Comparative molecular analyses of Eimeria Schneider (Apicomplexa: Eimeriidae) species from rock ptarmigan in Iceland, Svalbard-Norway, and Japan.

The rock ptarmigan (Lagopus muta) has a Holarctic breeding distribution and is found in arctic and sub-arctic regions. Isolated populations and glacial relicts occur in alpine areas south of the main range, like the Pyrenees in Europe, the Pamir mountains in Central Asia, and the Japanese Alps. In recent decades considerable effort has been made to clarify parasite infections in the rock ptarmigan. Seven Eimeria spp. have been reported parasitizing rock ptarmigan. Two of those species, E. uekii and E. raichoi parasitizing rock ptarmigan (L. m. japonica) in Japan, have been identified genetically. Here we compare partial sequences of nuclear (18S rRNA) and mitochondrial (COI) genes and we detail the morphology of sporulated oocysts of E. uekii and E. raichoi from Japan, E. muta and E. rjupa, from the rock ptarmigan (L. m. islandorum) in Iceland, and two undescribed eimerian morphotypes, Eimeria sp. A, and Eimeria sp. B, from rock ptarmigan (L. m. hyperborea) in Norway (Svalbard in the Norwegian Archipelago). Two morphotypes, ellipsoidal and spheroidal, are recognized for each of the three host subspecies. Our phylogenetic analysis suggests that the ellipsoidal oocyst types, E. uekii, E. muta, and Eimeria sp. A (Svalbard-Norway) are identical and infects rock ptarmigan in Japan, Iceland, and Svalbard-Norway, respectively. Eimeria uekii was first described in Japan in 1981 so that E. muta, described in Iceland in 2007, and Eimeria sp. A in Svalbard-Norway are junior synonyms of E. uekii. Also, phylogenetic analysis shows that the spheroidal oocyst types, E. rjupa and Eimeria sp. B (Svalbard-Norway), are identical, indicating that rock ptarmigan in Iceland and Svalbard-Norway are infected by the same Eimeria species and differ from E. raichoi in Japan.

Evaluation of the host specificity of Eimeria uekii and Eimeria raichoi for Japanese rock ptarmigans by oocyst transfer to taxonomically related birds.

Eimeria spp. are protozoan parasites that are commonly found in a broad range of vertebrate hosts. These parasites generally exhibit strict host specificity, but some Eimeria spp. can infect groups of closely related species such as species within a genus or family. Compared with Eimeria spp. that infect livestock, limited information is available about such infections in wild animals including data on host specificity, virulence, and prevalence. The Japanese rock ptarmigan, Lagopus muta japonica, is an endangered bird belonging to the family Phasianidae, order Galliformes, and inhabits only alpine areas of Japan. In conservation efforts for these birds, two Eimeria spp., E. uekii and E. raichoi, were frequently detected. Here, we examined cross-transmission of the parasites to other bird species to characterize their infectivity as well as the development of experimental bird models to contribute to conservation programs by the oocyst transfer. Consequently, among the examined eight bird species (chickens, Japanese pheasants, turkeys, chukar partridges, quails, helmeted guineafowls and ducks), only turkeys (family Phasianidae, order Galliformes) could be infected with E. raichoi. However, the number of oocysts per feces was relatively low, and few parasites in the intestinal mucosa could be found by histopathological analyses. These results might indicate that E. uekii and E. raichoi are highly adapted to Japanese rock ptarmigans that inhabit the alpine zone although further studies are anticipated.

Molecular identification of Eimeria species in liver and feces of naturally infected rabbits in Japan.

Among the 11 species of Eimeria in rabbits, some of which are known to be pathogenic and cause enteritis, E. stiedae induces severe liver lesions resulting in elevated mortality. Unlike in other countries, the incidence and prevalence of the parasites in rabbits have not been reported in Japan. In the present study, we histopathologically analyzed hepatic coccidiosis in a rabbit and attempted several primers to genetically identify the parasites and investigated the prevalence of Eimeria species at the same farm. In the liver of the affected rabbit, we observed fibrosis and edema around multiple bile ducts and epithelial cell hyperplasia of the bile ducts. Large numbers of developing parasites of Eimeria spp., mainly oocysts, were present in the bile ducts. PCR and sequencing analyses with the published primers for Cyclospora and Eimeria spp. were used to successfully identify the parasites in the liver as E. stiedae. The oocysts of Eimeria spp. were detected in 13 out of 20 fecal samples collected from other rabbits at the farm, and five Eimeria spp. (E. perforans, E. flavescens, E. exigua, E. magna, and E. vejdovskyi) were genetically confirmed. Our results provide the first indication that Eimeria spp., including highly pathogenic species, are present in Japan and the primer set used herein can be a useful tool for the identification of rabbit Eimeria spp.

Systemic and intestinal porcine epidemic diarrhea virus-specific antibody response and distribution of antibody-secreting cells in experimentally infected conventional pigs.

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.

Immunity against a Japanese local strain of porcine reproductive and respiratory syndrome virus decreases viremia and symptoms of a highly pathogenic strain.

BACKGROUND: The type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has spread throughout countries of southeast Asia, where it has caused severe economic losses. Even countries presently free of PRRSV are at high risk for infection and spread of this virus. Some of these countries, including Japan, have broad epidemics of the local type 2 PRRSV, creating chronic pathogenicity in the domestic pig population. The present study aimed to evaluate the protective efficacy of immunity by infection with a Japanese field isolate, EDRD1, against heterologous challenge with a Vietnamese HP-PRRSV field strain. To this end, four groups of PRRSV-negative crossbreed piglets were used for a challenge study. Groups 1 and 2 were inoculated with EDRD1 via the intranasal route. After 26 days, Groups 2 and 3 were inoculated with HP-PRRSV via the same route. Group 4 served as an uninfected control. Blood and oral fluid samples were taken every 3-4 days after HP-PRRSV challenge; on day 16 post-challenge, all pigs were euthanized, and examined pathologically. RESULTS: The nucleotide sequence analysis of nonstructural protein 2 gene of EDRD1 and comparison with Vietnamese HP-PRRSV showed that the 39 amino acid deletion sites of EDRD1 was nearly in the same region as the 29 amino acid deletion sites of HP-PRRSV. Immunity conferred by inoculation with EDRD1 dramatically reduced viral load in the sera and tissues besides viral shedding (Group 2) compared with those in pigs infected only with HP-PRRSV (Group 3). The clinical signs and rectal temperature were significantly reduced, and the average daily weight gain was significantly improved in the EDRD1-inoculated pigs (Group 2) compared with the Group 3 pigs. Notably, no viral RNA was detected in various organs of the Group 2 pigs 16 days post-infection with HP-PRRSV, except in one pig. Therefore, the immunity induced by EDRD1 and its genetically close field isolates may play a role in reducing viremia caused by HP-PRRSV. CONCLUSIONS: The results of the present study demonstrate that pigs are highly protected against heterologous Vietnamese HP-PRRSV challenge by immunity against a Japanese local strain, EDRD1.

Morphological and molecular identification of Eimeria tetartooimia oocysts from a Japanese green pheasant (Galliformes; Phasianidae; Phasianus versicolor) at a zoo in Japan.

We detected Eimeria oocysts from Japanese green pheasants (Phasianus versicolor) at a zoo in Osaka, Japan. The oocyst isolates were subspherical or ovoidal shaped and measured 17.2 (range 14.7-20.0) µm in length and 14.8 (13.3-16.7) µm in width with a length/width (L/W) ratio of 1.2 (1.0-1.4) and each had one polar granule. The oocysts lacked a residuum and micropyle. Sporocysts measured 9.8 (6.7-13.3) µm in length and 5.9 (4.7-7.3) µm in width, with a L/W ratio of 1.2 (1.1-1.4). Compared to previously published values, this strain shows morphological similarities with an isolate of E. teetartooimia from ring-necked pheasants from other countries. Phylogenetic analysis of the 18S rRNA and mitochondrial cytochrome c oxidase subunit I genes places the isolate in a clade related to chicken Eimeria spp., such as E. acervulina or E. brunetti. Although further analysis is needed, this information can be helpful for the diagnosis and determination of virulence of Eimeria spp. in pheasants.

Production of immunogenic recombinant L1 protein of bovine papillomavirus type 9 causing teat papillomatosis.

Bovine papillomavirus type 9 (BPV9) is a causative agent of severe teat papillomatosis. Considering the lack of efficient BPV culture methods, recombinant proteins such as virus-like particles developed through genetic engineering may serve as a useful tool for developing effective vaccines against BPV infection. In this study, we successfully produced immunogenic particles composed of recombinant L1 protein of BPV9 (rBPV9-L1), using a baculovirus expression system. rBPV9-L1-immunized mice produced BPV9-specific IgG, which did not cross-react with BPV type 6, which is another causative agent of teat papillomatosis. Hence, immunogenic rBPV9-L1 is potentially applicable as a vaccine candidate for teat papillomatosis.

Molecular identification of Eimeria hestermani and Eimeria prionotemni from a red-necked wallaby (Macropodidae; Macropus rufogriseus) in Japan.

To date, more than 50 Eimeria spp. have been isolated from marsupials of the family Macropodidae. Although 18 species of Eimeria have been previously detected from multiple animal species belonging to the genus Macropus of the family, limited genetic analyses of the parasites are available, and their pathogenicity remains unclear. Here, we report the isolation of Eimeria spp. from a zoo specimen of red-necked wallaby (Macropodidae; Macropus rufogriseus). Specifically, two distinct types of Eimeria oocysts were recovered, one from the feces before treatment with an anthelmintic and the second from the intestinal contents after death of the animal. The oocysts obtained from the two sources were morphologically identified as E. hestermani and E. prionotemni, respectively. We successfully determined partial gene sequences from the two isolates, including segments of the 18S rRNA genes, and for the first time have used phylogenetic analyses of these sequences to assign the species to distinct clades. In combination with further genetic data, these results are expected to help elucidate the pathogenicity and host ranges of Eimeria spp. within the respective family and genus.

Occurrence and genetic identifications of porcine Entamoeba, E. suis and E. polecki, at Tangerang in West Java, Indonesia.

Entamoeba suis and E. polecki subtype (ST) 1 and ST3 recently have been inferred to be virulent in pigs. However, because relevant molecular epidemiological surveys have been limited, the prevalences of these species remain unknown and their pathogenicities are still controversial. We surveyed 196 fecal samples of pigs (118 of adults, 78 of piglets) at Tangerang in West Java, Indonesia, in 2017, employing PCR using porcine Entamoeba-specific primers. E. suis was the more frequently detected species, observed in 81.1% of samples, while E. polecki ST1 and ST3 were detected in 18.4% and 17.3% of samples, respectively; mixed infections (harboring 2-3 species or subtypes of Entamoeba) were confirmed in 29.3% of positive samples. Statistically significant differences in the positive rates were not seen between adult pigs and piglets, except for those of E. polecki ST3. The prevalences of Eimeria spp. and/or Cystoisospora suis (79.1%), strongyles (55.6%), and Strongyloides spp. (6.1%) were also observed morphologically in the samples. Further chronological or seasonal investigations of pigs and humans in these high-prevalence areas are needed to assess the virulence of the Entamoeba parasites, including the effects on pig productivity, and to evaluate the zoonotic impacts of these organisms.

In vivo effect of a TLR5 SNP (C1205T) on Salmonella enterica serovar Typhimurium infection in weaned, specific pathogen-free Landrace piglets.

Toll-like receptor 5 is a pattern-recognition receptor for bacterial flagellin. We previously reported that a single nucleotide polymorphism (SNP) of swine TLR5, C1205T, impairs recognition of Salmonella typhimurium (ST) flagellin and ethanol-killed Salmonella Choleraesuis (SC). In the present study, weaned, specific pathogen-free (SPF) Landrace piglets with CC, CT or TT genotypes were orally infected with ST (L-3569 strain) to determine the effect of this specific SNP on ST infection in vivo. Eighteen ST-infected piglets (six each with CC, CT, or TT) exhibited fever and diarrhea for 1 week after infection. TT piglets had the longest duration of fever. TT piglets had the greatest mean diarrhea score during the experimental period, followed by CT and CC piglets. Fecal ST shedding was greater in CT and TT pigs than CC pigs from 2 days after infection. Serum haptoglobin concentration increased in ST-infected piglets and to greater extents in CT and TT pigs than CC pigs. Daily weight gain was lower in infected pigs, particularly TT piglets, than control pigs. To the best of our knowledge, this study is the first to demonstrate that impairment of TLR recognition affects pig susceptibility to disease in vivo. Thus, piglets with the T allele of swine TLR5 (C1205T) exhibit impaired resistance to ST infection. Furthermore, elimination of the T allele of this SNP from Landrace pigs would lead to enhancement of their resistance to ST infection.

First surveillance and molecular identification of the Cryptosporidium skunk genotype and Cryptosporidium parvum in wild raccoons (Procyon lotor) in Osaka, Japan.

Recent research suggests that raccoons (Procyon lotor) can transmit several important pathogens affecting humans, including protozoans. In Japan, the number of wild raccoons has increased since they were first introduced more than 50 years ago. Here, we report the first survey of Cryptosporidium infection using fecal swabs of raccoons captured in Osaka, Japan. Of 116 raccoons examined by PCR targeting of the Cryptosporidium 18S rRNA gene, 7 (6.03%; 2 adults and 5 young animals) were positive, and the isolates were identified as Cryptosporidium skunk genotype (subtype XVIa) and C. parvum based on sequence and phylogenetic analyses. Both species and the genotype are zoonotic; thus, our results suggest that raccoons could transmit Cryptosporidium infections to humans in Japan.

First detection and molecular identification of Entamoeba bovis from Japanese cattle.

Thus far, Entamoeba species have been classified based on morphology such as the number of nuclei in mature cysts and their hosts. Using recently developed molecular tools, ruminant Entamoeba spp. are currently classified into four species/genotypes: E. bovis and Entamoeba ribosomal lineages (RL) 1, 2, and 4. However, the distribution or pathogenicity of ruminant Entamoeba has not been well documented. In the present study, we examined a total of 25 fecal and seven environmental samples collected from six farms in Japan from 2016 to 2017 by the floatation method and PCR and sequencing analyses. Consequently, we detected Entamoeba cysts in 18 of 25 cattle samples and four of the seven environmental samples, including soil and drinking water, by microscopic examinations. In sequential examinations, Entamoeba-positive cattle were found to shed cysts without any clinical symptoms for more than 8 months. By PCR for molecular identification, isolates in ten cattle and one soil sample were successfully sequenced and formed a cluster of E. bovis, which was separated from those of other Entamoeba species/genotypes such as RL1-4 in phylogenetic analysis. To our knowledge, this is the first report about E. bovis in Japan, and our results may implicate that E. bovis is not pathogenic.

Pathogenic characteristics of a novel intranuclear coccidia in Japanese black calves and its genetic identification as Eimeria subspherica.

Bovine intranuclear coccidiosis is caused by the protozoans Eimeria alabamensis and Cyclospora spp. Here, we characterized the disease and genetically identified the causative species in Japanese black calves with chronic and refractory watery diarrhea. Histologic examinations revealed atrophy of the jejunal villi and numerous parasites in the nucleus of epithelial cells in the jejunum. Based on molecular analyses using 18S ribosomal RNA gene-specific primers that we designed, the parasites were found to be formed in the same cluster as Eimeria subspherica in the phylogenetic tree, which was separated from those of other related Eimeria spp. These results constitute the first report of E. subspherica as a cause of bovine intranuclear coccidiosis.

Development of molecular diagnostic protocols for detecting three types of Entamoeba from diarrheal and asymptomatic pigs and environmental moist soils.

Entamoeba suis and Entamoeba polecki subtypes (ST) 1 and 3 have recently been implicated in disease outbreaks in pigs. However, the distributions of these parasites in Japan and the potential sources of infection on farms still remain unclear. Here, we examined a farm of fattening/growing pigs with abnormal feces in Kagoshima Prefecture, Japan, and found the presence of parasites in the farm environment. Examination of intestinal tissues from pigs presenting with ulcerative colitis revealed a large number of trophozoites that had invaded the lesions. We identified single and mixed infections of E. suis and E. polecki ST1 and ST3 in paraffin sections or fecal samples from affected pigs. Two subtypes of Entamoeba were identified using four primer sets by PCR and sequencing. The parasites were detected in moist soil samples obtained around the drinking water source or puddles, implicating transmission of cysts via contaminated soils. Additionally, we found evidence of Entamoeba spp. and coinfections in surveyed pigs without any diarrhea at two neighboring farms. Our results establish methods for successfully identification of parasites, including cases in which multiple infections are present.

Pig epidemic diarrhoea virus S gene variant with a large deletion non-lethal to colostrum-deprived newborn piglets.

We previously identified a third porcine epidemic diarrhoea virus (PEDV) S variant with a large deletion of 582 nucleotides in the 5' terminal region of the S gene, in addition to the North American type and the S INDELs type. To investigate the pathogenicity of this variant, TTR-2/JPN/2014, we performed experimental infection using colostrum-deprived piglets and compared the results with those from the North American type PEDV, OKN-1/JPN/2013. Fifteen newborn piglets were divided into two groups of 7-8 piglets each and inoculated orally with one of PEDV isolates maintained at the eighth passage in Vero cell culture. Although all PEDV-inoculated piglets showed acute watery diarrhoea, lethality clearly differed between both PEDV-inoculated groups. Moreover, there were differences in virus distribution and lesions on the intestines between the two PEDV-inoculated groups. Therefore, our data suggest that the OKN-1/JPN/2013 PEDV isolate is virulent, whereas the TTR-2/JPN/2014 PEDV isolate is avirulent.

First isolation of Schizophyllum commune in a harbor seal (Phoca vitulina).

To date, Schizophyllum commune infection has been identified in only humans and dogs. A 7-year-old female harbor seal (Phoca vitulina) died after exhibiting corneal opacity, anorexia, and labored respiration. At necropsy, phthisis of the left eyeball was detected, and multiple nodular lesions were observed in the thoracic and abdominal regions, especially in the lung, heart, and lymph nodes. Histopathologically, numerous hyphae were seen in granulomatous lesions in the eyes, lung, heart, and lymph nodules. An isolate on potato dextrose agar from the eyes, lung, and sputum yielded a rapidly growing white woolly mycelia with basidiocarps (fruiting bodies) at 37°C. A suitable temperature for mycelial growth was obtained at 25°C, although sustained growth also occurred at 37°C. The fungal isolate, KH-JPN15-011, had distinctive features including hyphae bearing spicules and clamp connections, which were consistent with the characteristics of basidiomycete fungus. The sequence of the internal transcribed spacer region of nuclear ribosomal DNA showed 99.67% (617 bp) similarity with those of S. commune Phylogenetic analysis showed that the present isolate is most closely related to the samples from the Old World. This is the first report of a fatal disease caused by S. commune in exotic animals. Previously reported human and canine infections have not included granulomatous endophthalmitis and myocarditis. After considering these and previous findings, there is a possibility that S. commune from the Old World may include numerous highly pathogenic strains.

Genomic sequence and virulence evaluation of MN184A-like porcine reproductive and respiratory syndrome virus in Japan.

In this study, a porcine reproductive and respiratory syndrome virus (PRRSV) that was isolated from a 9-week-old diseased pig on a farm in Japan with a high mortality rate during 2007-2008 was characterized. This unique isolate, designated as Jpn5-37, did not have a high nucleotide identity in open reading frame 5 against any Japanese isolates. Among all available type 2 PRRSV complete genome sequences, Jpn5-37 shared the highest nucleotide identity (93.6%) with virulent strain MN184A. The genomic characteristics of Jpn5-37 were highly conserved with respect to the virulent MN184A, including a continuous eight amino acid deletion in the nonstructural protein 2 region. Moreover, virus distribution, viremia and the gross and microscopic characteristics of lesions were investigated in pigs 10 days post-inoculation to elucidate the pathogenicity of the isolate. Intranasal inoculation was found to rapidly result in viremia and dissemination of the Jpn5-37 isolate to several tissues in a similar manner to EDRD1; however, the amounts of Jpn5-37 RNA in serum were significantly greater. Similarly, the quantities of Jpn5-37 viral RNA in all organs tested tended to be higher than with EDRD1 infection. Mean rectal temperatures were significantly higher in the Jpn5-37-inoculated than in the control group at 4 and 6 days post infection (dpi) and in the EDRD1-inoculated group at 6 and 8 dpi. These results suggest that the Jpn5-37 strain replicates and is more efficiently distributed to the organs than is EDRD1 under the same conditions.

First molecular identification of Entamoeba polecki in a piglet in Japan and implications for aggravation of ileitis by coinfection with Lawsonia intracellularis.

Parasitic Entamoeba spp. are found in many vertebrate species including humans, as well as many livestock including pigs. In pigs, three Entamoeba spp., E. suis, and E. polecki and E. histolytica as zoonotic species, have been identified, but their pathogenicity has not been fully characterized. Here, we report the bacteriological, virological, and histopathological examination of three piglets with chronic diarrhea. Two animals appeared to be additionally infected with Lawsonia intracellularis, which caused a characteristic proliferative ileitis. In the piglet infected with Entamoeba spp., the trophozoites (approximately 10-15 µm with one nucleus in their cytoplasm) invaded into the lamina propria and the disease was worsened by the formation of ulcers and pseudomembranes. Genetic analysis identified the parasite as E. polecki (99.5% identity). Although E. polecki in humans or animals might be less pathogenic in the case of a single infection, coinfections with other pathogens including L. intracellularis may increase the severity of the disease.

Age-related detection and molecular characterization of Cryptosporidium suis and Cryptosporidium scrofarum in pre- and post-weaned piglets and adult pigs in Japan.

We investigated the distribution of Cryptosporidium in pigs in Japan by immunofluorescence staining of fecal samples and characterization of isolates by multilocus sequencing. The 344 animals sampled on eight farms included pre-weaned piglets (<1 month old; n = 55), weaned piglets (1-2 months old; n = 65), finished pigs (2-4 months old, n = 105) and of 4-6 months old (n = 67), sows (n = 36), and boars (n = 16). Average prevalence of Cryptosporidium on farms was 32.6%, ranging from 4.9 to 58.1%, decreasing with animal age (prevalences of <1 month old, 1-2 months old, 2-4 months old, 4-6 months old, sows, and boars were 27.3, 47.7, 41.9, 22.4, 11.1, 18.8%, respectively). Piglets (<1 and 1-2 months old) showing signs of diarrhea shed relatively more oocysts (5.28 in average log scale of oocysts per gram) in feces than piglets with normal or loose stools (those of 4.90). Thirty seven successful sequencing of the 18S ribosomal RNA gene among 62 examined samples revealed that all of the identified isolates were Cryptosporidium suis or Cryptosporidium scrofarum, which are generally specific to pigs, and that other species, such as zoonotic Cryptosporidium parvum, were absent. Interestingly, C. suis was frequently found in piglets younger than 2 months old, while C. scrofarum infection was more prevalent in older pigs which also showed increased prevalence of mixed C. suis and C. scrofarum infections. Sequencing of actin gene loci revealed the existence of variants of both Cryptosporidium species in pigs in Japan. Although the number of pigs examined in this study was relatively low, our results suggest that Cryptosporidium infection is widespread among pigs in Japan. In addition, the possibility of age-related specificity and pathogenicity in pig infections is also suggested.