Serologic evidence of Echinococcus granulosus in slaughterhouses in Pakistan: global alarm for butchers in developing countries.

INTRODUCTION: Cystic echinococcosis, caused by Echinococcus granulosus, is a neglected zoonosis that affects humans and livestock. This sero-survey was designed for the first time in Pakistan to assess the exposure of butchers to E. granulosus as there was no previous report in the country for this occupational group. METHODOLOGY: Blood samples were collected from registered butchers (n = 364) in five different slaughterhouses in Faisalabad and Bahawalnagar Districts. Sera were tested for anti-Echinococcus granulosus IgG with a commercially available ELISA kit (specificity, 100%; sensitivity, 97%). RESULTS: Overall, seroprevalence was 9.61% (35/364). Butchers >30 years of age (10.34%), those involved in small ruminants butchery (11.70%), >10 years' experience (10.04%), formal education level up to middle standard (10.28%), contact with dogs (12.71%), improper/unhygienic disposal of dog feces (11.87%), and those unaware of the consequences of eating with unwashed hands (13.80%) were more seropositive with significant statistical differences (p < 0.05). Variables like previous cyst encounter, no knowledge of zoonoses and/or cystic echinococcosis, living in rural areas and the presence of stray/feral dogs in surroundings did not show any significant association (p > 0.05) with seroprevalence in butchers. The binary logistic regression model also showed a statistically significant relationship (p < 0.05) for all risk factors found statistically significant (p < 0.05) in the univariate analysis. CONCLUSIONS: This study shows high prevalence of cystic echinococcosis among butchers in Pakistan and underscores the need for educating native slaughterhouse personnel on cystic echinococcosis. It also serves as a global warning, especially in developing countries.

Hydatigera taeniaeformis in urban rats (Rattus rattus) in Faisalabad, Pakistan.

Hydatigera taeniaeformis formerly referred to as Taenia taeniaeformis is a cestode of cats (definitive hosts) and rodents (intermediate hosts). The prevalence of the metacestode larval stage has been reported in rodents in many parts of the world even though the genetic polymorphisms or intraspecies variation is still understudied. Here, we report a prevalence of 22.09% (38/172) from an urban rodent population in Pakistan and a nucleotide diversity (cox1) of 0.00463 among the population. Infection was higher in male (27.85%) and adult (32.29%) rats than female and sub-adult/young rats. Interestingly, The median-joining network and phylogenetic construction comprising isolates from China, Japan, Kenya, Laos, Malaysia, Senegal, the United Arab Emirates, and countries in Europe demonstrated that Pakistani H. taeniaeformis are closer to Asian and African population than those of European origin. The results of the study will add-in preliminary data for H. taeniaeformis and will also contribute to understand the global molecular epidemiology and population structure of H. taeniaeformis.

First Report on Molecular Characterization of Taenia multiceps Isolates From Sheep and Goats in Faisalabad, Pakistan.

Coenurus cerebralis is the larval stage of Taenia multiceps commonly found in the brain (cerebral form), intramuscular and subcutaneous tissues (non-cerebral form) of ungulates. Globally, few reports exist on the molecular characterization and genetic diversity of C. cerebralis with none available for Pakistan. The current study molecularly characterized 12 C. cerebralis isolates surgically recovered from sheep (n = 4) and goats (n = 8) from a total of 3,040 small ruminants using a portion of the cytochrome c oxidase subunit 1 (cox1) mitochondrial (mt) gene. NCBI BLAST search confirmed the identity of each isolate. A high haplotype and a low nucleotide diversity with three haplotypes from the 12 isolates were observed. The findings suggest the existence of unique haplotypes of C. cerebralis in Pakistan. The negative value of Tajima's D and the positive value of Fu's Fs were inconsistent with population expansion, however, the sample size was small. Bayesian phylogeny revealed that all Pakistani isolates alongside the Chinese sequences (obtained from GenBank) constituted a cluster while sequences from other regions constituted another cluster. This is the first molecular study to determine the genetic diversity of C. cerebralis in Pakistan and serves as a foundation for prospective studies on the prevalence and population structure of C. cerebralis in the country. Furthermore, in this study, we amplified only a partial segment of the cox1 gene from a limited sample size. This could have implications on the interpretation of the actual population structure in reality. Thus, we recommend future studies to consider a larger sample size in a massive epidemiological survey for further insights.

In-House Developed ELISA Indicates High Prevalence of Anti-Echinococcus granulosus IgG in Sheep Population-An Update from Pakistan.

Cystic echinococcosis (CE) is a World Health Organization (WHO)-listed neglected tropical farm economy jeopardizing and public health concern disease. This study was aimed at furnishing sero-epidemiological baseline data of CE in sheep in Pakistan, where data are non-existent. For this purpose, two sheep-rich provinces of Pakistan were selected, and 728 sheep sera were collected using probability proportional to size (PPS) statistical technique. Epidemiological information was recorded on a questionnaire for the estimation of potential risk factors. The serum samples were analyzed for IgG antibodies against Echinococcus granulosus using an in-house-developed EgAgB-based ELISA kit. The overall seroprevalence recorded was 21.98% (160/728) in the tested sheep, suggesting higher seropositivity in sheep from Punjab (23.73%) as compared to Khyber Pakhtunkhwa (KPK) (19.04%). The overall apparent prevalence observed by this ELISA method was almost similar to the calculated true prevalence (21.77%). Prevalence was significantly different (p < 0.05) among sheep from different districts. Higher prevalence was found in females (22.54%, OR 1.41), age group > 5 years (29.66%, OR 1.64), crossbreeds (42.85%, OR 2.70), and sheep with pasture access (25.96%, OR 3.06). Being in age group > 5 years and having pasture access were the factors significantly associated with seropositivity (p < 0.05). This study provides serological evidence of E. granulosus infection in sheep and can be used as a model for ante-mortem screening of the sheep globally.

Preliminary information on the prevalence and molecular description of Taenia hydatigena isolates in Pakistan based on mitochondrial cox1 gene.

Taenia hydatigena is a cestode of veterinary importance. Infection with the metacestode larval stage results in cysticercosis, which poses a serious challenge to the livestock industry worldwide. Globally, there are numerous reports on cysticercosis caused by T. hydatigena in sheep and goat but a lack of data on the prevalence and genetic diversity exists for Pakistan. We designed this study to provide an insight into the disease status as well as investigate the genetic variation among the recovered isolates based on the mitochondrial cox1 gene. In this study, we examined small ruminants (sheep and goats) slaughtered in Faisalabad in eastern Punjab province of Pakistan for T. hydatigena metacestodes and described the population structure and genetic variation using the cytochrome c oxidase subunit 1 (cox1) mitochondrial gene. Overall, a prevalence of 4.40% (goat =4.67% sheep = 4.07%) from a total of 2225 small ruminant carcasses (sheep = 983, goats = 1242) was observed. Based on the NCBI BLAST search and Bayesian phylogeny, the identity of all isolates was confirmed via their nucleotide sequences. The diversity indices indicated a high haplotype and a low nucleotide diversity with 43 haplotypes from 98 isolates. The results also show the existence of unique haplotypes of T. hydatigena in Pakistan as demonstrated by the significant negative values of Tajima's D and Fu's Fs neutrality test suggesting a recent population expansion. The median-joining network of the partial cox1 sequence dataset showed the existence of two main haplotypes detected in both sheep and goat populations. This study shows that the prevalence of cycticercosis due to T. hydatigena is below 5% in sheep and goats in Faisalabad, Punjab, Pakistan. The molecular analysis of the partial cox1 gene also indicates a high degree of genetic variation with the existence of rare haplotypes. These findings represent a preliminary report on the prevalence and genetic variation of T. hydatigena in Pakistan and serve as baseline information for future studies on the prevalence and population structure of T. hydatigena in the country.

Echinococcus granulosus (sensu stricto) (G1, G3) and E. ortleppi (G5) in Pakistan: phylogeny, genetic diversity and population structural analysis based on mitochondrial DNA.

BACKGROUND: Cystic echinococcosis (CE) is a serious tapeworm infection caused by Echinococcus granulosus (sensu lato) which infects a wide range of animals and humans worldwide. Despite the millions of livestock heads reared in Pakistan, only a few reports on CE prevalence and even fewer on the genetic diversity are available for the country. Meanwhile, the available reports on the genetic diversity are predominantly based on short sequences of the cox1 gene. METHODS: To close this knowledge gap, this study was designed to investigate the genetic diversity and population structure of Echinococcus spp. in Pakistan using the complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. RESULTS: Based on BLAST searches of the generated cox1 and nad1 gene sequences from a total of 60 hydatid cysts collected from cattle (n = 40) and buffalo (n = 20), 52 isolates were identified as E. granulosus (s.s.) (G1, G3) and 8 as E. ortleppi (G5). The detection of the G5 genotype represents the first in Pakistan. The phylogeny inferred by the Bayesian method using nucleotide sequences of cox1-nad1 further confirmed their identity. The diversity indices indicated a high haplotype diversity and a low nucleotide diversity. The negative values of Tajima's D and Fu's Fs test demonstrated deviation from neutrality suggesting a recent population expansion. CONCLUSIONS: To the best of our knowledge, this report described the genetic variation of E. granulosus population for the first time in Pakistan using the complete cox1 and nad1 mitochondrial genes and confirms E. ortleppi as one of the causative agents of CE among livestock in Pakistan. While this report will contribute to baseline information for CE control, more studies considering species diversity and distribution in different hosts across unstudied regions of Pakistan are highly needed.