[Development and preliminary application of a multiplex PCR assay for simultaneous detection of four intestinal parasites in goats].

OBJECTIVE: To develop a multiplex PCR assay for simultaneous detection of four intestinal parasites, including Giardia duodenalis, Cryptosporidium parvum, Enterocytozoon bieneusi and Moniezia, and to preliminarily evaluate its detection efficiency. METHODS: Four pairs of specific primers were designed based on the conserved sequences of the corresponding genes of G. duodenalis (GenBank accession number: XM_001710026.2), C. parvum (GenBank accession number: XM_626998.1), E. bieneusi (GenBank accession number: KJ719492.1) and Moniezia (GenBank accession number: OM296991.1) retrieved from the GenBank database, and a multiplex PCR assay for simultaneous detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia was developed and optimized. A total of 116 fresh goat stool samples were collected from four goat farms in Zhanjiang City, Guangdong Province during the period from October to December 2022, including 96 samples used for evaluating the detection efficacy of the multiplex PCR assay, and 20 samples as baseline controls for sample testing. Genomic DNA extracted from 96 goat stool samples was tested using the single-target PCR assay and the developed multiplex PCR assay, and the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex PCR assay were evaluated for detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia DNA in goat stool samples with the single-target PCR assay as the gold standard. RESULTS: The multiplex PCR assay developed in this study allowed simultaneous amplification of specific gene fragments of G. duodenalis, C. parvum, E. bieneusi and Moniezia, with 1 400, 755, 314 bp and 585 bp in sizes, respectively, and the detection limit was 102 and higher copies of parasite DNA clones, while the multiplex PCR assay was negative for gene amplification of Schistosoma japonicum, Fasciola hepatica, Echinococcus granulosus, Blastocystis hominis and Homalogaster paloniae. Single-target PCR assay and the developed multiplex PCR assay were employed to test DNA samples extracted from 96 goat stool samples, and single-target PCR assay tested positive in 40 goat stool samples (41.67%), including 39 positive samples tested with the multiplex PCR assay, with a mean coincidence rate of 97.50% (39/40). The multiplex PCR assay tested positive for G. duodenalis DNA in 26 goat stool samples (27.10%), C. parvum DNA in 22 samples (22.90%), E. bieneusi DNA in 24 samples (25.00%), and Moniezia in 9 samples (9.40%), which was consistent with the detection using the single-target PCR assay. The sensitivity, negative predictive value, and positive predictive value of the multiplex PCR assay were 96.15%, 95.83%, 100.00% and 100.00%, 98.90%, 98.92%, 100.00% and 100.00%, 100.00%, 100.00%, 100.00% and 100.00% for detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia DNA in goat stool samples, respectively, if the single-target PCR assay served as the gold standard. CONCLUSIONS: A highly sensitive and specific multiplex PCR assay has been developed for simultaneous detection of G. duodenalis, C. parvum, E. bieneusi and Moniezia in goats, which is suitable for rapid, large-scale screening of intestinal parasites in sheep stool samples.

Environmental contamination with feces of free-roaming dogs and the risk of transmission of Echinococcus and Taenia species in urban regions of southeastern Iran.

BACKGROUND: Dogs are the most important definitive hosts of zoonotic taeniid helminths worldwide. Different Echinococcus and Taenia species of domestic and wild carnivores pose a potential risk to human population. High populations of free-roaming dogs (FRDs) in urban areas of Iran and widespread contamination of the environment with dog feces is a potential source of infecting people living in the urban regions with cystic echinococcosis (CE). Our knowledge on the risk of CE transmission in the urban settings in the endemic regions is limited. The present study surveyed the species and genotypes of E. granulosus sensu lato and other taeniids by examining feces of free-roaming dogs in the urban areas in the city of Kerman, southeastern Iran. METHODS: The city was divided into 100 consecutive blocks of which 25 blocks were randomly selected. Fecal samples of FRDs were counted, mapped and fresh samples were collected. Then Zinc chloride flotation, and sequential sieving was performed, and the samples were examined under an inverted microscope. Single individual taeniid eggs were isolated, partial nad1 gene was amplified and sequenced to identify species and genotypes. RESULTS: In total 5607 fecal samples of dogs were mapped and 83 fresh samples were collected. Taeniid eggs were detected in nine fecal samples (10.8%) from seven out of the 25 city blocks (28.0%). Echinococcus eggs were found in four samples (4.8%) from three city blocks, two samples containing E. granulosus sensu stricto (2.4%), two samples containing E. canadensis G6/7 (2.4%). In addition, three samples contained eggs of Taenia hydatigena (3.6%), and one sample of Taenia serialis (1.2%). CONCLUSIONS: This study documented the potential risk of CE transmission to humans resulting from the feces of dogs roaming freely in urban areas.

Effect of Hydroxyurea on Morphology, Proliferation, and Protein Expression on Taenia crassiceps WFU Strain.

Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.

The infection of Cysticercus fasciolaris in natural rats (Rattus species) residing in human residence areas, Nakhon Si Thammarat, Thailand.

Cysticercus fasciolaris (C. fasciolaris) is the larval stage of a cestode parasite named Taenia taeniaeformis (T. taeniaeformis). C. fasiolaris is found in small rodents, especially rats. Rattus species are listed as intermediate hosts of this parasite, and cats are the main definitive host of C. fasiolaris. The objective of this study was to study the pathological, microscopic, and molecular aspects of C. fasciolaris in rodents residing in human residence areas. One hundred and two rodents were trapped in human settlements and dissected for larva-containing cyst examinations in the body cavity. The larvae of C. fasciolaris were investigated using histopathological examination, microscopic observations under a stereomicroscope and scanning electron microscope, and molecular detection using polymerase chain reaction. The prevalence of hepatic cysts containing larvae was 8.91% (95% CI = 4.16-16.24). In addition, the older larvae also had longer micropapillae. Histopathological investigation revealed normal hepatic tissue containing larvae and a scanty fluid cyst. The cyst capsule contains mostly mononuclear cells and spindle cells in all infected rats. The molecular detection using two primer sets revealed the amplicons were similar to the clade of C. fasciolaris. In the future, more investigation is necessary to fully understand the parasite's molecular pathogenesis and virulent molecules, which are less obvious.

Taenia martis Neurocysticercosis-Like Lesion in Child, Associated with Local Source, the Netherlands.

A neurocysticercosis-like lesion in an 11-year-old boy in the Netherlands was determined to be caused by the zoonotic Taenia martis tapeworm. Subsequent testing revealed that 15% of wild martens tested in that region were infected with T. martis tapeworms with 100% genetic similarity; thus, the infection source was most likely local.

Parasitic findings on threatened pudu deer from Central Chile accounts first genetic characterization of lice parasitizing P. puda in Chile and the first molecular report of Taenia hydatigena metacestodes in this species.

Southern pudu (Pudu puda) is a threatened endemic deer of the temperate forests of Chile. In recent years pudu populations rates have decreased mainly due to anthropogenic causes including forest loss and landscape fragmentation. In this context, the parasitic fauna of Chilean pudu has been scarcely investigated. The aim of this study was to determine the parasitic status of rescued pudu n = 13 from its natural habitat in Central Chile (Maule region) during March 2022 and June 2023 by applying morphological, histopathological, and molecular analyses. As result, we report the presence of transmission of parasites from dogs to pudus as showed by the presence of metacestodes of the parasite Taenia hydatigena on omentum, liver, and pleura of pudus during postmortem examinations, being the first molecular report on the presence of this parasite on Chilean pudu. Meanwhile, ectoparasite examinations determined the presence of chewing and sucking lice on pudu exemplars here analysed. Molecular and phylogenetic analysis of lice revealed new insights on Bovicola and Anoplura lice parasitizing P. puda in Chile, equally being the first genetic characterization of lice parasitizing pudu exemplars in Chile. In addition, parasite loads of lice and metacestodes were analysed. However, no statistically significance was observed when comparing environmental and individual traits influence on parasite load variation. Overall, the study area is the northern limit of habitat distribution of this specie in Chile and we here provide novel information on pudu deer parasites, thus making a useful and valuable contribution to the parasitological knowledge on this threatened species.

Taenia martis in a white-headed lemur (Eulemur albifrons) from a zoological park in North Rhine-Westphalia, Germany.

We present the case of Taenia martis metacestode infection in a white-headed lemur (Eulemur albifrons) from a zoological park. A post-mortem examination was conducted on the unexpectedly perished animal and focal granulomatous pneumonia with metacestodic tissue was discovered. Identification of T. martis was conducted through amplification and sequencing of a 12S rRNA gene fragment. We discuss the possible sources of infection and underline the importance of this infection in public health and conservation.

Comparison of Kato-Katz, PCR and coproantigen for the diagnosis of Taenia solium taeniasis.

Four methods were compared for the diagnosis of human taeniasis caused by Taenia solium. Fecal samples from persons living in a T. solium endemic region of Madagascar were examined for taeniid eggs by the Kato­Katz method. Subsequently, samples positive (n = 16) and negative (n = 200) for T. solium eggs were examined by (i) amplification of the fragment of small subunit of the mitochondrial ribosomal RNA (rrnS) gene using conventional polymerase chain reaction (PCR) and (ii) a nested PCR of a fragment of the T. solium Tso31 gene. Additionally, 12 egg-positive and all egg-negative samples were tested for coproantigen detection. A further 9 egg-positive fecal samples were examined using both PCRs. Of the 12 egg-positive samples tested by PCRs and coproantigen methods, 9 (75%) were positive by rrnS PCR, 3 (25%) using Tso31-nested PCR and 9 (75%) by coproantigen testing. None of the 200 egg-negative fecal samples was positive in either rrnS or Tso31-nested PCR. Twenty of the 25 egg-positive samples (80%) were positive in rrnS PCR, and DNA sequencing of PCR amplicons was obtained from 18 samples, all confirmed to be T. solium. Twelve of the 25 egg-positive samples (48%) were positive in the Tso31-nested PCR, all of which were also positive by rrnS PCR. It is suggested that species-specific diagnosis of T. solium taeniasis may be achieved by either coprological examination to detect eggs or coproantigen testing, followed by rrnS PCR and DNA sequencing to confirm the tapeworm species in egg-positive or coproantigen-positive samples.

Global profiling of miRNA and protein expression patterns in rabbit peritoneal macrophages treated with exosomes derived from Taenia pisiformis cysticercus.

Infection of Taenia pisiformis cysticercus is very frequently found in lagomorphs and causes serious economic losses to rabbit breeding industry. T. pisiformis cysticercus has evolved numerous strategies to manipulate their hosts. The release of exosomes is of importance in the interaction between host and parasite. However, the mechanism by which T. pisiformis cysticercus evades the host immune system for long-term survival within the host remains unclear. Using small RNA sequencing and TMT labelling proteomic, we profiled the expression patterns of miRNAs and proteins in rabbit peritoneal macrophages treated with T. pisiformis cysticercus exosomes. Seven differentially expressed (DE)-miRNAs and six DE-proteins were randomly selected to validate the accuracy of the sequencing data by qRT-PCR or western blot. Functions of DE-miRNAs and proteins were analyzed using public data bases. And DE-miRNAs-DE-proteins correlation network were established. CCK-8 assay was used to evaluate the effect of exosomes on macrophages proliferation. Cell cycle of macrophages, isolated from T. pisiformis-infected rabbits, was determined using flow cytometry. A total of 21 miRNAs were significantly differentially expressed, including three worm-derived miRNAs. The expressions of miRNAs and proteins were consistent with the sequencing results. DE-miRNAs targets were related to cell proliferation and apoptosis. Exosomes treatment resulted in a decrease of macrophages proliferation. In vivo, T. pisiformis cysticercus significantly induced S phase cell arrest. Moreover, DE-proteins were related to production of interferon-gamma and interleukin-12, and immunoregulation. Correlation network analysis revealed a negative correlation relationship between DE-miRNAs and DE-proteins. Among them, novel334 and tpi-let-7-5p have potential regulatory effects on IL1ß and NFκB2 respectively, which imply that novel334-IL1ß/tpi-let-7-5p-NFκB2 axis may be an important way that T. pisiformis cysticercus modulates host immune response through exosomes. Further understanding of these potential regulatory mechanisms will contribute to clarify the mechanism of escape mediated by T. pisiformis exosomes.

Cryptic species Hydatigera kamiyai and other taeniid metacestodes in the populations of small mammals in Serbia.

BACKGROUND: Hydatigera (Cestoda: Taeniidae) is a recently resurrected genus with the description of a new species, Hydatigera kamiyai, a cryptic entity within the Hydatigera taeniaeformis species complex. Rodents are intermediate hosts and correct taxonomic identification of H. taeniaeformis sensu lato (s.l.) species is difficult without the use of molecular methods. The aim of this study was to identify and explore the genetic diversity of Hydatigera and other taeniid species. METHODS: Ten different small mammals species (856 individuals) (Rattus rattus, three Apodemus, three Arvicolinae and three Soricidae species) were examined from 2013 to 2023. Captured animals were visually examined for cysts and visible lesions. Two markers were used for amplification and sequencing: cox1 and 12S rDNA. RESULTS: Molecular analysis of cysts and visible lesions revealed four taeniid species: Hydatigera kamiyai, H. taeniaeformis sensu stricto (s.s.), Taenia martis and T. crassiceps. Hydatigera kamiyai was found in Apodemus flavicollis, A. agrarius, Microtus arvalis and Crocidrua leucodon, while H. taeniaeformis s.s. is registered in R. rattus. Hydatigera kamiyai cox1 sequences clustered with European populations and showed at least 25 nucleotid differences compared to Asian, African, Australian and one of our isolates of H. taeniaeformis s.s acquired from a rat, followed by large sequence distances (9.4% to 12.9%), indicating clear molecular distinction of two species. CONCLUSIONS: This is one of the few mitochondrial gene-based studies performed after the description of cryptic entities within the Hydatigera taeniaeformis s.l. complex and represents a valuable contribution to understanding of genetic diversity, host suitability and geographic distribution of these tapeworm species. Also, our study provides an important basis of molecular data from this part of Europe for further studies. We emphasize the importance of additional studies of intermediate hosts, especially rats from Europe and Apodemus spp. and voles from Asia and Africa.

Identification of wild rodents as intermediate hosts for Hydatigera taeniaeformis in Peru.

Hydatigera taeniaeformis is a cestode that uses felines and rodents as definitive and intermediate hosts, respectively. Its larval stage, or metacestode, infects a wide variety of rodent species and develops in the liver parenchyma into a cyst. The aim of this study was to evaluate the occurrence of H. taeniaeformis metacestode in various species of wild rodents from Peru. For this, the livers of 356 rodents were macroscopically examined for any parasitic form compatible with metacestodes. Metacestodes were identified by measuring characteristic morphological parameters, and the diagnosis was confirmed by molecular analysis of a fragment of the cytochrome c oxidase subunit 1 gene (cox1). Five rodents: two small-eared pygmy rice rats (Oligoryzomys microtis), two white-naped squirrels (Simosciurus nebouxii), and one pygmy rice rat (Oligoryzomys sp.) were infected with H. taeniaeformis metacestodes. The cox1 sequences from our metacestodes showed up to 100% identity with previous H. taeniaeformis sequences from the GenBank. These results demonstrated the occurrence of H. taeniaeformis in new intermediate hosts, as well as the first molecular contribution for H. taeniaeformis from Peru.

Subcutaneous Taenia crassiceps Cysticercosis in a Ring-Tailed Lemur (Lemur catta) in a Serbian Zoo.

BACKGROUND: Different rodent species serve as natural intermediate hosts for carnivore tapeworm Taenia crassiceps. However, this cestode occasionally infects various dead-end hosts including humans and other primates and may cause serious pathological implications with potentially fatal outcome. In this paper, we present subcutaneous cysticercosis caused by T. crassiceps, found in a previously healthy 17-years-old male ring-tailed lemur (Lemur catta) in a Serbian Zoo. CASE PRESENTATION: The animal was presented to a veterinarian with a history of periarticular subcutaneous swelling in medial right knee region. After fine needle aspiration revealed cycticerci-like structures, a surgery was performed for complete extraction of the incapsulated multicystic mass containing numerous cysticerci. Collected material was sent for parasitological, histological and molecular analysis. One month after surgery, the lemur died due to respiratory failure unrelated to cysticercosis. Based on morphological features of large and small hooks and characteristic proliferation of cysticerci, a metacestode of T. crassiceps was identified, which was confirmed after sequencing of obtained amplicons and comparing them to the GenBank database. CONCLUSIONS: This is one of the few reported cases of T. crassiceps cysticercosis in a ring-tailed lemur, and the first one in Serbia. This endangered species seem to be more sensitive for T. crassiceps than other non-human primates, which represents serious conservation challenge for captive animals. Due to zoonotic nature of the parasite, challenging diagnosis, severity of the disease, difficult treatment and possible fatalities, high biosecurity measures are of particular importance, especially in endemic regions.

The potential role of roaming dogs in establishing a geographically novel life cycle of taeniids (Echinococcus spp. and Taenia spp.) in a non-endemic area.

INTRODUCTION: Cystic Echinococcosis (CE) is endemic in humans and livestock in many pastoral communities in Kenya. The distribution of the disease is enhanced by several factors, including livestock trade, which has allowed for the spread of CE to non-endemic areas such as western Kenya. Dogs' roaming behaviour, with consequent contamination of the environment with intestinal parasites, could then lead to parasite establishment. This study examined dogs' infection levels with taeniid eggs and their potential role in contaminating the environment with intestinal parasites. METHODOLOGY: We selected sixteen ruminant slaughterhouses in Busia and Bungoma Counties, and around each slaughterhouse we identified ten homesteads owning free-roaming dogs. We administered a questionnaire on dog management practices to the homestead owner and collected a faecal sample from the dog's rectum. In homesteads around 8 of the 16 slaughterhouses, we collared dogs with a GPS tracker to assess their movement patterns. The faecal samples were examined microscopically following zinc-chloride sieving-floatation technique for the presence of taeniid eggs and other canine intestinal parasites. Polymerase Chain Reaction - Restriction Fragment Length Polymorphism of NADH dehydrogenase subunit 1 gene and sequencing were used to confirm taeniid eggs identified during microscopy. Additionally, the Coproantigen-ELISA was used to detect the presence of taeniid antigen in a sub-set of the faecal samples. RESULTS: Helminths detected in the 155 dogs sampled included hookworms (n = 92; 59.4%), ascarids (n = 15; 9.7%), and taeniids (n = 1; 0.6%). Through Copro-PCR, 13 eggs extracted from the sample of the only taeniid infected dog were sequenced and identified as E. canadensis (G6/7) [n = 1], Taenia multiceps [n = 1], and Taenia serialis [n = 6]; the remaining were indeterminate. Of the 77 faecal samples tested for E. granulosus sensu lato (s. l.) with the Copro-ELISA, 64 (83.1%) were negative, 12 (15.6%) were positive, while 1 (1.3%) was suspicious. The dogs travelled a median of 13.5 km daily, and 28 dogs visited the slaughterhouses during the 5-day recording period. CONCLUSION: The results indicate a relatively high carriage of zoonotic parasites by free-roaming domestic dogs in western Kenya, which poses a risk to human and livestock populations. We report for the first time a domestic lifecycle of Echinococcus canadensis and Taenia multiceps in western Kenya, as well as a presumptive sylvatic cycle of coenurosis by T. serialis. We recommend an extensive and ongoing Copro-antigen survey of dog faeces, broader assessment of dog parasites with zoonotic potential, adherence to slaughterhouse management practices, and dog-ownership programmes to highlight the importance of deworming and restricted dog movements.

Comparison of multiplex copro PCR with coproscopy followed by PCR on recovered eggs for the detection of Echinococcus granulosus and Taenia spp. infection in dogs.

Echinococcosis and taeniasis are important helminth diseases that carry considerable impact on human and animal health. Domestic dogs and other canids are definitive hosts for several parasites of this group, including Echinococcus granulosus, Taenia multiceps, T. ovis, T. hydatigena and E. multilocularis. Detection of infection in dog populations is imperative for estimating the risk to susceptible humans and animals, and for its mitigation through prevention measures in dogs, other animals and humans. To date, identification of taeniid eggs, antigens or DNA in fecal samples are the most practical diagnostic modalities available for canine definitive hosts. Although widely used for this purpose, there is limited information comparing copro PCR and combined coproscopy-PCR protocols for the detection of taeniids. In the current study, a widely used multiplex PCR was performed on a large number of dog fecal samples using DNA extracted directly from feces. The samples were also tested by fecal flotation and coproscopy, eggs were isolated from microscopically-positive samples and extracted DNA was tested using the same multiplex PCR. The total number of taeniid positive samples detected using both methods was 46/317 (14.5%), including 10/317 (3.2%) E. granulosus positive samples. Both copro PCR and coproscopy have identified an equal number of samples as taeniid positive (n = 32). However, for the purpose of identification to species level, the copro PCR was significantly more sensitive than coproscopy followed by PCR on isolated eggs (sensitivity 0.7 vs. 0.41, p = 0.012), with 32/317 (10.1%) and 19/317 (6%) positive samples identified, respectively. The difference in identification of E. granulosus was highly apparent, as the majority of the E. granulosus positive samples (8/10) were detected by the copro PCR only. Coproscopy and egg PCR have identified 5/317 (1.6%) positive samples not detected by the copro PCR, including only a single sample (0.3%) positive for E. granulosus. Adding these positive samples to those identified by the copro PCR did not significantly improve the overall sensitivity (p = 0.074). Therefore, using both copro PCR and coproscopy in parallel may not be advantageous for taeniid detection and identification, at least until the egg PCR is further optimized and performs better. These results should be weighed against the different advantages that coproscopy based approach may offer.

A rare case of human taeniasis caused by Taenia saginata with species undetermined cysticercosis.

Taeniasis and cysticercosis, which are caused by Taenia saginata, Taenia solium and Taenia asiatica, are zoonotic parasitic infections with a significant disease burden worldwide. There is consensus amongst experts that T. saginata is a common tapeworm that causes taeniasis in humans as opposed to cysticercosis. This case study of a middle-aged Tibetan man conducted in 2021 challenges the prevailing notion that T. saginata exclusively causes taeniasis and not cysticercosis by documenting symptoms and laboratory studies related to both taeniasis and multiple cysticercosis. The patient's medical record with the symptoms of taeniasis and cysticercosis was reviewed, and the tapeworm's proglottids and cyst were identified from the patient by morphological evaluation, DNA amplification and sequencing. The patient frequently experienced severe headaches and vomiting. Both routine blood screenings and testing for antibodies against the most common parasites were normal. After anthelmintic treatment, an adult tapeworm was found in feces, and medical imaging examinations suggested multiple focal nodules in the brain and muscles of the patient. The morphological and molecular diagnosis of the proglottids revealed the Cestoda was T. saginata. Despite the challenges presented by the cyst's morphology, the molecular analysis suggested that it was most likely T. saginata. This case study suggests that T. saginata infection in humans has the potential to cause human cysticercosis. However, such a conclusion needs to be vetted by accurate genome-wide analysis in patients with T. saginata taeniasis associated with cysts. Such studies shall provide new insights into the pathogenicity of T. saginata.

Identification and expression analysis of a new small ubiquitin-like modifier from Taenia pisiformis.

The small ubiquitin-like modifier (SUMO) plays important roles, with the SUMOylation pathway as one of its core components. In the present work, a single SUMO gene was initially identified from Taenia pisiformis and designated as TpSUMO. Bioinformatic analysis showed that the TpSUMO gene contained a 309 bp open reading frame (ORF), encoding 102 amino acids, and had a predicted molecular weight of ∼12 kDa. The amino acid sequence of TpSUMO was deduced and it shared 44.00% identity with human SUMO2 (HsSUMO2) and exhibited more than 97.78% identity with SUMOs from Taenia and Echinococcus. TpSUMO possessed a putative non-consensus site (FK11MG) within its N-terminus and a typical di-glycine (GG) motif at the C-terminus. Basic local alignment search tool (BLAST) analysis showed that only a single SUMO-related ortholog was present in each set of known genome data for fourteen tapeworm species. The precursor His-TpSUMO-FL, mature His-TpSUMO-GG and mutant His-TpSUMO-GGK11R proteins (∼18 kDa) were expressed in Escherichia coli Rosseta (DE3), and rabbit polyclonal anti-TpSUMO was generated with a high titer of 1.28 × 105. In vitro SUMOylation assay results showed that TpSUMO multimer formation in the His-TpSUMO-GG reaction could be catalyzed by the human SAE1/SAE2 and UBC9 conjugation system, but K11R mutation disrupted TpSUMO chain synthesis. Quantitative real-time PCR (qRT-PCR) further revealed that TpSUMO was ubiquitously expressed in different stages of T. pisiformis and in higher levels during an early development phase (day 14) of adult worms. Immunofluorescence localization showed that TpSUMO was detected in the bladder wall of cysticerci, in the testis in immature segment, and within eggs in the gravid proglottids. These findings indicated that TpSUMO is a new member of the SUMO protein family and may play a vital role in regulation of functions within proteins involved in worm growth and development.

Morphometry and genetic diversity pattern of Cysticercus tenuicollis, an important food-borne taeniid metacestode in goats in Bangladesh.

Cysticercus tenuicollis is a larval stage of Taenia hydatigena resulting in cysticercosis, and responsible for enormous economic loses, especially in livestock production. Here, we planned to determine the prevalence and explore genetic variation of C. tenuicollis isolated from goats based on small subunit ribosomal RNA (rrnS) and cytochrome oxidase subunit 1 (cox1). To do this, samples were collected from different slaughter houses of municipal areas such as Bramhapalli slaughterhouse, Jubileeghat slaughterhouse and Mesuabazar slaughterhouse at Mymensingh sadar, and tentatively identified by morphological and morphometrical analysis. To study genetic variation, DNA was extracted from C. tenuicollis, and amplified rrnS and cox1 genes using specific primers, and were sequenced. Among 1372 examined animals, 177 (12.9%) were infected with C. tenuicollis. Cysts were recovered from peritoneum (7.9%), liver (4.4%) and urinary bladder (0.6%) of the infected animals. Females (18.9%) and adults (20.7%) were significantly more susceptible than male (8.8%) and young (9.3%), respectively. Genetic analysis defined 8 distinct rrnS genotypes and 9 unique cox1 haplotypes among 20 C. tenuicollis isolates. The nucleotide diversities were 0.00283 and 0.00434 for rrnS and cox1 genes, respectively. Neighbor joining (NJ) trees of rrnS and cox1 gene were constructed and the studied sequences were clustered with reference sequences of T. hydatigena with strong nodal support (100%). To compare Bangladeshi isolates, a median joining network was constructed with the population from other geographical regions and hosts. This led to a clustering pattern, but the clusters were not built with unique geographical regions or hosts. In conclusion, this is the first study that describes the genetic variation of T. hydatigena population and suggests the existence of host-specific variants. Therefore, it is fundamental to dispose infected viscera, restrict dog movement and proper management of slaughter house for the prevention and control of cysticercosis.

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.

Global epidemiology and molecular biology of Taenia multiceps: a comparative meta-analysis and in silico analysis study.

In the present study, all published data on the epidemiology and molecular characters of Taenia multiceps were systematically collected from relevant databases (e.g. PubMed, Scopus, National Center for Biotechnology Information), and combined in various statistical and genetic analyses as a contribution to a better understanding of the epidemiology of this ubiquitous taeniid worldwide. While 5.8% of the key hosts (dogs) from various countries had T. multiceps, grey wolves displayed the highest prevalence (21.6%) among the definitive hosts. Small ruminants are the main intermediate hosts and carry the coenuri in various locations, but most commonly in the central nervous system (CNS). Cerebral coenuri were confirmed in 53% of sheep exhibiting neurological symptoms, and infected animals often had only a single coenurus in the brain. Sheep had a higher prevalence (8.8%) of CNS coenuri than goats (5.8%); however, extra-CNS coenuri were detected more frequently in goats than in sheep. In either case, the difference between sheep and goats was statistically insignificant. Analysis of 233 partial cytochrome oxidase subunit I nucleotide sections for T. multiceps revealed high haplotype and low nucleotide diversities. Fifty-one haplotypes were detected circulating in 6 geographic populations. China, Iran and Turkey had 2 major haplotypes, whereas Italy and Egypt shared 3. Haplotypes from Greece circulate worldwide, and displayed similar gene flow values when compared with the other populations. There were no distinct patterns for haplotype distribution in relation to the infected hosts or coenuri locations. The existence of genetic variants in T. multiceps was highlighted, but needs further studies.

Genomic palaeoparasitology traced the occurrence of Taenia asiatica in ancient Iran (Sassanid Empire, 2th cent. CE-6th cent. CE).

Palaeoparasitology investigates parasitological infections in animals and humans of past distance by examining biological remains. Palaeofaeces (or coprolites) are biological remains that provide valuable information on the disease, diet, and population movements in ancient times. Today, advances in detecting ancient DNA have cast light on dark corners that microscopy could never reach. The archaeological site of the Chehrabad salt mine of Achaemenid (550-330 BC) and Sassanid (third-seventh century AD) provides remains of various biotic and abiotic samples, including animal coprolites, for multidisciplinary studies. In the present work, we investigated coprolites for helminth eggs and larvae by microscopy and traced their biological agents' DNA by Next Generation Sequencing. Our results revealed various helminths, including Taenia asiatica, the species introduced in the 1990s. Implementing advanced modern molecular techniques like NGS gives a paramount view of pathogenic agents in space and time.