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.

Improved molecular identification of Strongyloides myopotami in nutrias using fecal samples.

Strongyloides myopotami is an intestinal nematode parasite of nutrias. Identification of S. myopotami is conducted based on the morphological characteristics of adult worms or cultured larvae. To widely and effectively understand the infection in nutrias, it would be preferable to develop the molecular identification using a few grams of the feces. Here, we attempted to identify S. myopotami using DNA extracted from eggs obtained from fecal samples. Among previously reported primer pairs targeting the 18S rRNA gene of Strongyloides spp., most could not be successful. We newly designed primers that successfully amplified the partial sequences in S. myopotami, resulting in being sequenced. Our simple protocol can be useful in nationwide surveys for clarifying the risk of human infection.

Experimental evaluation of pathogenicity and acquired immunity of Eimeria species, E. uekii and E. raichoi, infecting Japanese rock ptarmigans in a subspecies of the birds.

Japanese rock ptarmigans (Lagopus muta japonica) are birds that inhabit only alpine regions of central Honshu Island, Japan, known as the Japanese Alps. The number of these birds has recently declined, and in situ and ex situ national conservation programs for Japanese rock ptarmigans have been initiated. The infections of Eimeria spp. as protozoan parasites of the phylum Apicomplexa, E. uekii and E. raichoi, were frequently reported in the birds. However, the virulence of these Eimeria parasites has not been determined. Here, we analyzed the pathogenicity of these Eimeria parasites using experimental infections of a subspecies model of Japanese rock ptarmigans, Svalbard rock ptarmigans (Lagopus mutus hyperboreus), and evaluated acquired protective immunity against challenge in birds tolerant of low-dose inoculation with Eimeria parasites. Following inoculation with two Eimeria parasites derived from Japanese rock ptarmigans (dose range of 4 × 104 to 4 × 102 for E. uekii and 1.7 × 104 to 4 × 101 for E. raichoi), oocysts were detected at 6-8 days post-inoculation (PI), and the maximum number of oocysts per gram of feces was observed 7-10 days PI and then gradually decreased. The mortality rate and reduction in weight gain of chicks increased following high-dose inoculation of oocysts with abnormal feces (soft and diarrhea). Developmental zoites were detected histopathologically in epithelial tissues and sometimes the lamina propria from the duodenum to the colon. Chicks that survived low-dose inoculation did not show clear clinical symptoms after challenge inoculation. Our results suggest that the pathological characteristics of Eimeria parasites infecting Japanese rock ptarmigans include abnormal feces and reduction in weight gain, resulting in mortality in cases of heavy infection due to high-dose inoculation. These findings provide helpful data for Japanese rock ptarmigan conservation efforts.

Citrobacter koseri related abortion and fetal septicemia in cattle.

A 31-month-old Holstein dairy cow aborted at 224 days of gestation with ejection of cheese-like lochia. Citrobacter koseri, which commonly exists in the normal flora of human and animal digestive tracts, was isolated from aborted fetal tissues (liver, spleen, kidney, heart, lung, cerebrum, and skeletal muscle) and fetal membranes. Histopathological examination revealed suppurative fibrinous meningoencephalitis of the cerebrum, cerebellum, and brainstem; suppurative bronchopneumonia; suppurative chorioamnionitis; and fibrous splenic serositis. Numerous gram-negative bacilli were detected in the cytoplasm of macrophages and/or neutrophils in these lesions. Bacteriological investigation and immunohistochemical staining identified the bacilli as C. koseri. This is the first report of cattle abortion caused by C. koseri infection in dairy cattle.

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.

Occurrence, Histopathological Findings, and Molecular Identification of Pathogenic Eimeria Infections in Rabbits (Mammalia: Lagomorpha) in Japan.

PURPOSE: Eimeria spp. are commonly found among rabbits (Mammalia: Lagomorpha) worldwide. Among the 11 Eimeria species, several are highly virulent, including E. intestinalis and E. flavescens, which cause intestinal coccidiosis, and E. stiedae, which causes hepatic coccidiosis. Unlike other countries, the occurrence of Eimeria infections in rabbits in Japan remains unknown, except for one reported case of natural infection. METHODS: We surveyed Eimeria infections in clinically affected rabbits over the past approximately 10 years at Livestock Hygiene Centers in 42 prefectures. A total of 16 tissue samples (14 liver, 1 ileum, and 1 cecum) were collected from 15 rabbits in 6 prefectures. RESULTS: Characteristic histopathologic findings were observed, especially around the bile ducts, depending on the developmental stages of the parasites. Eimeria stiedae and E. flavescens were successfully identified by PCR and sequencing analyses in 5 liver samples and 1 cecum sample, respectively. CONCLUSION: Our results could enhance understanding of infection with Eimeria spp. in rabbits in Japan and contribute to pathological or molecular diagnoses.

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.

Bovine abortion and necrotic placentitis by Aspergillus terreus.

A 31-month-old Japanese Black cow (Bos taurus) aborted at 5 months of gestation with no clinical symptoms. Histopathological examination of the placenta and fetus revealed severe necrotic placentitis associated with numerous irregular degenerative fungi and inflammatory cells. Regular filamentous fungi were also detected, without inflammatory response in the fetal digestive and respiratory organs. Both fungi had aleurioconidia and septa in the placenta and fetal organs and immunohistochemically stained with antibodies against Aspergillus spp. Aspergillus terreus was isolated from the fetal lung and abomasal contents as confirmed using mycological and molecular methods. This is the first immunohistochemical, morphological, and molecular identification of A. terreus in bovine placenta and aborted fetuses.

Suppurative meningoencephalitis and perineuritis caused by Streptococcus gallolyticus in a Japanese Black calf.

A 179-day-old calf, which was weak and stunted, showed neurological signs and was euthanized. Postmortem examination revealed extensive and severe cloudy area in the meninges, and pleural pneumonia. Gram-positive cocci were isolated from systemic organs. Biochemical and 16S rRNA gene sequence analyses identified the isolate as Streptococcus gallolyticus, and its subspecies was suggested to be gallolyticus (SGG). The isolate was classified as a novel sequence type (ST115) by the multilocus sequence typing scheme for SGG and showed susceptibility to penicillin, ampicillin, amoxicillin, florfenicol, sulfamethoxazole-trimethoprim, and chloramphenicol. Histopathologically, suppurative meningoencephalitis and perineuritis were detected. As SGG has been isolated solely from a cow with mastitis in Japan, this is the first SGG infection in a calf with suppurative meningoencephalitis and perineuritis in this country.

Striatal necrosis caused by Lichtheimia ramosa in a neonatal calf.

A 12-day-old male calf that did not want breast milk from birth died following neurological signs such as staggering. Postmortem examination revealed bleeding and encephalomalacia in the left striatum and frontal lobe. Histopathologically, necrotic granulomatous encephalitis with numerous fungi was detected. The fungi were positively stained with anti-Rhizomucor mouse monoclonal antibodies. Lichtheimia ramosa was detected in formalin-fixed paraffin-embedded samples of the affected tissue by molecular methods. To the best of our knowledge, striatal necrosis caused by L. ramosa in a neonatal calf has not been reported. This study provides the first evidence of striatal necrosis caused by L. ramosa in a neonatal calf.

Swine abortion caused by Candida parapsilosis.

In July 2020, a sow in a breeding herd in the Chiba Prefecture, Japan, suffered abortion. A necropsy revealed pale pulmonary foci scattered in the two fetuses. Histologically, multifocal pulmonary necrosis was detected with numerous yeasts. The yeast was positively stained using the periodic acid-Schiff reaction and Grocott's silver stain. Molecular identification indicated that the yeast was Candida parapsilosis. In conclusion, our results suggested that C. parapsilosis caused multifocal necrotizing pneumonia in the two fetuses. This study is the first report of a swine abortion with C. parapsilosis infection.

Programmed death-ligand 1 expression in swine chronic infections and enhancement of interleukin-2 production via programmed death-1/programmed death-ligand 1 blockade.

INTRODUCTION: Chronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death-1 (PD-1). The inhibitory signal is produced by the interaction between PD-1 and its PD-ligand 1 (PD-L1) and impairs the effector functions of T cells. However, the expression dynamics of PD-L1 and the immunological functions of the PD-1/PD-L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD-L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD-1/PD-L1 pathway. METHODS: In the initial experiments, anti-bovine PD-L1 monoclonal antibodies (mAbs) were tested for cross-reactivity with swine PD-L1. Subsequently, immunohistochemical analysis was conducted using the anti-PD-L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti-PD-L1 mAb. RESULTS: Several anti-PD-L1 mAbs tested recognized swine PD-L1-expressing cells. The binding of swine PD-L1 protein to swine PD-1 was inhibited by some of these cross-reactive mAbs. In addition, immunohistochemical analysis revealed that PD-L1 was expressed at the site of infection in chronic infections of pigs. The PD-L1 blockade increased the production of interleukin-2 from swine PBMCs. CONCLUSIONS: These findings suggest that the PD-1/PD-L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways.

Molecular phylogenetic analysis of bovine papular stomatitis viruses detected in Saga, Japan.

In this study, we performed a molecular phylogenetic analysis of six bovine papular stomatitis virus (BPSV) field strains detected from Japanese beef calves kept on a farm in Saga prefecture, a southwest part of Japan, from 2017 to 2020. The phylogenetic analysis based on a partial B2L gene (554-nt) showed that these field strains were divided into two lineages, a lineage (A-lineage) constructed by a Saga strain and strains obtained from various regions of Japan and the world, and other lineage (B-lineage) constructed by five Saga strains and strains obtained from France, USA and Iwate prefecture (a north part of Japan). Furthermore, a Saga field strain named BPSV_SAGAbv2 and strains obtained from USA and Iwate prefecture belonged to a sub-lineage blanched from B-lineage. This is the first report elucidating molecular epidemiological characters of field BPSVs obtained from Saga prefecture. The existence of the multiple lineages was thought to be related to a history of calf introduction from various regions of Japan into Saga prefecture.

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.

Distribution of Eimeria uekii and Eimeria raichoi in cage protection environments for the conservation of Japanese rock ptarmigans (Lagopus muta japonica) in the Japanese Alps.

Japanese rock ptarmigans, Lagopus muta japonica, are classified as an endangered species in Japan and are found only in the Japanese Alps. The number of birds has decreased in the last half century and cage protection projects have been undertaken as in situ conservation strategies (one of the projects for the recovery plan of Japanese rock ptarmigan) in the mountains. During the period with cage protections, some chicks died and two Eimeria spp., E. uekii and E. raichoi, were identified in the chicks. Here, we examined the soil within the cages and in the surrounding environment to assess potential sources of infection between July to August 2020. We found high numbers of oocysts in the cages, especially at the back sides where the ptarmigan family frequently congregated, but soils in other areas outside the cages were less contaminated or not contaminated at all. The time required for more than 50% of the oocysts to sporulate at 15, 20 and 25 °C for E. uekii was 20, 11, and 5 h, respectively, and 72, 48 and 18 h, respectively, for E. raichoi. Our results cast some doubt that coprophagia by chicks is the source of infection because chicks consumed fresh cecal feces (approximately within 1 h) as far as we know, and instead, the protected chicks might be directly or indirectly infected by oocysts in soils or the environment.

Phylogenetic characterization of Isospora jaracimrmani oocysts from a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) reared at a zoo in Ishikawa, Japan.

Oocysts of Isospora sp. were detected in the feces of a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) kept at a zoo in Ishikawa, Japan. Phylogenetic analysis placed the sequence in the cluster of Isospora spp. isolated from reptiles. Based on a comparison of morphological data of ten previously reported Isospora species from the Chamaeleonidae family, this isolate was morphologically similar to I. jaracimrmani, which has been considered to be a virulent species. This case study suggests the possibility that species of Isospora might not always cause disease because the animal that shed these oocysts showed no symptoms for more than two months.

Fatal suppurative meningoencephalitis caused by Klebsiella pneumoniae in two calves.

One calf died (No. 1) and another was euthanized following astasia (No. 2). Histopathological examination revealed suppurative meningoencephalitis in these calves. Klebsiella pneumoniae antigens were detected in lesions. Thymocytes were decreased in the thymus cortex in both cases. 16S rRNA gene sequencing of the No. 1 isolate and bacterial extracts from formalin fixed paraffin embedded sections of No. 2 revealed that both samples were K. pneumoniae. The No. 1 isolate showed multidrug resistance against penicillin antibiotics, fosfomycin, streptomycin, macrolide antibiotics, tetracycline antibiotics, and clindamycin. Immunosuppression is a significant septicemic K. pneumoniae infection risk factor. Our study provides new aspects regarding K. pneumoniae infections in cattle, bacterial meningoencephalitis differentiation, and K. pneumoniae and bacterial meningoencephalitis treatments.

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.

Capsular polysaccharide switching in Streptococcus suis modulates host cell interactions and virulence.

The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.