Background: Cystic echinococcosis (CE) is one of the principal causes of economic loss to the livestock industry because of its morbidity and mortality of food-producing animals and condemnation of important visceral organs. Pakistan being an agricultural country having an extensive livestock sector, is mostly practiced by poor people, which has a fundamental role in the economy. The present study was aimed to conduct a cross-sectional survey and PCR based confirmation of Echinococcus granulosus in sheep, goats, cows, and buffaloes from southern regions (three districts: Lakki Marwat, Bannu, and Karak) of Khyber Pakhtunkhwa, Pakistan. During the study, a total of 2833 animals were examined randomly including; sheep (n = 529), goats (n = 428), cows (n = 1693), and buffaloes (n = 183). Hydatid cysts were collected and examined for the presence of protoscoleces using microscopy. Detection of DNA was performed by using PCR and two mitochondrial genetic markers namely; NAD-1 and COX-1 were amplified. Results: The overall prevalence of CE was found to be (9%) among the examined animals. The hydatid cyst infection was highly prevalent in buffaloes (12%), followed by sheep (10%), cows (9%), and goats (5.1%). Cystic echinococcosis was more prevalent (10%; 96/992) in district Lakki Marwat followed by district Bannu (9%; 112/1246) and Karak (7%; 39/595). Female animals were more likely to be infected with CE (11.6%) than male animals (5.3%) (p = 0.001). Similarly, the infection was higher in the older group of animals as compared to younger (p = 0.001). Mostly (52.2%; n = 129) of hydatid cysts were found in the liver, while (64.4%; n = 159) cysts of the infected animals were infertile. PCR based identification confirmed the presence of E. granulosus sensu stricto (s.s) in the study area. Conclusion: Cystic echinococcosis was found to be highly prevalent in southern regions of Khyber Pakhtunkhwa and could be a potential threat to human health. Moreover, molecular sequencing and phylogenetic analyses should be carried out in future to identify the prevailing genotype (s) of E. granulosus s.s.

Salmonella Typhimurium, a foodborne pathogen, is a major concern for food safety. Its MDR serovars of animal origin pose a serious threat to the human population. Phage therapy can be an alternative for the treatment of such MDR Salmonella serovars. In this study, we report on detailed genome analyses of a novel Salmonella phage (Salmonella-Phage-SSBI34) and evaluate its therapeutic potential. The phage was evaluated for latent time, burst size, host range, and bacterial growth reduction in liquid cultures. The phage stability was examined at various pH levels and temperatures. The genome analysis (141.095 Kb) indicated that its nucleotide sequence is novel, as it exhibited only 1–7% DNA coverage. The phage genome features 44% GC content, and 234 putative open reading frames were predicted. The genome was predicted to encode for 28 structural proteins and 40 enzymes related to nucleotide metabolism, DNA modification, and protein synthesis. Further, the genome features 11 tRNA genes for 10 different amino acids, indicating alternate codon usage, and hosts a unique hydrolase for bacterial lysis. This study provides new insights into the subfamily Vequintavirinae, of which SSBI34 may represent a new genus.

Cystic echinococcosis (CE) is a neglected zoonotic disease prevalent in Pakistan, but the genetic diversity of the cestode is largely unexplored in the country. This study investigated the molecular epidemiology of CE infecting the livestock population of the Malakand division, Khyber Pakhtunkhwa, Pakistan. A total of 1,200 livestock, including buffaloes, cattle, goats, and sheep, were examined for echinococcosis from November 2017–2018 at different slaughterhouses in the Malakand division. Hydatid cysts were collected from different organs, and hydatid cyst fluid (HCF) was examined microscopically and used for DNA extraction. The LSU (rrnl) and NAD1 genes were amplified and sequenced. The overall prevalence of CE was 17% (204/1,200), including cows (21.7%), buffaloes (17.4%), goats (10%), and sheep (9.6%). The infection was relatively more prevalent among males (17%) than females (16.9%) and animals of older age (>5 years) (p = 0.710). Liver (63.2%) and lungs (25%) were more affected as compared to kidneys (6.8%) and heart (4.9%). HCF analysis indicated that 52.0% of the cysts were sterile and (48.0%) were fertile. Sequencing and phylogenetic analyses confirmed 80.0% of the isolates as Echinococcus granulosus sensu stricto (G1-G3) in all animal species, while Echinococcus equinus (G4) and Echinococcus ortleppi (G5) were present in buffaloes. The present study concluded that CE is prevalent in the livestock population of Malakand. Besides E. granulosus s. s. (G1-G3), E. ortleppi genotype (G5) and E. equinus (G4) in livestock were also reported.

Cystic echinococcosis (CE), or hydatid cyst disease (HCD), is a zoonosis of significant importance caused by the cestode of Echinococcus granulosus sensu lato (s. l.) that affects mainly nomadic populations and has substantial economic consequences. Due to the 76% rural and nomadic population, Baluchistan is a highly endemic region in Pakistan for CE; however, it has not yet been investigated for CE. For this purpose, this study was carried out to investigate the molecular epidemiology of CE in this region. In total, 23 human hydatid cyst samples were collected from tertiary health care units in Baluchistan and processed for DNA extraction, which was then followed by sequencing of the cox1 mitochondrial gene of all 23 collected samples, genotyping, and phylogenetic and haplotype analysis. Most subjects were livestock owners (39.13%) in rural settings (73.91%). Most patients (73.19%) were pet owners (dogs) and used water from open sources for drinking. The liver was the most affected organ (52.17%), followed by the lungs (17.39%). Sequence analysis based on the cox1 gene revealed that EG genotype 1 (G1) was the most prevalent (56.52%), followed by G3 (34.78%), while some samples (8.7%) were identified as the Echinococcus canadensis (G6/7) genotype. A total of five haplotypes were detected with high haplotype diversity (0.80) and low nucleotide diversity (0.033). Phylogenetic analysis revealed two diverse sub-clades, each of G1 and G3 isolates from Baluchistan, that were evolutionarily related to previously reported G1 and G3 isolates from Pakistan and China. On the other hand, the G6/7 isolates of this study were evolutionarily identical to the already reported G6/7 isolates from Pakistan, Turkey, and Kazakhstan. This study concludes that diverse G1 and G3 EG isolates are present in this part of Pakistan, while the G6/G7 genotype was reported for the first time from Baluchistan.

Cystic echinococcosis (CE) is a worldwide zoonotic public health issue. The reasons for this include a lack of specific therapy options, increasing antiparasitic drug resistance, a lack of control strategies, and the absence of an approved vaccine. The aim of the current study is to develop a multiepitope vaccine against CE by in-silico identification and using different Antigen B subunits. The five Echinococcus granulosus antigen B (EgAgB) subunits were examined for eminent antigenic epitopes, and then the best B-cell and Major Histocompatibility Complex MHC-binding epitopes were predicted. Most significant epitopes were combined to create an effective multi-epitope vaccine, which was then validated by testing its secondary and tertiary structures, physicochemical properties, and molecular dynamics (MD) modelling. A multi-epitope vaccine construct of 483 amino acid sequences was designed. It contains B-cell, Helper T Lymphocyte (HTL), and Cytotoxic T Lymphocyte (CTL) epitopes as well as the appropriate adjuvant and linker molecules. The resultant vaccinal construct had a GDT-HA value of 0.9725, RMSD of 0.299, MolProbity of 1.891, Clash score of 13.1, Poor rotamers of 0.9, and qualifying features with Rama favoured of 89.9. It was also highly immunogenic and less allergic. The majority of the amino acids were positioned in the Ramachandran plot’s favourable area, and during the molecular dynamic simulation at 100 ns, no notable structural abnormalities were noticed. The resultant construct was significantly expressed and received good endorsement in the pIB2-SEC13-mEGFP expressional vector. In conclusion, the current in-silico multi-epitope vaccine may be evaluated in-vitro, in-vivo, and in clinical trials as an immunogenic vaccine model. It can also play a vital role in preventing this zoonotic parasite infection.

Introduction: Echinococcosis is a neglected tropical zoonotic infection that affects both the human and livestock populations. In Pakistan, the infection is long-standing, but data on its molecular epidemiology and genotypic characterization in the southern Punjab region are limited. The aim of the current study was the molecular characterization of human echinococcosis in southern Punjab, Pakistan.Methods: Echinococcal cysts were obtained from a total of 28 surgically treated patients. Patients’ demographic characteristics were also recorded. The cyst samples were subjected to further processing to isolate DNA in order to probe the Nad1 and Cyt-b genes, followed by DNA sequencing and phylogenetic analysis for genotypic identification.Results: The majority of the echinococcal cysts were from male patients (60.7%). The liver was the most commonly infected organ (60.71%), followed by the lungs (25%), spleen (7.14%), and the mesentery (7.14%). Molecular and genotypic identification through sequencing and phylogenetic tree analysis showed that most of the cysts (24/28, 85.7%) were caused by the species Echinococcus granulosus sensu stricto (E. granulosus s.s.) (G1 and G3), followed by Echinococcus multilocularis (E. multilocularis) and Echinococcus canadensis (E. canadensis) (G6/G7) (3/28, 10.8%, and 1/28, 3.5%, respectively).Conclusion: The current study concluded that the majority of human infections were caused by E. granulosus s.s., followed by the E. multilocularis and E. canadensis species (G6/G7). Genotypic characterization among both human and livestock populations is needed to explore the genetic diversity of echinococcosis.