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Objective: Alveolar echinococcosis is caused by Echinococcus multilocularis, a parasite of zoonotic significance with a wide range of intermediate and final hosts, and the parasite survives successfully in diversified conditions. Plentiful studies have been done to study the genetic structure of the population of the parasite and the level of intimate kinship using mitochondrial (mt) DNA. The present study was conducted to investigate the population structure, genetic variation, and phylogenetic relationship of various isolates of E. multiocularis submitted to GenBank worldwide. Sequences of mt genes (mt-cytochrome c oxidase (cox1), mt-NADH dehydrogenase (nad1)) of E. multilocularis were analyzed to achieve the set goals. Materials and Methods: A total of 275 and 124 gene sequences of mt-cox1 and mt-nad1 belonging to E. multilocularis, respectively, were retrieved from the National Center for Biotechnology Information GenBank. The retrieved sequences were subjected to alignment with respective reference sequences using MEGA software. The PopArt software was used to establish median-joining networks, while DnaSp was used to calculate neutrality and diversity indices. MrBayes software was used to investigate the phylogenetic association between haplotypes based on Bayesian phylogeny. Results: Approximately 13 and 20 distinctive haplotypes of nad1 and cox1 genes, respectively, were observed in the present study. In both of the mt genes, diversity indices indicated low haplotype (mt-cox1 = 0.140; mt-nad1 = 0.374) and nucleotide (mt-cox1 = 0.00111; mt-nad1 = 0.00287) diversities. The values of Tajima's D and Fu Fs for a population of both of the genes under study were found to be negative. Conclusion: This study is a maiden attempt to provide insights into the population structure and genetic variation of E. multilocularis on a global scale. However, it is suggested that to better understand the population structure and genetic diversity of E. multilocularis, more geographical locations and amplifications of full-length gene sequences should be considered, which could be helpful in widening the insights into the genetic diversity of E. multilocularis.

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Helminths are considered a significant threat to the livestock industry, as they cause substantial economic losses in small and large ruminant farming. Their morbidity and mortality rates are also increasing day by day as they have zoonotic importance. Anthelmintic drugs have been used for controlling these parasites; unfortunately, due to the development of resistance of these drugs in helminths (parasites), especially in three major classes like benzimidazoles, nicotinic agonists, and macrocyclic lactones, their use is becoming very low. Although new anthelmintics are being developed, the process is time-consuming and costly. As a result, nanoparticles are being explored as an alternative to anthelmintics. Nanoparticles enhance drug effectiveness, drug delivery, and target specificity and have no resistance against parasites. Different types of nanoparticles are used, such as organic (chitosan) and inorganic (gold, silver, zinc oxide, iron oxide, and nickel oxide). One of them, silver nanoparticles (AgNPs), has unique properties in various fields, especially parasitology. AgNPs are synthesized from three primary methods: physical, chemical, and biological. Their primary mechanism of action is causing stress through the production of ROS that destroys cells, organs, proteins, and DNA parasites. The present review is about AgNPs, their mode of action, and their role in controlling anthelmintic resistance against small and large ruminants.

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Monieziasis is a parasite-borne production-limiting disease of livestock. Moniezia expansa is the most important species having cosmopolitan distribution. Despite of numerous prevalence reports, very little information is available about the evolutionary biology and population genetics of M. expansa. To close this research gap, this study was undertaken to recognize and inspect the genetic variation of M. expansa populations around the world using the cox1 and nad1 genes and deduce phylogenetic relationships with M. expansa populations. The cox1 and nad1 gene sequences were downloaded from the NCBI GenBank database. Followed by sequence alignment, median-joining networks were constructed using PopArt software. Diversity and neutrality indices were computed through DnaSp software while MEGA software was used to draw the maximum-likelihood phylogenetic tree. Thirty-two cox1 sequences, from five different countries, and 9 nad1 sequences from three different countries, were among the sequences used in this study. The cox1 and nad1 gene sequences had mutations in 97 and 36 different places, respectively. Twenty and 7 unique haplotypes were discovered for the cox1 and nad1 gene sequences, respectively. Comparable haplotype diversities were observed for both the genes under study (cox1 = 0.950; nad1 = 0.944). Negative Tajima's D and Fu Fs were found for the cox1 gene while these indices were positive for the nad1 gene. Phylogenetic analysis also showed the existence of unique haplotypes for both the cox1 and nad1 genes. The results of this study indicate that there is the existence of a huge genetic diversity in M. expansa isolates. For future studies, it is recommended that longer gene sequences should be used to describe genetic variation among M. expansa isolates as the length of the gene under study affects the genetic variation. Moreover, additional mitochondrial markers should also be investigated because the assertive strength of a group of gene targets is superior to defining genetic diversity.

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Fasciola hepatica is a trematode leading to heavy economic setbacks to the livestock sector globally. The population's genetic information and intimate kinship level are frequently assessed using analysis of mitochondrial DNA. In this analysis, we retrieved cox1 (n = 247) and nad1 (n = 357) sequences of F. hepatica from the NCBI GenBank database and aligned the sequences with the respective reference sequences using MEGA software. The median joining network was drawn using PopArt software while neutrality and diversity indices were estimated with the help of DnaSp software. Neighbor-joining phylogenetic tree was constructed using the MEGA software package. A total of 46 and 98 distinctive haplotypes were observed for cox1 and nad1 genes, respectively. Diversity indices indicated high haplotype and nucleotide diversities in both genes. Positive Tajima's D and Fu's Fs values were found for the entire population of both the genes under study. The cox1 and nad1 gene segments in this study showed high Tajima's D values, suggesting a low likelihood of future population growth. The Tajima's D value of the nad1 gene sequence is lower (2.14910) than that of the cox1 gene sequence (3.40314), which suggests that the former is growing at a slower rate. However, the region-wise analysis revealed that both the cox1 and nad1 genes showed deviation from neutrality suggesting a recent population expansion as a result of an excess of low-frequency polymorphism. Furthermore, the overall host-wise analysis showed positive and significant Tajima's D values for the cox1 and nad1 gene sequences. To the best of our knowledge, this is the first attempt to provide insights into genetic variations and population structure of F. hepatica at a global scale using cox1 and nad1 genes. Our findings suggest the existence of specific variants of F. hepatica in different parts of the world and provide information on the molecular ecology of F. hepatica. The results of this study also mark a critical development in upcoming epidemiological investigations on F. hepatica and will also contribute to understanding the global molecular epidemiology and population structure of F. hepatica.

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The larval forms of taeniid cestodes belonging to the genus Echinococcus are the source of the zoonotic infection known as echinococcosis. Alveolar and cystic echinococcosis are caused by Echinococcus multilocularis and Echinococcus granulosus (s. s), respectively. It is endemic in several regions of the world. In this systematic review, we describe diagnosis, and the species (human, canids, livestock, and small rodents) affected by cystic (CE) and alveolar echinococcosis (AE). From 1999 to 2021, we searched the online directory through PubMed, SCOPUS, Web of Science, and google scholar. Among the 37,700 records found in the online databases, 187 publications met our eligibility requirements. The majority of investigations employed a range of diagnostic methods, such as ELISA, imaging, copro-PCR, necropsy or arecoline hydrobromide purgation, morphological cestode confirmation, and fecal sieving/flotation to detect and confirm Echinococcus infection. ELISA was the most commonly used method followed by PCR, and imaging. The research team retrieved data describing the incidence or assessment of the diagnostic test for E. multilocularis in humans (N = 99), canids (N = 63), small ruminants (N = 13), large ruminants (N = 3), camel (N = 2), pigs (N = 2) and small mammals (N = 5). This study was conducted to explore the diagnostic tools applied to detect echinococcosis in humans as well as animals in prevalent countries, and to report the characteristic of new diagnostic tests for disease surveillance. This systematic review revealed that ELISA (alone or in combination) was the most common method used for disease diagnosis and diagnostic efficacy and prevalence rate increased when recombinant antigens were used. It is highly recommended to use combination protcols such as serological with molecular and imaging technique to diagnose disease. Our study identified scarcity of data of reporting echinococcosis in humans/ animals in low-income or developing countries particularly central Asian countries. Study reports in small rodents indicate their role in disease dissemination but real situation in these host is not reflected due to limited number of studies. Even though echinococcosis affects both public health and the domestic animal sector, therefore, it is important to devise new and strengthen implementation of the existing monitoring, judging, and control measures in this estimate.

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Cystic echinococcosis is a zoonotic disease of livestock having serious economic setbacks. The etiological agents of the disease belong to Echinococcus granulosus sensu lato. Despite of worldwide distribution of the disease, the molecular studies mainly employ amplification of cox1, nad1 and nad5 genes. To further strengthen the knowledge about significance of other molecular markers and to investigate the genetic diversity and population structure of Echinococcus species in Pakistan, the current study was designed in which full length mitochondrial cytb, atp6 and nad2 genes were amplified. Based on BLAST searches of the generated cytb, atp6 and nad2 gene sequences from a total of 18 hydatid cysts collected from cattle, 12 isolates were identified as E. granulousus G3 and 6 as E. granulosus (G1). The phylogeny inferred by the Bayesian method using nucleotide sequences of cytb-atp6-nad2 further confirmed their identity. The diversity indices indicated a high haplotype 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. To the best of our knowledge, the present study described the genetic variation of E. granulosus population for the first time in Pakistan using full-length cytb, atp6 and nad2 mitochondrial genes. The findings on the genetic variation of E. granulosus in Pakistan will constitute useful baseline information for future studies on the prevalence and population structure of E. granulosus based on full-length cytb, atp6 and nad2.

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Cryptosporidium is a primary cause of waterborne epidemics, despite being previously considered only an opportunistic pathogen. The disease is associated with significant economic losses in humans and animals that are brought on by diarrhea, which frequently causes dehydration. Contact with diseased people or animals, as well as polluted water, is the major cause of infection. Different drugs are used to control the parasites. Nitazoxanide (NTZ), which is an anti-protozoan and anti-viral drug, can be used to control helminths, viruses, and protozoan parasites as a broad-spectrum antibiotic and has been approved by the food and drug authority (FDA). However, the problem is the development of resistance over a period of time in these parasites. Nanoparticles have received significant attention as possible anti-parasitic agents in recent years. By directing medications to specific cellular locations, targeted drug delivery minimizes the side effects of medications. Nanoparticles have demonstrated effectiveness against different Cryptosporidium species. Nanoparticles loaded with NTZ are found to be an effective remedy for C. parvum in young ones and decrease the oocyst count shed in the stools. Additionally, silver nanoparticles have proven to be effective against C. parvum by releasing silver ions that breach the cell wall of the oocyst, causing the escape of intracellular contents and the destruction of sporozoites within the oocyst. Implementing tiny particles for the purification of consuming water from Cryptosporidium is an economical and environmentally sustainable process. However, the use of nanoparticles in medicine requires more research.

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Parasitic infestations are one of the major threats to the livestock industry in Pakistan. These have a negative impact on the production of domesticated livestock species. Paramphistomes belong to the superfamily Paramphistomoidea and are involved in infecting ruminants all over the world. To date, there was no information on mitochondrial DNA-based molecular characterization of Paramphistomum epiclitum from Pakistan. To close this research gap, this study was designed to provide insights into the epidemiology of Paramphistomum species. Paramphistomum epiclitum isolates were recovered from the rumen of small ruminants slaughtered at an abattoir located in Faisalabad city and animal demographics were recorded. DNA was extracted and mitochondrial cox1 was amplified and sequenced. Prevalence was calculated along with a 95% confidence interval in various groups. The chi-square test was applied to determine the association between different variables under investigation. A phylogenetic tree was constructed based on the Bayesian method. Population diversity indices were calculated using DnaSP 4.5 software. A total of 43 mutations were observed among 7 haplotypes. Negative values of Fu's Fs values, and Tajima's D indicated population expansion. Deworming, season, and grazing were the variables that significantly correlate (p < 0.05) with the prevalence of P. epiclitum. The high prevalence of P. epiclitum demonstrates that more studies are indeed needed to further understand the prevalence and distribution of P. epiclitum in definitive and all potential intermediate hosts in addition to intraspecies variation and relationship with populations from other locations.

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Echinococcosis is a serious public health issue that affects people and livestock all over the world. Many synthetic and natural products have been examined in vitro and in vivo on Echinococcus species but only a few are used clinically, however, they may cause some complications and side effects. To overcome these limitations, new horizons of herbal drugs to cure echinococcosis are opening with every passing day. To summarize the developments during the last 21 years, we conducted this review of the literature to identify medicinal herbs utilized throughout the world that have anti-Echinococcus activity. From 2000 to 2021, data were carefully obtained from four English databases: Science Direct, PubMed, Scopus, and OpenGrey. Botanical name, extraction technique, extract quantities, efficacy, duration of treatment, year of publication, and half-maximal inhibitory concentration (IC50) values were all well noted. Ninety-one published papers, with 78 in vitro and 15 in vivo, fulfilled our selection criteria. Fifty-eight different plant species were thoroughly tested against Echinococcus granulosus. Zataria multiflora, Nigella sativa, Berberis vulgaris, Zingiber officinale (ginger), and Allium sativum were the most often utilized anti-Echinococcus herbs and the leaves of the herbs were extensively used. The pooled value of IC50 was 61 (95% CI 60−61.9) according to the random effect model and a large degree of diversity among studies was observed. The current systematic study described the medicinal plants with anti-Echinococcus activity, which could be investigated in future experimental and clinical studies to identify their in vivo efficacy, lethal effects, and mechanisms of action.

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Third-world countries have a higher prevalence of food-related disorders than developed nations. Millions of people in underdeveloped countries are seriously at risk from the potential water supply contamination with protozoan diseases. Toxoplasma gondii is one of the important protozoans causing diseases in livestock and humans. Despite the standard tests for diagnosing this parasite and different treatment methods, the spread of these parasites is uncontrollable and rising every year due to other management disorders. In this review, we summarize etiopathogenesis and prevalence in Pakistan. We looked for papers reporting the seroprevalence of T. gondii in people and animals between 2000 and 2022 in different databases: PubMed, Google Scholar, ScienceDirect, Scopus, and Web of Science. Data on the seroprevalence of T. gondii in Pakistan's domestic animals (sheep and goats, horses, donkeys, mules, cattle, and buffaloes), domestic pets (cats and dogs), poultry and rodents, and humans were gathered. According to the findings, sheep had an estimated pooled seroprevalence of T. gondii that varied from 11.20 to 26.50 %, and goats from 24.50 to 38.40%. Whereas in buffalo the opposite trend was followed, and the prevalence was observed is 0% in 2022, in horses, donkeys, and mules, only one study was reported according to which a high prevalence was observed in mules (28.60%) followed by donkeys (23.50%) and horses (23.50%), in cats 38.5% prevalence was observed in a recent study and in dogs 28.43% observed, and in humans from 22 to 60%. Human beings are found to be the most affected species showing high prevalence among all. According to our findings, animals and pets not only serve as a reservoir for the parasite but also serve as a direct route for human infection with T. gondii. The diagnostic techniques used in the observed studies were mostly serological testing whereas only a few studies have only been observed with molecular testing. To know the exact pattern of the disease for its control, the trend of molecular and advanced testing should be adopted as it is more reliable. Moreover, to decrease the transmission chances of T. gondii to humans, it is crucial to manage T. gondii infections in non-human species.

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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.

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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.

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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.

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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.

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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.

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