An epidemiological survey of echinococcosis in intermediate and definitive hosts in Qinghai Province, China.

In order to understand the epidemiological status of alveolar and cystic echinococcosis in intermediate and definitive hosts in Qinghai Province, China, during the period 2007-2011, we investigated the infection in humans and animals, including yaks, Tibetan sheep, Tibetan dogs, and wild foxes distributed in different counties around the province. Sera from local residents were examined using a rapid serodiagnostic kit to detect specific antibodies against Echinococcus. Seropositive samples were confirmed with B-scan ultrasonography and X-ray examinations. Yaks and Tibetan sheep were checked at slaughterhouses, and cysts and suspicious lesions were collected for analysis. A rapid diagnostic strip was used to detect Echinococcus adults in Tibetan dogs. Positive dogs were dewormed and the parasites collected. Wild foxes were trapped and necropsies performed with particular attention to the intestine. Forty-eight of 735 (6.4%) humans tested were positive and 475 of 854 (55.6%) Tibetan sheep and 85 of 352 (24.15%) yaks were infected with Echinococcus. Across different counties, 214 of 948 (22.57%) Tibetan dogs were positive, and five of 36 (13.9%) wild foxes were infected with Echinococcus. Molecular studies showed that all the infections detected in humans, domestic yaks, and Tibetan sheep were the G1 genotype (E. granulosus), whereas the parasites from Tibetan foxes and Tibetan dogs were E. shiquicus and E. multilocularis, respectively. In conclusion, Echinococcosis is hyperendemic in Qinghai Province in both its intermediate and definitive hosts and the G1 genotype of cystic Echinococcus is the dominant strain.

Campylobacter and Salmonella are prevalent in broiler farms in Kyushu, Japan: results of a 2-year distribution and circulation dynamics audit.

AIM: To elucidate the distribution and circulation dynamics of Campylobacter and Salmonella in Japanese chicken broiler flocks. METHODS AND RESULTS: A 2-year investigation of the distribution of Campylobacter and Salmonella was conducted in 25 broiler flocks at nine farms in Japan from 2013 to 2014. Campylobacter and Salmonella tested positive in 11 (44·0%) and 24 (96·0%) broiler flocks respectively. One hundred and ninety-five Campylobacter and 184 Salmonella isolates were characterized into 12 Campylobacter (including two novel genotypes) and three Salmonella MLST genotypes. Only Salmonella isolation between caecal and environmental samples were significantly correlated. Further, one litter sample tested positive for Salmonella before new chicks were introduced. The Campylobacter strains rapidly lost culturability within 2-18 days; in contrast, the Salmonella strains survived from 64-211 days in artificially inoculated water samples. CONCLUSION: No persistent circulation-mediated Campylobacter contamination was observed. In contrast, circulation of Salmonella in broiler houses was seen, apparently due to the litter excreted from broiler flocks, as well as Salmonella-contaminated water and feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the distribution, genotypic data and circulation dynamics of Campylobacter and Salmonella as recently observed in Japanese chicken broiler farms.

Molecular identification of the trematode Paragonimus in faecal samples from the wild cat Prionailurus bengalensis in the Da Krong Nature Reserve, Vietnam.

Conventional identification of Paragonimus species and their natural definitive hosts is based on the morphological features of adult parasites isolated from the lungs of wild mammalian hosts. However, wild animals are protected by strict regulations and sampling is not always possible. Recently, molecular techniques have been developed to identify the internal transcribed spacer (ITS) sequences of Paragonimus eggs in faeces/sputum of human patients. Also, mammalian hosts can be identified using the D-loop sequence of mitochondrial DNA in faecal samples. In this study, we used molecular techniques on faeces from wild animals collected in Da Krong Nature Reserve, Quang Tri province, central Vietnam, where Paragonimus metacercariae are highly prevalent in mountain crabs, to identify Paragonimus species and their natural definitive hosts. The results indicated that wild cats, Prionailurus bengalensis, were infected with at least three different Paragonimus species, P. westermani, P. skrjabini and P. heterotremus. Because all of these species can infect humans in Asian countries, human paragonimiasis should be considered in this area.

Increase of Clostridium perfringens in association with Eimeria in haemorrhagic enteritis in Japanese beef cattle.

A coprological survey with detailed clinical observation of naturally occurring haemorrhagic enteritis (HE) cases was conducted to understand the pathophysiology of HE by clarifying the infection status of Eimeria and enteropathogenic bacteria in cattle. Faecal samples from 55 cases of HE and 26 clinically normal animals were collected, and a quantitative examination of Eimeria and potential enteropathogenic bacteria was performed. The number of Eimeria species oocysts per gram of faeces (OPG) exceeded 10,000 in 69.1 per cent of HE cases with a maximum of 1,452,500 OPG and Eimeria zuernii was found to be overwhelmingly dominant. A significant increase in faecal coliform count was observed in HE cases compared with clinically normal animals. Among the animals shedding >10,000 OPG, 42.9 per cent showed a remarkable increase in Clostridium perfringens abundance (>104 CFU/g) in the faeces. In the cases with C. perfringens detected, its abundance was positively correlated with Eimeria OPG and high C. perfringens abundance was always accompanied by high Eimeria OPG. E. zuernii is likely to play a crucial role in massive multiplication of C. perfringens in HE in cattle.

Discovery of Paragonimus skrjabini in Vietnam and its phylogenetic status in the Paragonimus skrjabini complex.

Two members of the Paragonimus skrjabini complex, P. skrjabini and P. miyazakii, are now considered as two sub-species, P. skrjabini skrjabini and P. skrjabini miyazakii. They are well known as important pathogens for human paragonimiasis in China and Japan. Recently, members of this species complex have been reported from India. Here we report the first discovery of P. skrjabini from freshwater crab hosts in Thanh Hoa province, Vietnam. For morphological and molecular phylogenetic studies, adult worms were obtained by experimental infection in cats and dogs. Molecular analyses of metacercariae and adults revealed that the P. skrjabini population from Thanh Hoa, Vietnam was almost completely identical with that from Yunnan province, China. Those populations from Thanh Hoa, Vietnam and Yunnan, China and those from Manipur, India were significantly different from P. skrjabini populations reported from other localities of China in cytochrome oxidase subunit 1 (CO1) gene sequences, indicating considerable genetic variation within the P. skrjabini complex. Moreover, low bootstrap values in the CO1 tree suggested that more variant genotypes belonging to P. skrjabini complex may be found in other Asian countries in between Vietnam and India, such as Myanmar, Laos and Thailand. Since P. skrjabini is known as a pathogen for humans, paragonimiasis cases caused by P. skrjabini might be found in Vietnam and other Asian countries.

Genetically variant populations of Paragonimus proliferus Hsia & Chen, 1964 from central Vietnam.

Among about 50 nominal Paragonimus species, Paragonimus proliferus is rather a rare species, found only in Yunnan province, China, until our recent discovery of this species in Lai Chau province, northern Vietnam close to Yunnan, China. Here we add Quang Binh province, central Vietnam as a new endemic area of P. proliferus. Large excysted metacercariae found in mountainous crabs, Potamiscus tannanti, were morphologically identified as P. proliferus, which was confirmed further by molecular analyses. Second internal transcribed spacer (ITS2) sequences of the P. proliferus population in Quang Binh province were completely (100%) identical with those of P. proliferus populations in Lai Chau province, northern Vietnam and Yunnan province, China. However, cytochrome oxidase subunit 1 (CO1) gene sequences of Quang Binh population were significantly different (5.6%) from that of previously reported northern Vietnam and Yunnan, China populations. A phylogenetic tree revealed that all CO1 sequences of P. proliferus Quang Binh population formed a distinct group, which was clustered with northern Vietnam and Yunnan, China populations with the bootstrap value of 75%. This is the first record of the genetically variant population of P. proliferus, distribution of which is geographically remote from the previously reported endemic areas in the border between northern Vietnam and Yunnan, China, suggesting that P. proliferus may be much more widely distributed in the Indochina peninsula (or South-East Asia) than expected.

Coprological survey of alimentary tract parasites in dogs from Zambia and evaluation of a coproantigen assay for canine echinococcosis.

Faecal samples were collected from the rectum of 540 domestic dogs from four districts (Lusaka, Katete, Petauke and Luangwa) in Zambia between 2005 and 2006 and prevalences of canine alimentary tract parasites were determined by coprological examination. Thirteen different ova and parasites including strongyle (43.3%), Spirocerca lupi (18.7%), taeniid (13.1%), Toxocara canis (7.6%), Sarcocystis sp.* (7.5%), Isospora sp.* (5.7%), Physaloptera sp.* (4.6%), Capillaria sp.* (2.8%), Dipylidium caninum (2.2%), Mesocestoides sp.* (2.0%), Ascaris sp.* (1.7%), Trichuris vulpis* (0.4%) and Schistosoma mansoni* (0.4%) were detected, Ascaris and Schistosoma probably originating from coprophagy. The species with asterisks and later-described Taenia multiceps are for the first time reported from dogs in Zambia. A coproantigen enzyme-linked immunosorbent assay (CoproAg-ELISA) developed for Echinococcus spp. revealed 43 positive dogs and 37 of these harboured taeniid eggs. From 63 of the 71 taeniid egg-positive samples, eggs and DNA thereof were isolated and subjected to a multiplex polymerase chain reaction for differentiating E. granulosus sensu lato, E. multilocularis and Taenia spp. Amplicons indicative for Taenia spp. were obtained from 60 samples. Sequencing of amplicons spanning part of the mitochondrial cytochrome c oxidase subunit 1 gene, which was possible with 38 samples, revealed 35 infections with T. hydatigena and 3 with T. multiceps. Therefore, the CoproAg-ELISA showed some positives, but concrete evidence for the existence of canine E. granulosus infection could not be established. Comparison of the results of the CoproAg-ELISA and Taenia species identification indicated that the CoproAg-ELISA cross-reacts with patent infections of T. hydatigena (57%) and T. multiceps (33%).

Taenia taeniaeformis: fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats.

Fate and proliferation of gastric mucosal cells during hyperplasia of Taenia taeniaeformis eggs inoculated Wistar rats were investigated using PCNA immunohistochemistry, BrdU labeling and other histopathologic staining techniques. Results revealed marked cell proliferation in gastric corpus and antral mucosa of infected rats as evidenced by increased lengths of proliferative zones and indices of BrdU labeling. The gastropathy in corpus was characterized by massive accumulation of precursors, neck and intermediate cells following significant decreases in numbers of parietal and zymogenic cells. Gastropathy in antrum was described with significant increases in precursors and mucous cells. Our results suggested that T. taeniaeformis-induced gastric hyperplasia was initiated by depletion of parietal cells presumably due to the cestode's ES products. As a result, there was inhibition of zymogenic cell differentiation due to the disruption of normal development pathways of gastric mucosal lineages. These sequences of events were considered to cause the increase in cell proliferation and accumulation of intermediate cells resulting to the hyperplastic lesions.

Development of Taenia asiatica cysticerci to infective stage and adult stage in Mongolian gerbils.

The development of metacestodes and adult worms of Taenia asiatica in Mongolian gerbils (Meriones unguiculatus) were observed. Cysticerci were recovered from gerbils subcutaneously injected with hatched oncospheres. The recovery rate ranged from 0.1 to 3.2%. No cysticerci were recovered from the orally inoculated gerbils. The infectivity of the cysticerci recovered at 48 weeks post-infection was evaluated. Tapeworms were recovered on day 14 post-infection from the small intestine of 5 of 11 gerbils, with a recovery rate of 27% (6 worms recovered/22 worms inoculated). Three and four adult worms were recovered from two human volunteers who ingested five cysticerci after 4 months post-infection. In worms recovered from gerbils, segmentation and genital primordia in the posterior proglottids and hooklets in the residual rostellum were observed. The results indicate that gerbils can serve as an alternative intermediate host and that partial development of the adult worm stage occurs in gerbils.

Current control strategies targeting sources of echinococcosis in Japan.

The authors describe the current control strategies targeting definitive hosts of the most important zoonotic parasite in Japan, Echinococcus multilocularis. A dramatic increase in the prevalence of echinococcosis in foxes in Hokkaido (the second largest of Japan's islands), the invasion of wild foxes into urban areas, infection among pet and stray dogs, and the possibility of spreading the disease to the main island of Japan (Honshu)--all these pose significant threats to public health. Previous research findings and current strategies such as control measures against infections in wild foxes, suggest that it will be possible to eliminate echinococcosis in the future. The enforcement of a national reporting system for veterinarians, international collaboration, and the establishment of a Forum on Environment and Animals (FEA) give further reason to believe that success is possible. This is the first report of a multifaceted control strategy against echinococcosis in definitive hosts that includes collaborative efforts with local residents. This model might provide new ideas for Veterinary Services worldwide in their efforts to control other related zoonotic diseases.

Development of Taenia saginata asiatica metacestodes in SCID mice and its infectivity in human and alternative definitive hosts.

Development of Taenia saginata asiatica metacestodes in SCID mice, and its infectivity in humans, golden hamsters, and Mongolian gerbils as alternative definitive hosts, were investigated. Cysticerci were recovered from SCID mice that were subcutaneously injected with hatched oncospheres of T. s. asiatica. The morphological changes of metacestodes were observed. The recovered cysticerci were fed to gerbils, hamsters and humans, to check for their infectivity. Tapeworms were recovered from gerbils and hamsters fed with 20 to 45 week-old cysticerci, and proglottids excretions were observed in human volunteers fed with 45 week-old cysticerci. However, no tapeworms were recovered from gerbils fed with 10 week-old cysticerci. Our results suggest that T. s. asiatica oncospheres needed more than 20 weeks to develop to maturity in SCID mice to be infective to both their natural and alternative definitive hosts.

Potential remedy against Echinococcus multilocularis in wild red foxes using baits with anthelmintic distributed around fox breeding dens in Hokkaido, Japan.

The effect of bait-delivered anthelmintic to reduce the prevalence of Echinococcus multilocularis in wild red foxes was evaluated in Koshimizu, in the eastern part of Hokkaido, Japan. The study area (200 km2) was divided into baited and non-baited sections. The anthelmintic baits were distributed around fox den sites in the baited section every month for 13 months. After 1 year of the anthelmintic bait distribution, the prevalence of E. multilocularis in foxes, evaluated either by the parasite egg examination (from 27.1 to 5.6%) or coproantigen ELISA (from 59.6 to 29.7%), decreased in the baited section contrasting to that in the non-baited section (parasite egg: from 18.8 to 24.2%; ELISA: from 41.9 to 45.8%). The prevalence of E. multilocularis in grey red-backed vole Clethrionomys rufocanus, caught around fox dens, born after bait distribution also decreased and was significantly lower than that in non-baited section. However, within the study periods, the coproantigen-positive rate in fox faeces sporadically increased, while egg-positive rate constantly decreased. Since coproantigen ELISA can detect pre-patent infection, this observation indicates that reinfection pressure in the baited section was still high even after the 13 months of anthelmintic bait distribution. Therefore, the bait distribution longer than our study period is required for the efficient control of E. multilocularis in wild red fox population.

Assessment of the epidemiological status of Echinococcus multilocularis in foxes in France using ELISA coprotests on fox faeces collected in the field.

The aim of this study was to estimate the relevance of Echinococcus multilocularis coproantigen detection in fox faeces collected in the field to identify different levels of endemicity for Echinococcus multilocularis on a large scale (n x 10 km(2)). Six study sites were selected in a high endemicity area and two study sites in a low endemicity area in eastern France on the basis of landscape composition. Sampling was undertaken in the winters of 1996-97, 1997-98 and 1998-99. At each site, (i) necropsy and intestine examination was undertaken on a sample of shot foxes (total number of foxes, 222), and (ii) fox faeces were collected in the field along road verges, and scored for degradation status (total number of faeces, 625). Fox faeces were also sampled in a control area (n=30) in western France in the summer of 1998. Intestines were examined according to the sedimentation method. Echinococcus multilocularis coproantigens were detected by using two ELISA tests: EM-ELISA and EmA9-ELISA. The necropsy prevalence in high and low endemicity areas was 63.3% and 19.4%, respectively, and the distribution of adult worms in the fox population was highly overdispersed (75.5% of the total biomass was harboured by 11.6% of foxes). Using the two ELISA tests, there was no difference in the detection of E. multilocularis coproantigens in field faeces, regardless of the degradation status. The medians of EM- and EmA9-ELISA OD values of field faeces in high endemicity area were significantly higher than in low endemicity area (P<0.001 for both ELISA). The distribution of EM-ELISA OD values in low endemicity area was significantly higher (P=0.002) than in the control area. Moreover, for the two ELISA, the observed ELISA OD value distributions in high endemicity area, low endemicity area and control area seemed representative of the distribution of adult worms in fox populations. These results indicate that E. multilocularis coproantigen detection in field faeces could serve for large-scale surveillance, as an alternative to necropsy.

Morphometric analysis of the human trigeminal nerve.

We evaluated the components of nerve fibers of the motor and sensory roots of the trigeminal nerve by morphometric analysis. Trigeminal nerves were obtained from 5 cadavers (males, aged 67-95) were stained by Masson-Goldner-Goto method and examined under the microscope using a morphometric image-analyzer. The area and perimeter of axons were larger in the motor root than in the sensory root. The size distribution of axons was wider in the motor root than in the sensory root and the distribution pattern was unimodal type. These findings suggested that nerve fibers of the human trigeminal nerve gave similar findings to those of other peripheral nerves, regarding axonal size distribution and relative size of motor and sensory nerve axons.

A survey of canine echinococcosis in Gobi Altai Province of Mongolia by coproantigen detection.

Few studies have been carried out for the prevalence of canine echinococcosis in Mongolia. This study was designed to elucidate a preliminary information of the prevalence from feces collected in the field. Sixty-seven fecal samples from dogs and 2 red foxes in Altai town were collected and examined for Echinococcus coproantigen and eggs. Coproantigen detection was performed by a sandwich ELISA using a monoclonal antibody EmA9 raised against Echinococcus multilocularis somatic antigen. Of the dog samples examined, 17 (25.4%) were positive by the ELISA. One out of two foxes was positive, too. Taeniid egg-positive feces were recognized in 12 dog feces. Only 6 samples were both coproantigen and egg positive. Eggs of Ancylostoma sp., Trichuris sp.; and Capillaria sp.; were also registered.

Nerve fiber analysis and age-related changes of the human mandibular nerve.

We morphometrically analyzed nerve fibers of the human mandibular nerve with a discriminative staining method that makes it possible to separate nerve fibers into myelinated and unmyelinated fibers. We counted numbers and transverse areas of myelinated axons under the microscope using an on-line image-analyzer. This study revealed the morphometric changes which affect the human mandibular nerve during the aging process.

Inhibitory effect of different UV lamps on the infectivity of taeniid eggs.

The expanding distribution of alveolar echinococcosis in Hokkaido, Japan necessitates a search for control measures, particularly for killing Echinococcus eggs; and UV radiation is a potential tool. To determine the effective UV lamp and its optimum duration of exposure to inhibit the infectivity of taeniid eggs, three UV lamps (UVA, UVB and UVC) with peak emissions at different wavelengths were used to irradiate eggs of Taenia taeniaeformis. Irradiation was done in durations of 30, 90, 270, 810, 2,430 and 7,290 s. Infectivity of the irradiated eggs was tested by oral inoculation into rats. The inhibitory effect of the UV lamps at different exposure durations was determined by the reduction in the larval cyst establishment and by the delay of larval development. The UVC lamp (narrow peak at 254 nm) was found to be the most effective lamp at an exposure duration of more than 2,430 s (2,430 mJ/cm2), attaining 100% reduction. Also, shorter exposure durations produced a significant effect on the development of irradiated eggs in a greater percentage of small cysts (> 1 mm) among the metacestodes. Irradiation delayed the development of some cysts, as observed in histopathological sections. UVA and UVB lamps were not able to attain 100% reduction. It is concluded that the UVC lamp can be used as a tool in sterilizing taeniid eggs and is thus a promising method to control echinococcosis, although it may require a higher intensity of exposure.

Short report: seroprevalence of Taenia solium cysticercosis in pigs in Bahia State, northeastern Brazil.

To determine the prevalence and etiologic factors of Taenia solium cysticercosis in pigs in the state of Bahia, northeastern Brazil, we performed serological surveys in Salvador, the capital of the state, and in two local towns, Santo Amaro and Jequié. Residents raising free-roaming pigs in the study areas were randomly selected. Sera were obtained from the pigs and examined by the enzyme-linked immunoelectrotransfer blot assay (EITB). The prevalence of antibodies to T. solium was 4.4% (2 of 45) in Salvador, 3.2% (3 of 93) in Santo Amaro, and 23.5% (24 of 102) in Jequié. A significantly high seroprevalence in Jequié was associated with poor sanitary conditions, such as an open sewer system and no inspection process of pork before marketing.

First report of Trichinella nativa in red foxes (Vulpes vulpes schrencki) from Otaru City, Hokkaido, Japan.

Forty-three red foxes (Vulpes vulpes schrencki) and nine raccoon dogs (Nyctereutes procyonoides viverrinus) were captured in Otaru City, Hokkaido, Japan and examined by muscle digestion for the presence of Trichinella sp. larvae. Of the foxes, five (11.6%) were positive for larvae of Trichinella nativa while none of the raccoon dogs were found to be positive. This finding suggests that the red foxes are important reservoir hosts of sylvatic trichinellosis in Otaru, Hokkaido. This is the first report on Trichinella nativa in wild red foxes of Hokkaido, Japan.

Morphological differentiation of nerve fibers: central, peripheral, myelinated and unmyelinated.

We have developed a new technique for the morphological differentiation of various nerve fibers which is especially suitable for the morphometric study of nerve fibers of the human nervous system with the help of an image-analyzer. The knowledge from findings by this technique, which is based on several study methods, may be of importance in promoting further neuromorphologic studies and in properly understanding various aspects of neurological symptomatology and the aging process of the nervous system including nerve fibers.