Molecular identification of the rodent-borne pathogen Rodentolepis nana using the genetic markers of ITS-1, 18 S, and 28 S rDNA.

BACKGROUND: Rodentolepis nana (syn. Hymenolepis nana), the most common cyclophyllid tapeworm infecting rodents, is a well-studied gastrointestinal parasite in mice and belongs to the family Hymenolepididae. METHODS: The present study focuses on the molecular analysis for the nuclear genes (ITS-1, 18 S, and 28 S rDNA) used for the accurate recognition of the recovered Rodentolepis species. RESULTS: The annotated partial ITS-1, 18 S, and 28 S rDNA gene regions were deposited in GenBank (gbǀ MW310394.1, gbǀ MW327585.1, and gbǀ MW324479.1, respectively) and further used in the maximum likelihood method (ML) to clarify their genetic relationships at the species level. The interrogation sequence of R. nana was aligned and belonged to the family Hymenolepididae, in the same group as all Hymenolepis species, which were distinct from Cyclophyllidea cestodes, especially species belonging to Anoplocephalidae and Taeniidae. Sequence data support the paraphyly of Hymenolepis species. CONCLUSIONS: The phylogeny supports the availability of the ITS-1, 18 S, and 28 S rDNA genes as reliable genetic markers for evolutionary relationships.

Intestinal parasitosis, anaemia and risk factors among pre-school children in Tigray region, northern Ethiopia.

BACKGROUND: Intestinal parasitic infections (IPIs) and anaemia are major health problems. This study assessed the prevalence of intestinal parasitic infections, anaemia and associated factors among pre-school children in rural areas of the Tigray region, northern Ethiopia. METHODS: A community based cross-sectional study was conducted among 610 pre-school children in rural communities of Northern Ethiopia from June 2017 to August 2017. Stool specimens were examined for the presence of trophozoites, cysts, oocysts, and ova using direct, formal-ethyl acetate concentration, Kato-Katz, and Ziehl-Neelsen techniques. Haemoglobin was measured using a HemoCue spectrometer. RESULTS: Among the 610 participating pre-school children in the study, the prevalence of IPIs and anaemia were 58% (95% conference interval (CI): 54.1-61.9%) and 21.6% (95% CI: 18.5-25.1%), respectively. Single, double, and triple parasitic infections were seen in 249 (41, 95% CI: 37-45%), 83 (14, 95% CI: 11-17%), and 22 (3.6, 95% CI: 2.4-5.4%) children, respectively. Of the seven intestinal parasitic organisms recorded from the participants, Entamoeba histolytica/dispar was the most prevalent 220 (36.1%) followed by Giardia lamblia 128 (20.1%), and Hymenolepis nana 102 (16.7%). Mixed infections were common among G. lamblia, E. histolytica/dispar and Cryptosporidium spp. oocyst. Intestinal parasitic infection prevalence increased from 47% in children aged 6-11 months to 66% in those aged 48-59 months; the prevalence ratio (PR) associated with a one-year increase in age was 1.08 (95% CI: 1.02-1.14, p = 0.009). Age-adjusted prevalence was higher in children who had been dewormed (PR = 1.2; 95% CI: 1.00-1.4, p = 0.045), and lower in households having two or more children aged under five (PR = 0.76, 95% CI: 0.61-0.95, p = 0.015). Anaemia rose from 28% in children aged 6-11 months to 43% in those aged 12-23 months, then fell continuously with age, reaching 7% in those aged 48-59 months. Age adjusted, anaemia was more prevalent in households using proper disposal of solid waste (PR = 1.5, 95% CI: 0.1-2.10, p = 0.009) while eating raw meat (PR = 0.49, 95% CI: 0.45-0.54, p = 0.000), any maternal education (PR = 0.64 95% CI: 0.52-0.79, p = 0.000), and household water treatment (PR = 0.75, 95% CI: 0.56-1.0, p = 0.044) were associated with lower prevalence of anaemia. CONCLUSIONS: More than half of the children were infected with intestinal parasites, while anaemia prevalence was concentrated in the 12-23 month age group. This study has identified a number of potentially modifiable risk factors to address the significant prevalence of IPIs and anaemia in these children. Improvements in sanitation, clean water, hand hygiene, maternal education could address both short and long-term consequences of these conditions in this vulnerable population.

Morphological, Molecular, and Pathological Appraisal of Hymenolepis nana (Hymenolepididae) Infecting Laboratory Mice (Mus musculus).

Hymenolepis nana, typically a parasite found in conventionally established mouse colonies, has zoonotic potential characterized by autoinfection and direct life cycle. The objective of this study was to determine the rate of parasite infection in laboratory mice. The hymenolepidide cestode infected 40% of the 50 mice sampled. The rate of infection in males (52%) was higher than in females (28%). Morphological studies on the cestode parasite showed that worms had a globular scolex with four suckers, a retractable rostellum with 20-30 hooks, and a short unsegmented neck. In addition, the remaining strobila consisted of immature, mature, and gravid proglottids, irregularly alternating genital pores, lobulated ovaries, postovarian vitelline glands, and uteri with up to 200 eggs in their gravid proglottids. The parasite taxonomy was confirmed by using molecular characterization based on the sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (mtCOX1) gene. The parasite recovered was up to 80% identical to other species in GenBank. High blast scores and low divergence were noted between the isolated parasite and previously described H. nana (gb| AP017666.1). The phylogenetic analysis using the COX1 sequence places this hymenolepidid species of the order Cyclophyllidea.

The dwarf tapeworm Hymenolepis nana in pet rodents in Slovakia-epidemiological survey and genetic analysis.

Rodents are popular companion animals and are often kept as pets for children. However, they can be reservoirs of a variety of zoonotic pathogens. As little attention is being paid to the possibility of acquiring parasitic infections from pet rodents, the occurrence of Hymenolepis nana in rodents from pet shops and breeding clubs of Slovakia was surveyed, with parallel genetic analyses to type isolates from rodent species. In 2016-2018, pooled faecal samples from 119 boxes with 228 mice, 191 rats, 124 hamsters and 25 Mongolian gerbils were collected from 12 pet shops and 3 breeding clubs in five cities of eastern Slovakia. H. nana eggs were detected in 25 (21.0%) boxes. Animals from pet shops were infected more frequently (24.6% positive boxes) than those from breeding clubs (17.2%), without statistical significance. The highest prevalence was recorded in rats from pet shops, where 41.7% of boxes contained parasite eggs. Hamsters and mice in pet shops were also frequently infected; in 23.8% and 25% of boxes, respectively, H. nana eggs were observed. Prevalence in rats and hamsters from breeding clubs was lower, but in mice surpassed 40%. Nine samples with positive PCR products in any of the four DNA regions, mitochondrial cox1 and nuclear pmy, ITS1 and ITS2 targets, gave profiles characteristic of H. nana. The results imply the risk of zoonotic transmission of hymenolepiasis in Slovakia. Particular attention should be given to hygiene level maintained while keeping rodents. Furthermore, rodents intended for sale should be tested for parasites and then dewormed.

Molecular characterization of Hymenolepis nana based on nuclear rDNA ITS2 gene marker.

INTRODUCTION: Hymenolepis nana is a zoonotic tapeworm with widespread distribution. The goal of the present study was to identify the parasite in the specimens collected from NorthWestern regions of Iran using PCR-sequencing method. METHODS: A total of 1521 stool samples were collected from the study individuals. Initially, the identification of hymenolepis nana was confirmed by parasitological method including direct wet-mount and formalin-ethyl acetate concentration methods. Afterward, PCR-sequencing analysis of ribosomal ITS2 fragment was targeted to investigate the molecular identification of the parasite. RESULTS: Overall, 0.65% (10/1521) of the isolates were contaminated with H. nana in formalin-ethyl acetate concentration. All ten isolates were succefully amplified by PCR and further sequenced. The determined sequences were deposited in GenBank under the accession numbers MH337810 -MH337819. CONCLUSION: Our results clarified the presence of H. nana among the patients in the study areas. In addition, the molecular technique could be accessible when the human eggs are the only sources available to identify and diagnose the parasite.

Gene-based molecular characterization of cox1 and pnad5 in Hymenolepis nana isolated from naturally infected mice and rats in Saudi Arabia.

Mice and rats are animals commonly used in research and laboratory testing. Compared with other animal species, they harbor many more zoonotic agents. Hymenolepis nana (H. nana) is a common tapeworm that parasitizes both humans and rodents. Although this tapeworm is of socio-economic importance worldwide, information related to its mitochondrial genome is limited. The present study examined the sequence diversity of two mitochondrial (mt) genes, subunit I of cytochrome oxidase (cox1) and NADH dehydrogenase subunit 5 (pnad5), of H. nana in mice and rats from two geographical regions of Saudi Arabia (Makkah and Riyadh). Partial sequences of cox1 and pnad 5 from individual H. nana isolates were separately amplified using polymerase chain reaction (PCR) and sequenced. The GC contents of the sequences ranged between 31.6-33.5% and 27.2-28.6% for cox1 and pnad5, respectively. The genomic similarity among specimens determined via cox1 primer and pnad5 primer was 97.1% and 99.7%, respectively. Based on these primers, our data did not indicate any differences between H. nana from rat and mice isolates. Results demonstrated that the present species are deeply embedded in the genus Hymenolepis with close relationship to other Hymenolepis species, including H. nana as a putative sister taxon, and that the isolates cannot be categorized as belonging to two different groups with origins in Makkah and Riyadh.

Hymenolepis nana-An Emerging Intestinal Parasite Associated with Anemia in School Children from the Bolivian Chaco.

Tropical anemia can have multiple causes, whether socioeconomic, dietary, or infectious. In the Bolivian Chaco, soil-transmitted helminthiases (STH), malaria, and Chagas disease are potential infectious causes of anemia among school-aged children (SAC). Following years of preventive chemotherapy with mebendazole, the prevalence of STH among SAC living in that area is now negligible, whereas protozoan infections are still highly prevalent (81%); Hymenolepis nana is the most frequent intestinal helminth (∼13%). We present results of hemoglobin (Hb) assessment and the association between parasitic infections and Hb levels of that SAC population. Overall, 511 SAC (girls:boys ratio 1:1, mean age 9.4 years [95% confidence interval {CI}: 9.3-9.5]) had Hb levels measured by using a point of care testing (HemoCue® Hb 301 System; HemoCue, Angelhome, Sweden). The prevalence of anemia was 23% (117/511), with mean and median Hb level = 12.2 g/dL (95% CI: 12.1-12.3; range 9.2-15.4 g/dL). By multivariate analysis, H. nana infection was associated with an increased risk of anemia (odds ratio 2.9, 95% CI: 1.5-5.7, P = 0.002). Two samples (0.5%) were positive for Trypanosoma cruzi and none for Plasmodium spp. by polymerase chain reaction of the 439 children tested. Anemia is still a concern among SAC living in the Bolivian Chaco. Our findings call for a greater attention to fecal-oral emerging pathogens, such as H. nana, and highlight the importance of water, sanitation, and hygiene improvements for disadvantaged population such as those living in the Bolivian Chaco.

Prevalence of Hymenolepis nana and H. diminuta from Brown Rats (Rattus norvegicus) in Heilongjiang Province, China.

Hymenolepis nana and Hymenolepis diminuta are globally widespread zoonotic cestodes. Rodents are the main reservoir host of these cestodes. Brown rats (Rattus norvegicus) are the best known and most common rats, and usually live wherever humans live, especially in less than desirable hygiene conditions. Due to the little information of the 2 hymenolepidid species in brown rats in China, the aim of this study was to understand the prevalence and genetic characterization of H. nana and H. diminuta in brown rats in Heilongjiang Province, China. Total 114 fecal samples were collected from brown rats in Heilongjiang Province. All the samples were subjected to morphological examinations by microscopy and genetic analysis by PCR amplification of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal RNA gene. In total, 6.1% (7/114) and 14.9% (17/114) of samples were positive for H. nana and H. diminuta, respectively. Among them, 7 and 3 H. nana isolates were successfully amplified and sequenced at the COX1 and ITS2 loci, respectively. No nucleotide variations were found among H. nana isolates at either of the 2 loci. Seventeen H. diminuta isolates produced 2 different COX1 sequences while 7 ITS2 sequences obtained were identical to each other. The present results of H. nana and H. diminuta infections in brown rats implied the risk of zoonotic transmission of hymenolepiasis in China. These molecular data will be helpful to deeply study intra-specific variations within Hymenolepis cestodes in the future.

Differential diagnosis and molecular characterization of Hymenolepis nana and Hymenolepis diminuta (Cestoda: Cyclophyllidea: Hymenolepididae) based on nuclear rDNA ITS2 gene marker.

Given the widespread distribution and medical implication of members of the genus Hymenolepis, specific identification of the aetiological agent becomes imperative. For precise diagnosis of the species, molecular techniques such as PCR and RFLP of the nuclear ribosomal internal transcribed spacer 2 (rDNA-ITS2) gene marker were carried out. The results showed distinct restriction patterns for both Hymenolepis nana and Hymenolepis diminuta when digested with either of the enzymes RsaI, HaeIII or HhaI. The annotated rDNA-ITS2 sequences from the two species revealed differences in the length; the folded secondary structure also depicted clear demarcation between the two species with variations in length of the helices, pyrimidine-pyrimidine mismatches and sites where motifs occur. In phylogenetic analysis of the evolutionary relationship between the two species as well as with other members of the family Hymenolepididae, the species causing human hymenolepiasis were found to be distantly related as they diverged independently from the ancestral lineage.

Genetic variability among Hymenolepis nana isolates from different geographical regions in China revealed by sequence analysis of three mitochondrial genes.

Hymenolepis nana is a common tapeworm that parasitizes in the small intestine of rodent animals and humans. The present study examined the sequence diversity of three mitochondrial (mt) genes namely NADH dehydrogenase subunits 5 (nad5), small subunit ribosomal RNA (rrnS), and ATPase subunit 6 (atp6) of H. nana from mice in different geographical regions of China. A part of the nad5 (pnad5), complete rrnS and atp6 genes were amplified separately from individual H. nana isolates using polymerase chain reaction (PCR) and then sequenced. The sequences of pnad5, rrnS, and atp6 were 710 bp, 704-711 bp, and 516 bp in length, respectively. The A + T contents of the sequences were 70.1-73.5% (pnad5), 70.1-71.7% (rrnS), and 76.6-77.9% (atp6). Sequence variation within H. nana was 0-1.4% for atp6, 0-1.7% for rrnS, and 0-0.7% for pnad5. The inter-specific sequence differences between H. nana and Hymenolepis diminuta were significantly higher, which was 31.6-31.7% (pnad5), 16.1-17.6% (rrnS), and 26.5-27.1% (atp6). Phylogenetic analysis based on the combined three sequences using the maximum parsimony (MP) method supported that H. nana is a species complex or "cryptic" species. These findings demonstrated clearly the usefulness of the three mtDNA sequences for population genetics and systematic studies of H. nana of human and animal health significance.

The complete mitochondrial genome of the dwarf tapeworm Hymenolepis nana--a neglected zoonotic helminth.

Hymenolepis nana, commonly known as the dwarf tapeworm, is one of the most common tapeworms of humans and rodents and can cause hymenolepiasis. Although this zoonotic tapeworm is of socio-economic significance in many countries of the world, its genetics, systematics, epidemiology, and biology are poorly understood. In the present study, we sequenced and characterized the complete mitochondrial (mt) genome of H. nana. The mt genome is 13,764 bp in size and encodes 36 genes, including 12 protein-coding genes, 2 ribosomal RNA, and 22 transfer RNA genes. All genes are transcribed in the same direction. The gene order and genome content are completely identical with their congener Hymenolepis diminuta. Phylogenetic analyses based on concatenated amino acid sequences of 12 protein-coding genes by Bayesian inference, Maximum likelihood, and Maximum parsimony showed the division of class Cestoda into two orders, supported the monophylies of both the orders Cyclophyllidea and Pseudophyllidea. Analyses of mt genome sequences also support the monophylies of the three families Taeniidae, Hymenolepididae, and Diphyllobothriidae. This novel mt genome provides a useful genetic marker for studying the molecular epidemiology, systematics, and population genetics of the dwarf tapeworm and should have implications for the diagnosis, prevention, and control of hymenolepiasis in humans.

Malignant Transformation of Hymenolepis nana in a Human Host.

Neoplasms occur naturally in invertebrates but are not known to develop in tapeworms. We observed nests of monomorphic, undifferentiated cells in samples from lymph-node and lung biopsies in a man infected with the human immunodeficiency virus (HIV). The morphologic features and invasive behavior of the cells were characteristic of cancer, but their small size suggested a nonhuman origin. A polymerase-chain-reaction (PCR) assay targeting eukaryotes identified Hymenolepis nana DNA. Although the cells were unrecognizable as tapeworm tissue, immunohistochemical staining and probe hybridization labeled the cells in situ. Comparative deep sequencing identified H. nana structural genomic variants that are compatible with mutations described in cancer. Invasion of human tissue by abnormal, proliferating, genetically altered tapeworm cells is a novel disease mechanism that links infection and cancer.

The chromosomes of Rodentolepis nana (Siebold, 1852) Spasskii, 1954 obtained from naturally infected mice conventionally maintained in a Braziuan laboratory animal house.

The karyotype of Rodentolepis nana obtained from mice in Rio de Janeiro, Brazil, was described. The diploid chromosome number obtained by the division of embryonic cells was 2n = 12. The first and the third pairs presented subterminal centromeres and the other pairs were all acrocentric. The studied species differed in chromosome morphology when compared to previous description by Mutafova and Gergova (1994) in Bulgaria, suggesting an intraspecific variation.