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.

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.

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.

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%).

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.

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.

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.

Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay.

A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotinylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67,700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, showing a sensitivity of 100% and a specificity of 96%.

Time course of coproantigen excretion in Echinococcus multilocularis infections in foxes and an alternative definitive host, golden hamsters.

Coproantigen excretion during experimental infections of Echinococcus multilocularis in foxes and an alternative definitive host, golden hamsters, was evaluated by a sandwich ELISA using a monoclonal antibody. A sigmoidal increase of antigen excretion from the developing parasites was observed in in vitro incubation of the parasites collected on different days during the first 21 days post-infection (DPI). In hamsters, the ELISA O.D. value of faeces became positive at 4 DPI. Thereafter, the O.D. value increased in semi-sigmoidal fashion in the first 42 DPI, probably reflecting the development of the parasites. In foxes, the O.D. value became positive at 6 DPI. However, contrary to that in hamsters, after the initial steep rise, the O.D. value suddenly decreased to 1/2 the level during 15-17 DPI, indicating that a large number of worms might have been expelled. The parasite eggs were detected by the sugar centrifugal-flotation technique (Ito, Yagi & Ishige, 1989) from 29 to 84 DPI but not thereafter to 125 DPI, although mature parasites were detected at 125 DPI. In contrast, positive O.D. values were obtained almost constantly until 125 DPI, indicating that the coproantigen detection assay was more sensitive than the egg detection assay. The detection limit of the coproantigen assay was roughly estimated to be around 100 worms. These observations, along with the fact that the assay was designed to detect a heat-resistant coproantigen in heat-sterilized fecal samples, indicate that the coproantigen detection assay is a safe and useful method, not only for diagnosis in the definitive host of E. multilocularis, but also for monitoring parasite development and change in parasite burden during an experimental infection.

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.

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.

Development and sexual maturation of Echinococcus vogeli in an alternative definitive host, Mongolian gerbil (Meriones unguiculatus).

An alternative definitive host model for Echinococcus vogeli was developed using Mongolian gerbils. The animals treated with prednisolone tert-butylacetate were orally inoculated with protoscoleces of E. vogeli and the development of worms in the small intestine was observed until 50 days post infection (DPI). The parasites were recovered most frequently from the anterior part of the small intestine. The band formation (the sign of proglottisation) of the worm was observed at 7 DPI, the second proglottid and tests formation at 14 DPI, spermatozoa in seminal receptacle and cleavage of ova in uterus at 21 DPI, the hook formation of oncospheres at 28 DPI and the embryophore formation at 34 DPI. Eggs in the feces were first detected at 35 DPI. The coproantigen detection method developed for E. multilocularis could detect the E. vogeli coproantigen early in the infection, at 7 DPI during the prepatent period. Therefore, this technique could be used for the diagnosis of E. vogeli, replacing ordinary fecal egg examination.

Natural course of unoperated intracranial arteriovenous malformations: study of 50 cases.

The clinical course of 50 patients with conservatively treated intracranial arteriovenous malformations (AVM's) was followed, most of them for more than 5 years. The average follow-up period was 13.4 years. The initial symptom was intracranial bleeding in 29 patients (58%) and seizure in 15 patients (30%). Small and deep-seated AVM's were associated with a high incidence of bleeding; however, repeated hemorrhages were not necessarily indicative of a poor prognosis. Children younger than 15 years had a better prognosis than adults. There was no correlation between pregnancy and bleeding. In the hemorrhage group, the incidence of rebleeding was 6.9% in the 1st year after initial rupture, 1.91% per year after 5 years, and 0.92% per year after 15 years. The overall incidence of rebleeding was 34.5% in the hemorrhage group. Of the 50 patients, 37 (74%) had a good clinical outcome, four (8%) had a fair outcome, and four (8%) had a poor outcome; five patients died.

Coproantigen survey for Echinococcus multilocularis prevalence of red foxes in Hokkaido, Japan.

An epidemiological survey was conducted on the seasonal variation of Echinococcus multilocularis prevalence in red foxes from 1997 to 1998, using a monoclonal antibody-based detection of the tapeworm coproantigen. Thirty-six breeding dens of reproductive fox families were identified in the endemic area of Koshimizu, eastern Hokkaido, Japan. Fecal samples from each site were examined by coproantigen detection assay and fecal egg examination. Whereas the prevalence of coproantigen positive feces showed no seasonal fluctuation (51.6-66.7%), variation was found in the prevalence of egg positive feces in which a higher prevalence was observed in the summer and winter (31.1 and 38.7%) than spring and autumn (13.3 and 13.5%). Significant differences were observed between juveniles and adult foxes in both examinations. Samples from juvenile foxes gave higher coproantigen positive results and taeniid egg intensity. Those results suggest more juveniles infected with the cestode than adults in the same period. The practical use of coproantigen assay as a survey tool and factors which affect the prevalence and host age-related difference are discussed.

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.

TMC-2A, -2B and -2C, new dipeptidyl peptidase IV inhibitors produced by Aspergillus oryzae A374. II. Isolation and structure determination.

New dipeptidyl peptidase IV inhibitors, TMC-2A, -2B, and -2C, were isolated from the fermentation broth of Aspergillus oryzae A374. On the basis of chemical, spectroscopic and X-ray crystallographic analyses, their structures were established to be peptide-like compounds composed of three moieties, L-tryptophan, mono- or dihydroxy-L-leucine and highly substituted isoquinoline.

TMC-2A, -2B and -2C, novel dipeptidyl peptidase IV inhibitors produced by Aspergillus oryzae A374. I. Taxonomy of producing strain, fermentation, and biochemical properties.

TMC-2A(1), -2B (2) and -2C (3), novel dipeptidyl peptidase IV (DPIV) inhibitors, were isolated from the fermentation broth of Aspergillus oryzae A374. TMC-2A, -2B and -2C inhibited rat kidney DPIV with IC50 value of 8.1 microM, 17 microM, and 20 microM, respectively, as well as human DPIV prepared from mononuclear cells and adenocarcinoma cells. TMC-2 compounds inhibited only DPIV among the proteases tested, indicating their high selectivity for DPIV. The kinetic analyses revealed that TMC-2A was an uncompetitive inhibitor. Taxonomy and fermentation of the producing strain are also described.

TMC-66, a new endothelin converting enzyme inhibitor produced by Streptomyces sp. A5008.

A new endothelin converting enzyme (ECE) inhibitor, TMC-66 was isolated from the fermentation broth of Streptomyces sp. A5008. The structure of TMC-66 was elucidated by spectroscopic analyses to be a new member of benzo[a]naphthacenequinone class of antibiotics. TMC-66 had a highly selective inhibitory activity for ECE with an IC50 value of 2.9 microM. Taxonomy of the producing strain is also described.

In vitro maintenance of Eperythrozoon suis.

In vitro maintenance of Eperythrozoon suis was attempted using a Petri dish erythrocyte culture system. In preliminary experiments, the optimal conditions for maintaining E. suis attachment to erythrocytes during incubation were anticoagulation with heparin or citrate solution, incubation with 5 or 10% CO2 at 37 degrees C, and incubation with reduced or non-reduced Eagle's minimum essential medium. Using heparin, a CO2 incubator and reduced Eagle's medium (rEM), E. suis metabolic activity was evaluated by measuring glucose consumption, and lactate and pyruvate production. Glucose consumption and lactate production were measurable while pyruvate production was not detected. Erythrocyte integrity was improved by the addition of inosine although no effect was observed on maintenance of E. suis attachment to erythrocytes or the rate of glucose consumption. To determine whether the glucose consumption observed in culture was due to E. suis glycolytic activity or enhanced erythrocyte glycolytic activity, the effect of E. suis killing by EDTA addition to medium was evaluated using rEM containing inosine (rEMI). Glucose consumption decreased proportionally with the decline in the percentage of parasitized erythrocytes induced by EDTA, indicating that glucose consumption was due to E. suis. In a subsequent experiment, the effect of different types of serum (pig or fetal calf serum) and different gaseous environments (5% CO2 incubator or candle jar) were evaluated using rEMI. Glucose consumption by E. suis was significantly increased by the addition of fetal calf serum; however, no difference in the maintenance of E. suis attachment to erythrocytes and in E. suis glycolytic activity was observed between a 5% CO2 incubator and a candle jar. Finally, the effect of medium refreshment (rEMI containing fetal calf serum) was evaluated. Maintenance of E. suis parasitism on erythrocytes and E. suis glycolytic activity were significantly improved by frequent medium refreshment. The maintenance system developed enabled successful metabolic radiolabeling of E. suis for protein/antigen analysis.