Echinococcus granulosus is the causative agent of cystic echinococcosis (CE) and is an endemic disease in different areas of the world. CE is responsible of having a significant impact on human health and causes economic losses in endemic countries (Budke et al., 2017). CE is now listed as one of the neglected zoonotic diseases (NZDs) by World Health Organization (WHO) (Kern et al., 2017).

CE life cycle is maintained through dogs (definitive hosts), harboring the adult tapeworm, and a range of livestock species (cattle, sheep, goats and camels) as intermediate hosts. Dogs excrete the infective eggs via faeces, in soil and water (Gemmell, 1990). The accidental host (human) and intermediate hosts can be infected by intake of parasite eggs by contaminated food and water (Eckert et al., 2000; McManus et al., 2003; McManus and Yang, 2017).

Epidemiological studies conducted in central Asia so far on CE showed that it is a public health challenge. Out of 270 million people, 58% were at risk of infected with CE from different countries such as Mongolia, Kyrgyzstan, Tajikistan, Kazakhstan, Uzbekistan, Turkmenistan, Iran, Afghanistan, western China, and Pakistan. The majority of residents of these regions are at risk of developing CE, especially herdsman and farmers (Zhang et al., 2015).

Pakistan is an agricultural dependent country; around 8 million families of rural area are affiliated to agriculture, deriving 35–40% income from livestock production (Pakistan Economic Survey, 2020). The agricultural sector contributes 21% to national gross domestic product (GDP) of which 11.9% is contributed by the livestock sector. Thus, Pakistan's GDP and economic stability are greatly influenced by livestock. The latest Pakistan Economic Survey (2019–2020) reported that there were 78.2 million goats, 31.2 million sheep, 49.6 million cattle, and 41.2 million buffallo. The anticipated meat production was estimated to be around 4708 tons. Although, various non-infectious and infectious diseases including CE are main hurdle in growth of livestock sector. The economic loss causes by parasitic diseases was reported 26.5 million/annum (Pakistani Rupees), while CE-associated losses in sheep and goats were estimated US$ 276.20/100 and US$ 165.72/100 in cattle, buffalo and camels (Latif et al., 2010).

Using different molecular biological techinques, investigations have reported Echinococcus granulosus as a species complex, showing intraspecific variabilty worlwide. Mitochondrial DNA (mt-DNA) is extensively used to understand the evolutinary link among closely related species due to its rapid sequence evolution (Brown et al., 1979). Futhermore, large datasets derived from mitochondrial genomes have the potential to resolve Echinococcus taxonomy issues (Nakao et al., 2010). Consequently, a greater number of researchers are focusing on mt-CO1 and NADH dehydrogenase 1 (nad1) regions of the parasite as genetic markers (Sharbatkhori et al., 2009; Simsek et al., 2010; Nakao et al., 2010; Khan et al., 2020a). For gentyping of E. granulosus s.l. many researchers usually prefer mitochondrial markers. Despite, Saarma et al. (2009) stated that nuclear DNA analysis provides more reliable approach to inferring phylogenetic relationships within Echinococcus than mt-DNA, (Kesik et al., 2021, 2022; Khan et al., 2021d). Therefore, E. granulosus s.l. is recently divided into E. granulosus sensu stricto (G1, G3), Echinococcus equinus (G4), Echinococcus ortleppi (G5), Echinococcus canadensis (G6/G7, G8 and G10), and Echinococcus felidis (Vuitton et al., 2020).

During the past decades, extensive studies have identified different E. granulosus s.l. genotypes/species responsible for CE globally. However, between 1980 and 2022 only 15 molecular investigations have been conducted on Echinococcus spp in Pakistan. E. granulosus s.s. (G1, G3) (Khan et al., 2020a; Muqaddas et al., 2020), E. canadensis (G6/G7) (Khan et al., 2020a; Muqaddas et al., 2020), and E. multilocularis (Khan et al., 2020a) were reported from human isolates. Besides, E. granulosus s.s. (G1, G3) (Latif et al., 2010; Ehsan et al., 2017; Alvi et al., 2020; Mehmood et al., 2020; Khan et al., 2021a, 2021b), E. ortleppi (G5) (Alvi et al., 2020; Khan et al., 2020b), and E. equinus (G4) (Khan et al., 2021b) were found in animals in Pakistan. E. granulosus s.l. molecular identification has great impact on epidemiological findings and control strategies. Poor knowledge regarding parasite life cycle, lack of veterinary meat inspection and proper disposal of offal, significantly contributes to the parasite life cycle and transmission (Ahmed et al., 2017; Khan et al., 2019; 2020c, 2021c).

The purpose of current study was to use PCR and mt-CO1 gene sequence analysis to determine E. granulosus s.l. genetic diversity and haplotypes from cattle and buffaloes isolates.