nucleotide variations, such as G1/G3 GenBank sequences BE1(G1/98.10%; G3/98.88%), BE2(G1/97.83%; G3/98.60%), BE3(G1/97.61%; G3/97.13%), BE4(G1/96.40%; G3/97.12%), and BE5 (G1/97.20%; G3/99.15%). The presence of notable micronucleotide variations suggests local strain adaptation, which may influence transmission dynamics and host compatibility. These findings demonstrate that buffaloes, particularly older females play a critical role in sustaining CE transmission in the study area. Their infection status, combined with close interactions among livestock, Canidae, and humans, creates a high-risk interface for environmental contamination and spill-over of CE into human populations. Targeted control measures focusing on slaughter hygiene, dog access to infected offal, and surveillance of high-risk livestock groups are therefore essential for reducing zoonotic transmission.