Molecular identification of Spirometra infections in companion animals and wildlife in Japan.

Spirometra infections in companion animals and wildlife in Japan have been diagnosed based on the morphology of the adult worms and eggs, and the etiological agent has been mainly ascribed to Spirometra erinaceieuropaei. However, recent studies have revealed that two other species, Spirometra mansoni and Spirometra asiana, coexist in Japan. Spirometra asiana is a new species recently discovered in Japan. Although morphological discrimination between these two species is difficult, molecular identification is useful. Therefore, to understand which species commonly parasitizes companion animals and wildlife in Japan, a preliminary study was performed based on mitochondrial DNA analysis. Eleven adult worms examined were identified as S. mansoni, suggesting that S. mansoni infects companion animals and wildlife commonly than S. asiana in Japan.

Development and evaluation of an immunochromatography-based point-of-care test kit for a rapid diagnosis of human cysticercosis.

Human cysticercosis is a life-threatening zoonotic disease caused by infection with larvae (cysticerci) of the pork tapeworm, Taenia solium. This can affect the nervous system causing chronic headache and intracranial hypertension, potentially leading to epileptic seizures and paralysis. The disease is found in developing countries, especially in Southeast and South Asia, Sub-Saharan Africa, and Central and South America where porcine cysticercosis is endemic and people have a habit of eating undercooked pork. An immunochromatography-based test (ICT) kit, using T. solium cyst fluid as antigen, was manufactured to detect anti-T. solium IgG antibodies in human serum. To evaluate the kit, we used 187 serum samples including 24 from proven/confirmed cysticercosis cases, 133 from cases with other parasitosis and 30 healthy controls. Diagnostic efficiencies were calculated. The sensitivity, specificity, and accuracy were 83.3%, 92.0%, and 90.9%, respectively. Moreover, the ICT was positive before treatment but became negative after treatment, implying that this kit is also useful for follow-up monitoring post-treatment. In conclusion, we have successfully developed and present preliminary evaluation of an easy-to-handle rapid diagnostic tool for human cysticercosis in the form of an ICT platform using as antigen fluid from T. solium cysticerci.

Origin of the pork tapeworm Taenia solium in Bali and Papua, Indonesia.

Global distributions of zoonotic pathogens have been strongly affected by the history of human dispersal and domestication of livestock. The pork tapeworm Taenia solium is distributed worldwide as the cause of neurocysticercosis, one of the most serious neglected tropical diseases. T. solium has been reported in Indonesia but only endemic to restricted areas such as Bali and Papua. Previous studies indicated the distinctiveness of a mitochondrial haplotype confirmed in Papua, but only one isolate has been examined to date. In this study, genetic characterization of T. solium and pigs in Bali and Papua was conducted to clarify the distributional history of the parasite. Mitochondrial haplotype network analysis clearly showed that Indonesian T. solium comprises a unique haplogroup which was the first to diverge among Asian genotypes, indicating its single origin and the fact that it was not introduced in the recent past from other area in Asia in which it is endemic. Although phylogenetic analysis based on the mitochondrial D-loop revealed multiple origins of pigs in Bali and Papua, the majority of pigs belonged to the Pacific Clade, which is widely dispersed throughout the Island Southeast Asia (ISEA) and Oceania due to Neolithic human dispersal. Given the results of our network analysis, it is likely that the Pacific Clade pigs played a key role in the dispersal of T. solium. The data suggest that T. solium was introduced from mainland Asia into Western Indonesia, including Bali, by modern humans in the late Pleistocene, or in the early to middle Holocene along with the Pacific Clade pigs. Introduction into New Guinea most likely occurred in the late Holocene through the spread of Pacific Clade pigs. Over time, T. solium has been eradicated from most of Indonesia through the middle to modern ages owing to religious and cultural practices.

Taenia solium, Taenia saginata, Taenia asiatica, their hybrids and other helminthic infections occurring in a neglected tropical diseases' highly endemic area in Lao PDR.

Most part of Southeast Asia is considered endemic for human-infecting Taenia tapeworms; Taenia solium, T. saginata, and T. asiatica. However, until now there was no report of the occurrence of human cases of T. asiatica in Lao PDR. This study, conducted in Savannakhet Province, Lao PDR, microscopically examined a total of 470 fecal samples by Kato Katz method and found 86% of people harboring at least one helminth. Hookworms were detected in 56% of the samples besides Opisthorchis like eggs (42%), Trichuris trichiura (27%), Ascaris spp. (14%), and Taenia spp. (4%) eggs. Serology for cysticercosis showed 6.8% positives with results varying from 3% to 14.3% in Ethnic School students and Kalouk Kao village respectively. Species-specific PCR targeting mitochondrial DNA (mtDNA) of 28 tapeworms, recovered from 16 patients, revealed T. solium (n = 2), T. saginata (n = 21), and T. asiatica (n = 5). Two patients were confirmed to be coinfected with T. saginata and T. asiatica, indicating the endemicity of the 3 human Taenia in Lao PDR. However, nucleotide sequencing of a nuclear DNA gene, DNA polymerase delta (pold) revealed that all the tapeworms identified as T. asiatica using mtDNA had T. saginata type allele at pold locus, demonstrating that they are not "pure T. asiatica" but the hybrid descendants between the two species, confirming the wide distribution of hybrids of T. saginata/ T. asiatica in Southeast Asia. The high prevalence of several helminthic NTDs in east Savannakhet area even with conventional control measures indicates the importance to establish wide and multifaceted health programs to sustainably improve the quality of life of the populations living in these communities.

Genetic and morphological characterization of Thysaniezia tapeworms from cattle and sheep in Senegal.

Genetic and morphological diversity of Thysaniezia tapeworms from cattle and sheep in Senegal was investigated using light and scanning microscopic observations and molecular analysis based on mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear small subunit ribosomal RNA gene (SSU rDNA). A total of 52 adult tapeworms were collected from sheep and cattle. Although the tapeworms of the two hosts were morphologically very close, phylogenetic analysis based on cox1 and SSU rDNA gene sequences showed that they were divided into two clades corresponding each to a host. The maximum pairwise divergence between the clades were 12.1% in cox1 and 2.9% in SSU rDNA, indicating they are distinct species. The tapeworms collected from sheep were morphologically identified as Thysaniezia ovilla, a cosmopolitan species in domestic ruminants. Detailed morphological observations revealed a consistent difference between the tapeworms obtained from sheep and those from cattle. The latter were identified as Thysaniezia connochaeti. The present study highlights presence of two species of Thysaniezia among domestic ruminants in Senegal: T. ovilla specific to sheep and T. connochaeti specific to cattle. Our work is the first report of T. connochaeti from domestic animals.

The putative serine protease inhibitor (serpin) genes encoded on Echinococcus multilocularis genome and their expressions in metacestodal stage.

Two putative serpin genes were identified in Echinococcus multilocularis, in addition to the already reported serpinEmu, and were designated as serpin2Emu and serpin3Emu. Western blot analysis using polyclonal antibodies against serpinEmu, putative serpin2Emu protein, and putative serpin3Emu protein indicated that all three proteins were localized in both intracellular and excretory-secretory (ES) fractions of E. multilocularis metacestodes. In addition, immune staining of parasite tissue indicated that all three proteins were localized at the germinal layer.

Seroprevalence and risk factors of human cysticercosis and taeniasis prevalence in a highly endemic area of epilepsy in Bangoua, west Cameroon.

Cysticercosis caused by the larvae of Taenia solium is a serious and emerging threat to public health in the endemic areas as well as in the non-endemic areas.　Neurocysticercosis, an affection of the central nervous system is a leading cause of epilepsy in endemic areas. This study was carried out to investigate human cysticercosis, taeniasis and risk factors, and also their association with epilepsy in Bangoua, west Cameroon where epilepsy is highly prevalent. Out of 384 people investigated, 12 (3.1%) exhibited antibody response against low molecular weight antigens of T. solium by ELISA. Immunoblot revealed that six persons (1.6%) were seropositive with the same antigens. Among 61 epileptic patients, only one was seropositive by immunoblot and the study did not find any statistically significant difference (P>0.05) in seropositivity to T. solium between epileptic persons (1/61, 1.6%) and non-epileptic group (5/323, 1.5%). In addition, cysticercosis was associated with households eating pork meat from pigs slaughtered at home, but not with other factors. The risk factors including pig farming, the consumption of pork meat, vegetables, and non-drinkable water were attenuated by the relatively good hygiene and pig husbandry practices of the population. No egg of Taenia was found in stool by microscopic examination. All data obtained in this study suggested that cysticercosis might not be the principal causative agent of epilepsy in this area.

Serological validation of an alveolar echinococcosis rat model with a single hepatic lesion.

Serology is important for the diagnosis and follow-up of human alveolar echinococcosis (AE). However, patient conditions are highly variable among those with AE, and antibody responses in serological follow-up have not been well-defined. We recently described a new AE rat model established by implantation of small AE tissue into a single arbitrary location in the liver; no metastasis and dissemination were observed. In the present study, we examined the serological characteristics in our rat model before and after surgical treatment. The results showed that antibody responses against crude antigens were increased at one month after transplantation and similar to those of other model animals. For the antigen Em18, antibody responses were slower in our rat model than in other animal models. After surgical resection, changes in antibody responses against Em18 were similar to those observed in human patients with AE. Because of the slow growth of lesions, establishment of a single hepatic lesion and patterns of antibody responses, our rat model may be useful for clarifying follow-up serodiagnoses in human AE and determining the mechanisms of multi-organ involvement by primary infection with oncospheres rather than metastasis.

Swine cysticercosis in the Karangasem district of Bali, Indonesia: An evaluation of serological screening methods.

A serological assessment was undertaken on pigs from the Kubu and Abang sub-districts of Karangasem on the island of Bali, Indonesia, where earlier studies had detected patients with cysticercosis. Antigens purified from Taenia solium cyst fluid by cation-exchange chromatography were used to evaluate antibody responses in the pigs and the serological tests were also evaluated using sera from pigs experimentally infected with T. solium eggs. A total of 392 serum samples from naturally exposed pigs were tested using an ELISA that could be read based on both a colour change perceptible by the naked eye and an ELISA based on absorbance values. Twenty six (6.6%) pigs were found seropositive by the naked-eye ELISA and were categorized into three groups: strongly positive (absorbance values >0.8, n=6), moderately positive (absorbance values between 0.2 and 0.8, n=7), and weakly positive (absorbance values <0.2, n=13). Necropsies performed on 11 strongly and moderately positive pigs revealed that six strongly positive pigs were infected either solely with T. solium cysticerci (n=3), or co-infected with both T. solium and Taenia hydatigena (n=3). Four moderately positive pigs were infected solely with T. hydatigena. No cysticerci were found in one pig that was moderately positive by the naked-eye ELISA. Two experimentally infected pigs became antibody positive by 6 weeks post-infection, whereas eight control pigs remained negative. An additional 60 pigs slaughtered at authorized abattoirs on Bali were tested using the same ELISA. All 60 pigs were seronegative with no evidence of Taenia infection at necropsy. The results confirm the presence of porcine cysticercosis on Bali and, while the serological responses seen in T. solium infected animals were much stronger than those infected with T. hydatigena, the diagnostic antigens are clearly not species specific. Further studies are necessary to confirm if it is possible to draw a cut off line for differentiation of pig infected with T. solium from those infected with T. hydatigena.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical.

Comparative Study of Paired Serum and Cerebrospinal Fluid Samples from Neurocysticercosis Patients for the Detection of Specific Antibody to Taenia solium Immunodiagnostic Antigen.

Neurocysticercosis (NCC) is an important disease of the central nervous system caused by infection with Taenia solium metacestodes. In addition to the clinical findings and the imaging analysis, the results of immunological tests are informative for the diagnosis of NCC. To compare the usefulness of serum and cerebrospinal fluid (CSF) samples for antibody detection, paired serum and CSF samples from patients with NCC and other neurological diseases were examined by an enzyme-linked immunosorbent assay with low-molecular-weight antigens purified from T. solium cyst fluid in a blinded fashion. The sensitivity of both serum and CSF samples was 25.0% in inactive NCC cases (n = 4) and 90.9% in active NCC cases (n = 33), and the specificity of serum and CSF was 100% and 95.8%, respectively. When the serum and CSF samples were combined, the sensitivity in active NCC cases became 100%. There was no difference in test performance between serum and CSF samples. Based on these results, we recommend the detection of specific antibodies in serum for the diagnosis of active NCC because of the ease of collection. When the antibody test is negative, however, CSF should be used to confirm NCC and to rule out other medical disorders of the central nervous system. Antibody detection test using only serum or CSF has a limited diagnostic value and cannot be recommended for the diagnosis of suspected inactive NCC cases.

The present situation and towards the prevention and control of neurocysticercosis on the tropical island, Bali, Indonesia.

Neurocysticercosis (NCC), which is caused by accidental ingestion of eggs of the pork tapeworm, Taenia solium, was common in Bali, Indonesia until the early 1990s. However, improved education on hygiene and sanitation, a move to keeping pigs indoors, and improvement of economic and living conditions have substantially reduced the occurrence of NCC in Bali. Since 2011, T. solium tapeworm carriers (T. solium taeniasis) and heavily infected pigs and dogs have exclusively been detected from villages in mountainous regions of northeastern Bali where NCC and ocular cysticercosis (OCC) cases have also been identified. In response to this continued area of high infection, a one-day workshop was convened to discuss how to prevent and control this potentially lethal zoonotic parasitic infection in Bali. This review presents an overview of the current status of T. solium taeniasis and cysticercosis in Indonesia and proposes a strategy for the prevention and control of this zoonosis in Bali.

Genetic characterization of Moniezia species in Senegal and Ethiopia.

Genetic diversity of Moniezia spp. from domestic ruminants in Senegal and Ethiopia was investigated based on the nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear small subunit ribosomal RNA gene (SSU rDNA). A total of 64 adult tapeworms were collected from sheep, goat and cattle, and the tapeworms from cattle were all morphologically identified as Moniezia benedeni. On the other hand, the tapeworms obtained from sheep and goat were identified as Moniezia expansa or could not be identified because of the lack of diagnostic morphologic character, i.e. interproglottidal glands (IPGs). Phylogenetic analysis based on cox1 gene sequences revealed that the worms from sheep/goat and cattle formed distinct clades, and three mitochondrial lineages were confirmed within the sheep/goat tapeworms. The maximum pairwise divergences among the three mitochondrial linages were about 3% in cox1 and 0.1% in SSU rDNA, while that between the worms from sheep/goat and cattle reached 13% in cox1 and 2.7% in SSU rDNA. All of the three mitochondrial lineages contained tapeworms morphologically identified as M. expansa, and the tapeworms without IPGs were confirmed in one of the three lineages, indicating the tapeworms without IPGs were also M. expansa.

Genetics of the pig tapeworm in madagascar reveal a history of human dispersal and colonization.

An intricate history of human dispersal and geographic colonization has strongly affected the distribution of human pathogens. The pig tapeworm Taenia solium occurs throughout the world as the causative agent of cysticercosis, one of the most serious neglected tropical diseases. Discrete genetic lineages of T. solium in Asia and Africa/Latin America are geographically disjunct; only in Madagascar are they sympatric. Linguistic, archaeological and genetic evidence has indicated that the people in Madagascar have mixed ancestry from Island Southeast Asia and East Africa. Hence, anthropogenic introduction of the tapeworm from Southeast Asia and Africa had been postulated. This study shows that the major mitochondrial haplotype of T. solium in Madagascar is closely related to those from the Indian Subcontinent. Parasitological evidence presented here, and human genetics previously reported, support the hypothesis of an Indian influence on Malagasy culture coinciding with periods of early human migration onto the island. We also found evidence of nuclear-mitochondrial discordance in single tapeworms, indicating unexpected cross-fertilization between the two lineages of T. solium. Analyses of genetic and geographic populations of T. solium in Madagascar will shed light on apparently rapid evolution of this organism driven by recent (<2,000 yr) human migrations, following tens of thousands of years of geographic isolation.

Comparison of the serological tests ICT and ELISA for the diagnosis of alveolar echinococcosis in France.

Serological diagnosis of alveolar echinococcosis (AE) is a key element for efficient patient treatment management. A rapid immunochromatography test kit (ICT) using the recombinant Em18 antigen (rEm18) was recently developed. The aim of our study was to assess this test on a panel of sera from French patients with alveolar echinococcosis and control patients. In a blind test, a total of 112 serum samples were tested including samples of AE (n = 30), cystic echinococcosis [CE] (n = 15), and polycystic echinococcosis [PE] (n = 1). For the comparison, 66 sera from patients with hepatocarcinoma, fascioliasis, toxocariasis, Caroli's disease, or autoimmune chronic active hepatitis were used. The diagnostic test sets we used were the rEm18-ICT and two validated ELISAs with rEm18 and Em2-Em18 antigens, respectively. For the ICT, 27/30 sera from AE patients, 4/15 sera from CE patients and the PE patient serum were positive. One serum from the control panel (toxocariasis) was positive for the ICT. The rEm18-ICT sensitivity (90.0%) and specificity (92.7%) for detection of Em18-specific antibodies confirmed it as a relevant tool for AE diagnosis. The rEm18-ELISA had a sensitivity of 86.7% and specificity of 91.5%, and the Em2-Em18-ELISA had a sensitivity of 96.7% and specificity of 87.8%. However, when AE patient sera are recorded as weak in intensity with the ICT, we recommend a double reading and use of a reference sample if the ICT is used for patient follow-up.

Cystic echinococcoses in Mongolia: molecular identification, serology and risk factors.

BACKGROUND: Cystic echinococcosis (CE) is a globally distributed cestode zoonosis that causes hepatic cysts. Although Echinococcus granulosus sensu stricto (s.s.) is the major causative agent of CE worldwide, recent molecular epidemiological studies have revealed that E. canadensis is common in countries where camels are present. One such country is Mongolia. METHODOLOGY/PRINCIPAL FINDINGS: Forty-three human hepatic CE cases that were confirmed histopathologically at the National Center of Pathology (NCP) in Ulaanbaatar (UB) were identified by analysis of mitochondrial cox 1 gene as being caused by either E. canadensis (n=31, 72.1%) or E. granulosus s.s. (n=12, 27.9%). The majority of the E. canadensis cases were strain G6/7 (29/31, 93.5%). Twenty three haplotypes were identified. Sixteen of 39 CE cases with data on age, sex and province of residence were citizens of UB (41.0%), with 13 of the 16 cases from UB caused by E. canadensis (G6/7) (81.3%). Among these 13 cases, nine were children (69.2%). All pediatric cases (n  =  18) were due to E. canadensis with 17 of the 18 cases (94.4%) due to strain G6/7. Serum samples were available for 31 of the 43 CE cases, with 22 (71.0%) samples positive by ELISA to recombinant Antigen B8/1 (rAgB). Nine of 10 CE cases caused by E. granulosus s.s. (90.0%) and 13 of 20 CE cases by E. canadensis (G6/7) (65.0%) were seropositive. The one CE case caused by E. canadensis (G10) was seronegative. CE cases caused by E. granulosus s.s. showed higher absorbance values (median value 1.131) than those caused by E. canadensis (G6/7) (median value 0.106) (p  =  0.0137). CONCLUSION/SIGNIFICANCE: The main species/strains in the study population were E. canadenis and E. granulossus s.s. with E. canadensis the predominant species identified in children. The reason why E. canadensis appears to be so common in children is unknown.

Recent situation of taeniasis in Mongolia (2002-2012).

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia's 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia.

Serological and molecular characteristics of the first Korean case of Echinococcus multilocularis.

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient's serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.

Genotypic relationships between Taenia saginata, Taenia asiatica and their hybrids.

Partial sequences of the DNA polymerase delta (pold) gene from Taenia saginata-like adult worms were sequenced. Phylogenetic analysis revealed that pold gene sequences were clearly divided into two clades, differing from each other in five to seven nucleotides. There is little doubt that T. saginata and Taenia asiatica were once separated into two distinct taxa as has been concluded in previous studies. On the other hand, most of the adult worms, which were identified as T. asiatica using mitochondrial DNA, were homozygous for an allele that originated from the allele of T. saginata via single nucleotide substitution. These results indicate that most of the adult worms, which had been called T. asiatica, are not actually 'pure T. asiatica' but instead originated from the hybridization of 'pure T. saginata' and 'pure T. asiatica'.

Loop-mediated isothermal amplification method for a differential identification of human Taenia tapeworms.

Loop-mediated isothermal amplification (LAMP), which employs a Bst DNA polymerase with strand-displacement activity and four primers (two inner primers and two outer primers) recognizing six distinct regions on the target DNA, is a highly sensitive, specific, simple, and rapid nucleotide amplification method. Moreover, because the Bst DNA polymerase resists much DNA polymerase inhibitors present in biological specimens, the LAMP method is suitable for the detection of infectious agents from clinical material such as fecal samples. Here, we describe the LAMP method which can differentially detect and identify human Taenia tapeworms, Taenia solium, T. saginata, and T. asiatica, using DNA specimens prepared from parasite tissue and human fecal sample.