Infection of sheep by Echinococcus multilocularis in Gansu, China: evidence from mitochondrial and nuclear DNA analysis.

BACKGROUND: In the normal life cycle of the parasite (Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China. METHODS: From 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed. RESULTS: The isolates displayed ≥ 98.87% similarity to E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis. CONCLUSIONS: As a result of the discovery of E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area.

Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, ß-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). ß-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

Past and present of diagnosis of echinococcosis: A review (1999-2021).

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.

Update on the genetic diversity and population structure of Echinococcus granulosus in Gansu Province, Tibet Autonomous Region, and Xinjiang Uygur Autonomous Region, Western China, inferred from mitochondrial cox1, nad1, and nad5 sequences.

The identification of additional Echinococcus granulosus sensu lato (s.l.) complex species/genotypes in recent years raises the possibility that there might be more variation among this species in China than is currently understood. The aim of this study was to explore intra- and inter-species variation and population structure of Echinococcus species isolated from sheep in three areas of Western China. Of the isolates, 317, 322, and 326 were successfully amplified and sequenced for cox1, nad1, and nad5 genes, respectively. BLAST analysis revealed that the majority of the isolates were E. granulosus s.s., and using the cox1, nad1, and nad5 genes, respectively, 17, 14, and 11 isolates corresponded to Elodea canadensis (genotype G6/G7). In the three study areas, G1 genotypes were the most prevalent. There were 233 mutation sites along with 129 parsimony informative sites. A transition/transversion ratio of 7.5, 8, and 3.25, respectively, for cox1, nad1, and nad5 genes was obtained. Every mitochondrial gene had intraspecific variations, which were represented in a star-like network with a major haplotype with observable mutations from other distant and minor haplotypes. The Tajima's D value was significantly negative in all populations, indicating a substantial divergence from neutrality and supporting the demographic expansion of E. granulosus s.s. in the study areas. The phylogeny inferred by the maximum likelihood (ML) method using nucleotide sequences of cox1-nad1-nad5 further confirmed their identity. The nodes assigned to the G1, G3, and G6 clades as well as the reference sequences utilized had maximal posterior probability values (1.00). In conclusion, our study confirms the existence of a significant major haplotype of E. granulosus s.s. where G1 is the predominant genotype causing of CE in both livestock and humans in China.

Phylogeny and population structure of Echinococcus granulosus (sensu stricto) based on full-length cytb-nad2-atp6 mitochondrial genes - First report from Sialkot District of Pakistan.

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.

Comparison of mitochondrial genetic variation of Taenia hydatigena cysticerci from China and Mongolia.

Parasitic infection is one of the many challenges facing livestock production globally. Cysticercosis tenuicollis is a common parasitic disease in domestic and wild ruminants (intermediate host) caused by the larval stage of Taenia hydatigena that primarily infects dogs (definitive host). Although genetic studies on this parasite exist, only a few describe the genetic variation of this parasite in Mongolia. Our aim was thus, to identify the mitochondrial differences in ovine isolates of Cysticercus tenuicollis entering China from Mongolia and comparison with existing Chinese isolates from sheep and goats based on the recently described PCR-RFLP method and mitochondrial genes of NADH dehydrogenase subunit 4 (nad4) and the NADH dehydrogenase subunit 5 (nad5). Sixty-nine isolates were collected during routine veterinary meat inspections from sheep that originated from Mongolia, at the modern slaughterhouses in Erenhot City, Inner Mongolia. Additional 114 cysticerci were also retrieved from sheep and goats from northern (Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region, and Gansu Province), western (Tibet Autonomous Region), and southern (Jiangxi Province and Guangxi Province) China. The PCR-RFLP approach of the nad5 showed nine mitochondrial subclusters A1, A2, A3, A5, A8, A9, A10, A11, and B of T. hydatigena isolates from sheep and goats from Mongolia and China. Meanwhile, haplogroup A1 RFLP profile was more widespread than other variants. These data supplements existing information on the molecular epidemiology of T. hydatigena in China and Mongolia and demonstrate the occurrence of similar genetic population structures in both countries.

Echinococcus granulosus sensu lato in animal intermediate hosts: What is with the organ location?

Organ or tissue tropism/preference in many parasites is essential for their establishment, survival and pathogenesis. In protozoan parasites e.g. Trypanosoma and Plasmodium, tissue tropism is associated with many important aspects such as transmission, treatment, and pathogenesis. In these parasites, tissue tropism is achieved by mechanisms such as sequestration, extravasation, transcellular migration, and vascular permeability. In contrast, little is known about the mechanism behind organ tropism/preference and the potential implication in parasitic metazoans like Echinococcus. Since intraspecies/genetic variation was described for Echinococcus, the organ preference of currently known species and subspecies among animal intermediate hosts remains unclear or perhaps poorly investigated. Here, analysis of 89,359 Echinococcus cysts from different animal intermediate hosts across 47 countries suggests a potential organ distribution pattern in different animal intermediate hosts. For example, E. granulosus s.s. (G1, G3) and E. canadensis (G7) were found more in the liver of sheep and pigs, respectively than in other organs, whereas E. ortleppi and E. canadensis G6 were significantly higher in the lung of cattle and camels, respectively. While the mechanism of organ tropism in Echinococcus and whether it is species/genotype-dependent, host-dependent, or a combination of both is still a subject of future investigations, further and detailed investigation could provide significant information that may be applicable in the diagnosis and treatment of organ-specific cystic echinococcosis or designing host or genotype-specific interventions.

PCR-RFLP assay confirms the existence of different mitochondrial lineages of Taenia hydatigena including a possible geographically restricted group.

Taenia hydatigena is a widespread tapeworm of canids (primarily dogs) that causes cysticercosis in ruminants (domestic and wild) and manifests as depression and weakness secondary to various hepatic damages and sometimes mortality in young animals, although, commonly encountered cases are asymptomatic. In most taeniids, genetic polymorphism has been found to impact host preferences, distribution, disease epidemiology and management. Recently, we identified two main mitochondrial lineages of T. hydatigena in China, and here, we examined the mitochondrial nad4-nad5 genes of T. hydatigena from China, Nigeria, Pakistan and Sudan to assess the intraspecies variation of isolates from these countries and also the distribution of the distinct mitochondrial groups. In addition to China, haplogroup B variant was found in Pakistan, while haplogroup A demonstrated a widespread distribution. We then designed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay using XmiI (AccI) and RsaI (AfaI) restriction enzymes to differentiate members of both haplogroups. This result provides more molecular evidence supporting the existence of distinct mitochondrial variants of T. hydatigena. The epidemiological significance of these different mitochondrial groups remains to be explored further. The current PCR-RFLP assay offers a useful molecular approach for investigating the genetic population structure of T. hydatigena in enzootic regions and in identifying/discriminating the different mitochondrial groups (haplogroups A and B).

A systematic review and meta-analysis on prevalence and distribution of Taenia and Echinococcus infections in Ethiopia.

BACKGROUND: Tapeworm infections are among the tropical neglected parasitic diseases endemically occurring in Ethiopia. This systematic review and meta-analysis aims at estimating the pooled prevalence and distribution of Taenia and Echinococcus infections in humans and animals from reports from Ethiopia. METHODS: The systematic search was conducted in four bibliographic databases (PubMed, Google Scholar, Africa Journal Online and Science Direct). Additional data were retrieved from grey literature. Studies that met the inclusion criteria were considered for the systematic review and meta-analysis. The meta-analysis was conducted using MetaXL add-in for Microsoft Excel. Heterogeneity and inconsistency were evaluated using Cochran's Q and I2 statistics, respectively. RESULTS: The study provides a country-based database of Taenia and Echinococcus infections consisting of 311 datasets from 201 publications which were mostly abattoir surveys; of these, 251 datasets were subjected to meta-analysis. Most of the studies were from Oromia (32.8%) followed by Amhara (22.9%) regional states. The pooled prevalence of cystic echinococcosis in intermediate and accidental hosts was calculated as 22% (95% CI 18-26%) and high study variability (Q = 24,420.65, I2 = 100%, P = 0.000). Moreover, a pooled prevalence of Echinococcus infections in final hosts was calculated as 33% (95% CI 20-48%) and low study variability (Q = 17.24, I2 = 65%, P = 0.001). Similarly, study subjects (human, cattle, sheep, goat and wolf) were infected by Taenia spp. with pooled prevalence of 3% (95% CI 2-4%) and moderate study variability (Q = 279.07, I2 = 89, P = 0.000). Meanwhile, the pooled prevalence of Taenia hydatigena, T. ovis and T. multiceps infections in intermediate hosts were calculated as 38%, 14% and 5%, respectively. The random effect meta-analysis of bovine cysticercosis showed a pooled prevalence of 7% (95% CI 5-9%) and high study variability was of (Q = 4458.76; I2 = 99%, P = 0.000). Significant differences in prevalence of Taenia and Echinococcus infections between study sites or different livestock origins have been reported. CONCLUSION: The study evidenced a comprehensive dataset on the prevalence and distribution of Taenia and Echinococcus infections at different interfaces by regions and hosts and hence can aid in the design of more effective control strategies.

Comparative Transcriptome Analyses of the Developmental Stages of Taenia multiceps.

Cerebral coenurosis, caused by the larvae of Taenia multiceps (Coenurus cerebralis), is a fatal central nervous system disease in sheep and other herbivores and occasionally humans. Comparative transcriptomic profiles of the developmental stages of the parasite remain unknown. In this study, RNA sequencing was used to determine the transcriptome profiles of different stages of the life cycle of T. multiceps, including Oncosphere, Coenurus cerebralis (Pro with Cyst), and Adult (Adu), as well as scolex-neck proglottids (Snp), immature-mature proglottids (Imp), and gravid proglottids (Grp) of the adult stage. A total of 42.6 Gb (average 6.1 Gb) Illumina pair-end reads with a 125-bp read length were generated for seven samples. The total number of differentially expressed genes (DEGs) in the various life stages ranged from 2,577 to 3,879; however, for the tissues of the adult worm, the range was from 1,229 to 1,939. Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs mainly participated in cellular and metabolic processes, binding and catalytic activity, genetic information processing, and environmental information processing. In addition, a large number of genes related to development and parasite-host interaction were identified. Quantitative reverse transcription-polymerase chain reaction confirmed that the levels of 28 selected DEGs were consistent with those determined using RNA sequencing. The present study provides insights into the mechanisms of the development and parasitic life of T. multiceps.

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.

Echinococcus shiquicus in Qinghai-Tibet plateau: population structure and confirmation of additional endemic areas.

Echinococcus shiquicus is currently limited to the Qinghai­Tibet plateau, a large mountainous region in China. Although the zoonotic potential remains unknown, progress is being made on the distribution and intermediate host range. In this study, we report E. shiquicus within Gansu and Qinghai provinces in regions located not only around the central areas but also the southeast edge of the plateau and describe their genetic relationship with previous isolates from the plateau. From 1879 plateau pikas examined, 2.39% (95% CI 1.79­3.18) were infected with E. shiquicus. The highest prevalence of 10.26% (4.06­23.58) was recorded in Makehe town, Qinghai province. Overall the prevalence was marginally higher in Qinghai (2.5%, CI 1.82­3.43) than in Gansu (2%, CI 1.02­3.89). The cox1 and nad1 genes demonstrated high and low haplotype and nucleotide diversities, respectively. The median-joining network constructed by the cox1­nad1 gene sequences demonstrated a star-like configuration with a median vector (unsampled haplotype) occupying the centre of the network. No peculiar distinction or common haplotype was observed in isolates originating from the different provinces. The presence of E. shiquicus in regions of the southeast and northeast edges of the Qinghai­Tibet plateau and high genetic variation warrants more investigation into the haplotype distribution and genetic polymorphism by exploring more informative DNA regions of the mitochondrial genome to provide epidemiologically useful insight into the population structure of E. shiquicus across the plateau and its axis.

A new molecular nomenclature for Taenia hydatigena: mitochondrial DNA sequences reveal sufficient diversity suggesting the assignment of major haplotype divisions.

Cysticercosis caused by the metacestode larval stage of Taenia hydatigena formerly referred to as Cysticercus tenuicollis is a disease of veterinary importance that constitutes a significant threat to livestock production worldwide, especially in endemic regions due to condemnation of visceral organs and mortality rate of infected young animals. While the genetic diversity among parasites is found to be potentially useful in many areas of research including molecular diagnostics, epidemiology and control, that of T. hydatigena across the globe remains poorly understood. In this study, analysis of the mitochondrial DNA (mtDNA) of adult worms and larval stages of T. hydatigena isolated from dogs, sheep and a wild boar in China showed that the population structure consists of two major haplogroups with very high nucleotide substitutions involving synonymous and non-synonymous changes. Compared with other cestodes such as Echinococcus spp., the genetic variation observed between the haplogroups is sufficient for the assignment of major haplotype or genotype division as both groups showed a total of 166 point-mutation differences between the 12 mitochondrial protein-coding gene sequences. Preliminary analysis of a nuclear protein-coding gene (pepck) did not reveal any peculiar changes between both groups which suggests that these variants may only differ in their mitochondrial makeup.

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.

First report on the phylogenetic relationship, genetic variation of Echinococcus shiquicus isolates in Tibet Autonomous Region, China.

BACKGROUND: Cystic or alveolar echinococcosis caused by the larval stages of Echinococcus spp. is a very severe zoonotic helminth infection. Echinococcus shiquicus is a newly discovered species that has only been reported in the Qinghai and Sichuan provinces of the Qinghai-Tibet plateau, China where, to date, it has only been confirmed in Tibetan foxes and wild small mammal populations of the Tibetan plateau. Information on its genetic and evolutionary diversity is scanty. The aim of this study was to investigate the prevalence of E. shiquicus in plateau pikas (Ochotona curzoniae), a known intermediate host, and to determine the genetic variation and phylogenetic relationship of the E. shiquicus population in the Tibet region of China based on mitochondrial DNA. METHODS: Echinococcus shiquicus samples were collected from Damxung and Nyêmo counties (located in Tibet Autonomous Region, China). The mitochondrial cox1 and nad1 gene sequences were analyzed, and the genetic diversity and epidemiology of E. shiquicus in the region were discussed based on the results. RESULTS: The prevalence of E. shiquicus in pikas in Damxung and Nyêmo counties was 3.95% (6/152) and 6.98% (9/129), respectively. In combination with previous public sequence data, the haplotype analysis revealed 12 haplotypes (H) characterized by two distinct clusters (I and II), and a sequence distance of 99.1-99.9% from the reference haplotype (H1). The diversity and neutrality indices for the entire E. shiquicus populations were: haplotype diversity (Hd) ± standard deviation (SD) 0.862 ± 0.035; nucleotide diversity (Hd ± SD) 0.0056 ± 0.0003; Tajima's D 0.876 (P > 0.05); and Fu's F 6.000 (P > 0.05). CONCLUSIONS: This was the first analysis of the newly discovered E. shiquicus in plateau pikas in the Tibet Autonomous Region of China. The neutrality indices suggest a deficiency of alleles, indicative of a recent population bottleneck.

Trichinella spiralis Thioredoxin Peroxidase 2 Regulates Protective Th2 Immune Response in Mice by Directly Inducing Alternatively Activated Macrophages.

Trichinella infection can induce macrophages into the alternatively activated phenotype, which is primarily associated with the development of a polarized Th2 immune response. In the present study, we examined the immunomodulatory effect of T. spiralis thioredoxin peroxidase-2 (TsTPX2), a protein derived from T. spiralis ES products, in the regulation of Th2 response through direct activation of macrophages. The location of TsTPX2 was detected by immunohistochemistry and immunofluorescence analyses. The immune response in vivo induced by rTsTPX2 was characterized by analyzing the Th2 cytokines and Th1 cytokines in the peripheral blood. The rTsTPX2-activated macrophages (MrTsTPX2) were tested for polarization, their ability to evoke naïve CD4+ T cells, and resistance to the larval infection after adoptive transfer in BALB/c mice. The immunolocalization analysis showed TsTPX2 in cuticles and stichosome of T. spiralis ML. The immunostaining was detected in cuticles and stichosome of T. spiralis Ad3 and ML, as well as in tissue-dwellings around ML after the intestines and muscle tissues of infected mice were incubated with anti-rTsTPX2 antibody. Immunization of BALB/c mice with rTsTPX2 could induce a Th1-suppressing mixed immune response given the increased levels of Th2 cytokines (IL-4 and IL-10) production along with the decreased levels of Th1 cytokines (IFN-γ, IL-12, and TNF-α). In vitro studies showed that rTsTPX2 could directly drive RAW264.7 and peritoneal macrophages to the M2 phenotype. Moreover, MrTsTPX2 could promote CD4+ T cells polarized into Th2 type in vitro. Adoptive transfer of MrTsTPX2 into mice suppressed Th1 responses by enhancing Th2 responses and exhibited a 44.7% reduction in adult worm burden following challenge with T. spiralis infective larval, suggesting that the TsTPX2 is a potential vaccine candidate against trichinosis. Our study showed that TsTPX2 would be at least one of the molecules to switch macrophages into the M2 phenotype during T. spiralis infection, which provides a new therapeutic approach to various inflammatory disorders like allergies or autoimmune diseases.

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.

Complete mitochondrial genome analysis confirms the presence of Echinococcus granulosus sensu lato genotype G6 in Nigeria.

Cystic echinococcosis (CE) is common in Africa especially in northern and eastern countries where it is highly prevalent and mainly caused by Echinococcus granulosus sensu stricto and Echinococcus granulosus sensu lato (G6/7). In a recent epidemiological survey in Nigeria, the G6/7 genotype was reportedly responsible for the majority of CE infection. In this study, considering the taxonomic challenges of the G6/7 genotype and the limitation of the cox1/nad1 genes in resolving the differences, we sequenced the complete mitochondrial (mt) genome of seven larval isolates of E. granulosus s.l. G6/7 haplotypes recently reported in Nigeria to correctly assign them to either (G6/7) genotype and to understand the mt genome variation with isolates from other geographical regions. On analysis, a total of 13,731 bp in a covalently closed circular molecule were realized. The total mt length was ≥10 bp than previously reported G6 and G7 mt genome sequences. Also, the median-joining network and phylogeny based on the 12 protein-coding sequences correctly identified them as G6 genotype. Since longer mt genome sequences have shown some advantage over individual genes in resolving taxonomic challenges, we confirm that the genotype responsible for the majority of CE infection in livestock in Nigeria is the G6 genotype and the availability of the complete mt genome from different Nigerian intermediate hosts will prove useful in future genetic population studies across the country and the West African sub-region.

Prevalence and distribution of Echinococcus spp. in wild and domestic animals across Africa: A systematic review and meta-analysis.

Cystic echinococcosis (CE), a worldwide zoonosis, is highly prevalent in Africa particularly in northern and eastern Africa where data are more abundant than other regions. However, harmonization of available data through systematic review and meta-analysis may foster improved transboundary cooperation for the control of CE in Africa. Using the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines, research articles (from 2000 to 2019) were retrieved from ScienceDirect, PubMed, African Journals OnLine and Google Scholar databases. A total of 98 studies of 806,624 animals from 13 countries comprising 264,016 goats, 247,326 sheep, 251,106 cattle, 28,314 camels, 4,764 buffaloes, 2,920 equids, 1,966 pigs, 408 wild boars and 50 Norway rats were available for systematic review and meta-analysis of pooled prevalence including 5,048 dogs, 345 lions, 220 hyenas, 94 wolves and 47 jackals/foxes analysed for Echinococcus infection. In total, 46,869 animals were infected and pooled prevalence of CE in intermediate hosts was highest in camels (17.1%; 95% CI: 12.1-22.8) and lowest in pigs (0.3%; 95% CI: 0.1-0.6). Results also showed uneven species/genotype distribution across the continent such that Echinococcus granulosus sensu stricto (G1, G3) constituted 74.45% of the total isolates from East Africa, E. canadensis (G6/7) accounted for 60.3% and 97.4% in North and West Africa, respectively, while 81.3% of E. ortleppi (G5) were recorded for southern Africa. The comparatively higher prevalence estimates for eastern and northern Africa than other regions indicate where efforts on CE management should now be given greater attention in Africa. Additionally, this study also advocates for better cooperation between countries within the same sub-region and the establishment of joint CE control programmes.