Raillietina cesticillus infection causes reduced egg production in chickens in a windowless poultry house.

In a windowless poultry house raising layer chickens in Kanagawa prefecture, Japan, a slight increase in the mortality of chickens and a decrease in egg production were observed. Necropsy revealed numerous tapeworms and proglottids in chicken intestines. Histopathologically, gut-associated lymphoid tissues were observed in the lamina propria of the jejunum; however, no significant changes were observed in the other organs. Numerous hide beetles, Dermestes maculatus DeGeer, intermediate hosts of Raillietina cesticillus, were observed in the poultry house. Following a decline in beetle numbers, egg production increased and chicken mortality decreased. The life cycle of a tapeworm was easily established in a closed space, such as a windowless house, which led to severe infections.

Toxocara tanuki larval distribution in mice and the infectivity of tissue larvae.

Toxocara tanuki is a common large roundworm in raccoon dogs. Experimental infection studies of T. tanuki in mice were conducted to clarify the distribution and infectivity of larvae in tissue. Groups of BALB/c and C57BL/6 mice (n = 5 mice/group) were each inoculated with 1000 embryonated T. tanuki eggs and necropsied at 7, 31, 91, and 182 days post inoculation (dpi). The number of larvae in the central nervous system, heart, lungs, kidneys, spleen, gastrointestinal tract, liver, and carcass was examined. Larvae obtained from the aforementioned mice on different days of the necropsy were orally inoculated into four groups of ICR mice (n = 6 mice/group) that were then necropsied at 21 dpi. Larvae were recovered from all mice. In the BALB/c and C57BL/6 mice, most of the larvae (> 88.7%) were recovered from the liver and the remainder from other tissues. The total number of larvae recovered from C57BL/6 mice was significantly higher than that from BALB/c mice, but no difference in the relative larval distribution within the viscera between the two mouse strains was observed. The mean recovery percentage of larvae from ICR mice infected with 182-day-old tissue larvae was 3.3%. Our findings showed that T. tanuki larvae migrated predominantly to the liver of mice and that the larvae maintained their infectivity for at least half a year.

Detection of Toxocara species larvae in four Iranian free-range broiler farms.

BACKGROUND: The epidemiology of Toxocara canis and Toxocara cati in food animals, associated products, and their zoonotic potential are poorly understood. A cross sectional study was designed to determine the prevalence of Toxocara spp. larvae from free-range broiler chickens in traditional farms using conventional techniques and molecular method. Eight-hundred tissue samples including liver, gizzard, lungs and heart were collected from 200 chickens belonging to different regions of Zanjan Province, Iran and were processed by conventional and molecular methods. RESULTS: Out of 800 chicken tissues, 49 samples (6.1%) were positive for nematode larvae. Polymerase chain reaction was performed to identify species-specific of Toxocara larvae. The findings showed that 10.5% (21 out of 200) chickens were infected with Toxocara species, so that 57.1% (12 out of 21) of the samples were positive for Toxocara canis and 42.9% (9 out of 21) of the samples were positive for Toxocara cati. CONCLUSION: Considering the significant contamination/infection of free-range broiler chickens with Toxocara larvae, the consumption of chicken meat and viscera, especially liver and gizzards, can play an important role in the transmission of infection to humans. Prevention and control measures focused on regular deworming of dogs and cats, increasing public awareness of Toxocara infection are recommended.

Larva migrans in BALB/c mice experimentally infected with Toxocara cati ensured by PCR assay.

BACKGROUND: Toxocara cati, the cat roundworm, is a parasitic nematode that known to cause toxocariasis in intermediate hosts and humans. In this study, we characterized the dynamics of T. cati larvae migration in BALB/c mice after inoculation with eggs and ensured the migration detecting the larval DNA by a PCR. To evaluate the dynamics of larval migration and distribution, twenty-four BALB/c mice were orally inoculated with 2500 T. cati infective eggs and the visceral organs of the infected animals were examined by pepsin digestion and microscopic parasite counts, followed by PCR at day 1 to 28 post-inoculation. RESULTS: The PCR assays were successfully used for detection of T. cati larvae in tissue samples and T. cati larvae and the DNAs were found in the liver, lungs, heart, kidneys and the brain. We detected T. cati in 92.2% of tissue samples by PCR, 30% higher than the conventional pepsin digestion technique. CONCLUSION: Our findings demonstrated that the PCR assay is a sensitive and specific for the detection of T. cati larvae. Therefore, it could become a useful tool for the investigation of the dynamics of larval migration and Toxocara infection in murine model.

Seasonal and geographical differences in the ruminal microbial and chloroplast composition of sika deer (Cervus nippon) in Japan.

To understand the nutritional status of culled wild sika deer (Cervus nippon), we compared the ruminal microbes of deer living in habitats differing in food composition (Nagano winter, Nagano spring, and Hokkaido winter) using next-generation sequencing. Twenty-nine sika deer were sampled. Alpha and beta diversity metrics determined via 16S and 18S rRNA amplicon-seq analysis showed compositional differences. Prevotella, Entodinium, and Piromyces were the dominant genera of bacteria, fungi and protozoa, respectively. Moreover, 66 bacterial taxa, 44 eukaryotic taxa, and 46 chloroplastic taxa were shown to differ significantly among the groups by the linear discriminant analysis effect size (LEfSe) technique. Total RNA-seq analysis yielded 397 significantly differentially expressed transcripts (q < 0.05), of which 48 (q < 0.01) were correlated with the bacterial amplicon-seq results (Pearson correlation coefficient > 0.7). The ruminal microbial composition corresponded with the presence of different plants because the amplicon-seq results indicated that chloroplast from broadleaf trees and Stramenopiles-Alveolates-Rhizaria (SAR) were enriched in Nagano, whereas chloroplast from graminoids, Firmicutes and the dominant phylum of fungi were enriched in Hokkaido. These results could be related to the severe snow conditions in Hokkaido in winter and the richness of plants with leaves and acorns in Nagano in winter and spring. The findings are useful for understanding the nutritional status of wild sika deer.

Helminthes detected from wild sika deer (Cervus nippon centralis) in Kanto-Chubu region, Japan.

The helminth fauna of 105 sika deer (Cervus nippon centralis) captured in Yamanashi, Kanagawa and Nagano Prefectures, Japan was investigated during 2014-2019. As a result, 12 helminthes, i.e. 3 digeneans (Ogmocotyle sikae, Dicrocoelium chinensis and D. dendriticum), 8 nematodes (Gongylonema pulchrum, Dictyocaulus sp., Pygarginema sp., Spiculopteragia houdemeri, Chabaudstrongylus ninhae, Trichuris discolor, Oesophagostomum sikae and Oes. asperum), and 1 cestode (Moniezia sp.) were detected. To our knowledge, this is the first report of Pygarginema sp., Cha. ninhae, and Oes. asperum from sika deer in Japan. Some helminthes detected in the present study can infect livestock. Considering the possibility of the spread of the helminthes to livestock through deer excrement, it is important to promote understanding the parasite fauna in wild deer.

Trichinella pseudospiralis infection in red-eared slider, Trachemys scripta, influenced by environmental temperature.

Experimental infection with Trichinella pseudospiralis larvae in red-eared slider Trachemys scripta was carried out. Ten turtles were divided into 3 groups and kept at different temperature (38, 32 and 28 °C). The turtles were fed mice muscles which were infected with T. pseudospiralis larvae (444 - 23,013 larvae/mouse), kept in the aforementioned temperature and necropsied at day 30 after infection. At necropsy, the tongue, forelimbs, hindlimbs and spinal column muscles of turtles were removed separately for pepsin digestion, and the larvae counted. Larvae were found from all 3 turtles kept at 38 °C. Larvae per gram of muscle were highest in the tongue. No larvae were recovered from turtles kept at 32 and 28 °C. The result suggested that T. pseudospiralis is able to infect the red-eared slider in a high temperature environment. We speculated that environmental temperature play an important role in altering the physiological condition of the turtle to facilitate the infection of T. pseudospiralis.

Detection of larvae of Toxocara cati and T. tanuki from the muscles of free-ranging layer farm chickens.

Although raw or undercooked livestock meat or viscera has been suggested to be a source of human toxocariasis, there have been few reports on the prevalence of Toxocara larvae in the tissue of livestock animals. To investigate the presence of Toxocara larvae in chickens, we examined 50 culled chickens from a commercial layer farm. The liver, breast meat, and thigh meat were separated individually and artificially digested to examine for the presence of larvae. Nematode larvae were detected in 2 out of 50 chickens. One larva was detected from the breast meat, and it was molecularly identified as Toxocara tanuki. The other from the thigh meat of another chicken was molecularly identified as Toxocara cati. The present study demonstrated for the first time that T. tanuki larvae do infect chickens in the natural environment. The fact that Toxocara spp. larvae were found in muscles of farm chickens suggests that consumption of raw or undercooked chicken meat may present a risk for human toxocariasis.

Toxocara cati larval migration to mouse fetuses through transplacental infection.

To examine the Toxocara cati larval migration in mouse fetuses through vertical transmission, 7 pregnant mice were orally inoculated with 5000 embryonated eggs at day 4 and again with the same dose at day 9 of gestation (total 10,000 eggs / mouse). Seven non-pregnant mice of the same age were also inoculated likewise. All mice were necropsied 12 days after the first inoculation with eggs. The carcass, viscera, uterus, placenta and fetuses were individually removed and digested with pepsin for larval counts. Larvae were recovered from all pregnant and non-pregnant mice. The majority of the larvae were recovered from the carcass, and some from the viscera, uterus, placenta and fetuses. There were no differences between the pregnant and non-pregnant mice for the number of larvae in the carcass, viscera and uterus. Of a total of 103 fetuses from the 7 pregnant mice, larvae were recovered from 74 fetuses (71.8 %). The mean number of larvae recovered per infected fetus was 2.6 (n = 103), and the maximum number of larvae recovered in a fetus was 9. This study showed that most mouse fetuses were infected with T. cati larvae through intrauterine transmission, although the number of larvae was low. Since no significant difference in the number of larvae recovered from the pregnant and non-pregnant mice was observed, it is suggested that pregnancy does not directly affect the vertical transmission of T. cati larvae.

Prevalence of Blatticola blattae (Thelastomatidae) in German cockroaches Blattella germanica in Japan.

A survey for nematode infection in German cockroaches captured in restaurants in various areas of Japan as well as the laboratory-bred colony was carried out. The nematodes were then identified morphologically and molecularly. Of the 320 German cockroaches collected at 79 restaurants in 26 prefectures in Japan, 66.6% (213/320) were found to be parasitized by a single species of pinworm in the hindgut. The mean number of pinworms per cockroach was less than 1.6. Of the three laboratory-bred lines of cockroaches examined, 2 lines (NIID and NK) were found to be infected with a single species of nematode. The prevalence was 93.0% (40/43) and 84.8% (39/46), respectively. The other laboratory line (WAT) was found to be free of the nematode infection. The nematode detected in this study was identified as Blatticola blattae. This is the first report of B. blattae infection in German cockroaches in Japan. Our study showed that B. blattae is distributed all over Japan together with its host Blattella germanica. An experimental infection with B. blattae in nematode-free cockroach by contaminating the rearing environment with infected-cockroach feces showed that once the environment of the cockroach is contaminated with B. blattae eggs, the pinworm infection could spread easily.

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture, Japan revealed by ELISA using recombinant cathepsin L1.

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.

Isolation, Evaluation, and Identification of Angiotensin I-Converting Enzyme Inhibitory Peptides from Game Meat.

Game meat has been underutilized, while it offers the potential to diversify not only the human diet but also increase food production and the nutritional value of meat products. This study aimed to determine the angiotensin I-converting enzyme (ACE) inhibitory activities of the digested game meats (venison and boar meat) compared with those of livestock meats (beef and pork). Through the sodium dodecyl sulfate polyacrylamide gel electrophoresis and size chromatography results, we found that the digested products from each meat had different molecular weights. The ACE inhibitory ratio in all tested samples had gradually increased following by the enzyme treatments. ACE inhibitory ratios and the half maximal inhibitory concentration values indicated that digested venison was the most potent inhibitor of ACE activity, followed by the digested boar meat. The level of anserine in digested venison was higher than that in the other meats, but the carnosine level was lower. Through fractionations and liquid chromatography-tandem mass spectrometry analysis, five ACE inhibitory peptides were identified from the digested venison. Of these peptides, Isoleucine-Lysine- Glutamic Acid-Valine-Threonine-Glutamic Acid-Arginine (IKEVTER) demonstrated the highest ACE inhibitory activity. Therefore, the game meat is food that is believed potentially to offer high bioactivities, particularly antihypertensive forces.

Effects of anthelmintics on the pinworm Blatticola blattae in laboratory-reared German cockroaches Blattella germanica.

The efficacy of pyrantel pamoate, pyrvinium pamoate, ivermectin, and piperazine citrate against pinworm in cockroach was evaluated. Laboratory-reared German cockroaches naturally infected with Blatticola blattae were treated with the anthelmintics and necropsied at 3 to 35 days after treatment. Ivermectin at over 5 ppm and piperazine citrate at over 2000 ppm killed all the treated cockroaches. Pinworms were still detected in cockroaches given lower concentration of the aforementioned drugs. Administration of pyrantel pamoate (100-1000 ppm) and pyrvinium pamoate (2000 ppm) did not kill the cockroaches, and no pinworms were detected at 3 and 17 days after treatment. Thus, pyrantel pamoate and pyrvinium pamoate were found to be effective for deworming B. blattae in the German cockroaches, without causing mortality for the host. Our results showed that anthelmintics selection is essential for eradication of pinworms in cockroaches because of the toxicity for the host such as ivermectin or piperazine citrate. This is the first report of piperazine citrate toxicity in cockroaches.

Presence of Sarcocystis sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory and its phylogenetic relationship with Sarcocystis nipponi.

The first study reporting the morphological characterization of Sarcocystis sybillensis was performed in 1983; however, without any molecular analysis. Sarcocystis nipponi has been recently described as a species synonymic to S. sybillensis. We reconfirmed the presence of S. sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory; and performed its molecular and phylogenetic characterization. The morphological characteristics of the sarcocysts were consistent with those of S. nipponi and S. sybillensis described in the first report. However, the nucleotide sequence of 18S rRNA gene of S. sybillensis showed only 91.9% identity to that of S. nipponi, suggesting low homology among the concerned Sarcocystis spp. Accordingly, S. sybillensis was found to occupy a clade distinct from that of S. nipponi in a phylogenetic tree of Sarcocystis. Therefore, the present study provides essential information on 18S rRNA-based molecular characterization of S. sybillensis and disproves the existing notion of morphology-based species synonymity of S. sibillensis and S. nipponi. These results also suggest that S. sybillensis belongs to type 2 Sarcocystis.

Phylogenetic relationships among Toxocara spp. and Toxascaris sp. from different regions of the world.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

PREVALENCE OF YERSINIA AMONG WILD SIKA DEER (CERVUS NIPPON) AND BOARS (SUS SCROFA) IN JAPAN.

We examined the prevalence of Yersinia, including pathogenic species such as Yersinia enterocolitica and Yersinia pseudotuberculosis, among wild sika deer (Cervus nippon) and boars (Sus scrofa) captured in Japan. The prevalence of Yersinia in the wild deer was 75% (207/277) and in the boars was 74% (40/54). A total of 417 isolates of nine Yersinia species were isolated from the animals examined: the largest number of isolates (48%, 200/417) were Y. enterocolitica biotype 1A. Pathogenic Y. enterocolitica 1B/O:8 were also isolated from two deer, and Y. pseudotuberculosis serogroups 3 and 4 were isolated from two boars and a deer, respectively. The pathogenic Y. enterocolitica 1B/O:8 isolates carried four virulence genes (ail, ystA, yadA, and virF), and Y. pseudotuberculosis serogroups 3 and 4 isolates carried three virulence genes (inv, yadA, and lcrF). Although the Y. enterocolitica 1B/O:8 and Y. pseudotuberculosis isolates were sensitive to almost all the antimicrobials tested, the two Y. enterocolitica 1B/O:8 isolates were resistant to azithromycin and ampicillin, and the three Y. pseudotuberculosis isolates were resistant only to azithromycin. These findings suggested that wild deer and boars might be important reservoirs for the agent causing human yersiniosis.

The role of the American cockroach (Periplaneta americana) as transport host of Eimeria tenella to chickens.

The role of the American cockroach, Periplaneta americana as transport host for Eimeria tenella was evaluated. Twenty-four cockroaches were orally fed with sporulated oocysts of E. tenella. Their feces and digestive tract were examined for oocysts by sugar centrifugal flotation technique and PCR. Infectivity of the oocysts recovered from the digestive tract of infected cockroaches as well as from their feces was evaluated by orally inoculating them into Boris Brown chickens. E. tenella oocysts were found in the digestive tract and feces of infected cockroaches up to day 4 after ingestion of oocysts. Furthermore, oocysts that were recovered from the digestive tract and feces of cockroaches remained infective for 4 and 3 days after ingestion of oocysts, respectively. Presence of oocysts in the feces of chicken that had been inoculated with either digestive tract or feces of P. americana demonstrated the infectivity of E. tenella oocysts from digestive tract or feces, suggesting that P. americana may play a role in the transmission of E. tenella among chicken and between chicken flocks.

Detection of Sarcocystis spp. and Shiga toxin-producing Escherichia coli in Japanese sika deer meat using a loop-mediated isothermal amplification-lateral flow strip.

Game meat potentially harbors a number of parasitic and bacterial pathogens that cause foodborne disease. It is thus important to monitor the prevalence of such pathogens in game meats before retail and consumption to ensure consumer safety. In particular, Sarcocystis spp. and Shiga toxin-producing Escherichia coli (STEC) have been reported to be causative agents of food poisoning associated with deer meat consumption. To examine the prevalence of these microbiological agents on-site at a slaughterhouse, the rapid, simple and sensitive detection method known as the "DNA strip" has been developed, a novel tool combining loop-mediated isothermal amplification and a lateral flow strip. This assay has achieved higher sensitivity and faster than conventional PCR and is suitable for on-site inspection.

Eimeria tenella oocyst excretion and riboflavin supplement in infected chicken.

The effect of riboflavin supplement in Eimeria tenella-infected chickens was evaluated. Mortality, fecal consistency, and oocysts per gram of feces were monitored for groups of E. tenella-infected chickens administered a basal diet supplemented with either riboflavin, the anti-coccidial drug amprolium, or with both compounds. The number of oocysts shed per chicken in the riboflavin-treated group was significantly higher than the positive non-treated control group. No significant difference in oocyst number between the amprolium-treated group and riboflavin plus amprolium-treated group was observed. Thus, the addition of 0.8 g/kg of riboflavin to basal diet can increase oocyst number in E. tenella-infected chicken, but has no effect on the efficacy of amprolium.