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The Babesia divergens/B. capreoli group includes parasites with confirmed or possible zoonotic potential to cause human babesiosis. Currently, diagnostic antigen of the group has not been established. In this study, we investigated the ortholog of Bd37, a glycosylphosphatidylinositol (GPI)-anchored major merozoite surface protein of B. divergens sensu stricto, in the Asia lineage of the group. From two genomic isolates from sporozoite/sporoblast stages, three Bd37 gene variants, namely Bd37 JP-A, JP-B, and JP-C, were isolated with 62.3-64.1% amino acid sequence identity. Discriminative blood direct PCR revealed that Bd37 JP-A was encoded in all parasites infecting wild sika deer examined (n=22). While Bd37 JP-B and JP-C genes were randomly detected in 12 and 11 specimens, respectively. Sequencing of all JP-A variants revealed that the gene was polymorphic, with a low ratio of non-synonymous to synonymous substitutions (dN/dS) and that a highly polymorphic region was not related to predicted B-cell epitopes. A recombinant JP-A-based ELISA showed an overall positive rate of 13.9% in sika deer in Japan from north (Hokkaido) to south (Kyushu island) across 24 prefectures (n=360). This positive rate was twice as high as that examined by 18S rRNA-based PCR (6.6%). The geographical trends in infection rates were consistent. This study demonstrated that direct examination was informative for revealing genetic background and selecting antigen candidates. Bd37 orthologs may serve diagnostic purposes in combination with indirect fluorescence assay, which requires biological isolates.

Borna disease virus (BoDV) is a neurotropic virus that causes several infections in humans and neurological diseases in a wide range of animals worldwide. BoDV-1 has been molecularly and serologically detected in many domestic and wild animals in Japan; however, the genetic diversity of this virus and the origin of its infection are not fully understood. In this study, we investigated BoDV-1 infection and genetic diversity in samples collected from animals in Hokkaido between 2006 and 2020. The analysis was performed by focusing on the P region of BoDV-1 for virus detection. The presence of BoDV-1 RNA was observed in samples of brain tissue and various organs derived from persistently infected cattle. Moreover, after inoculation, BoDV-positive brains were isolated from neonatal rats. The gene sequences of the P region of BoDV obtained from the rat brain were in the same cluster as the P region of the virus isolated from the original bovine. Thus, genetic variation in BoDV-1 was extremely low. The phylogenetic analysis revealed that BoDV-1 isolates obtained in this study were part of the same cluster, which suggested that BoDV-1 of the same cluster was widespread among animals in Hokkaido.

We compared the Mx expression and anti-viral function and the 3D structure of Mx protein in four species: chicken (Gallus gallus), whooper swan (Cygnus cygnus), jungle crow (Corvus macrorhynchos), and rock dove (Columba livia). We observed different mortalities associated with highly pathogenic avian influenza virus (HPAIV) infection to understand the relationship between Mx function as an immune response factor and HPAIV proliferation in bird cells. Different levels of Mx were observed among the different bird species after virus infection. Strong Mx expression was confirmed in the rock dove and whooper swan 6 hr after viral infection. The lowest virus copy numbers were observed in rock dove. The virus infectivity was significantly reduced in the BALB/3T3 cells expressing rock dove and jungle crow Mx. These results suggested that high Mx expression and significant Mx-induced anti-viral effects might result in the rock dove primary cells having the lowest virus copy number. Comparison of the expected 3D structure of Mx protein in all four bird species demonstrated that the structure of loop L4 varied among the investigated species. It was reported that differences in amino acid sequence in loop L4 affect antiviral activity in human and mouse cells, and a significant anti-viral effect was observed in the rock dove Mx. Thus, the amino acid sequence of loop L4 in rock dove might represent relatively high anti-viral activity.

Laparoscopic cholecystectomy (LC) is widely accepted as the standard treatment for benign gall bladder diseases in humans because it has proven to be less invasive and safer than are traditional methods. However, the efficacy of LC in dogs remains unclear. The present study aimed to examine the short-term outcome of LC for benign gall bladder diseases in dogs. We enrolled 76 consecutive dogs that underwent LC for benign gall bladder diseases at our hospital between April 2008 and October 2016. Dogs with jaundice, gall bladder ruptures, abdominal effusion, or extrahepatic biliary obstruction were not excluded from the indication. Factors including age, body weight, sex, clinical sign, disease, operative time, conversion to open surgery, perioperative complications, and postoperative hospital stay were investigated. The median age of the dogs was 11 years, and the median body weight was 5.4 kg. Fifty percent of the dogs exhibited no symptoms at the initial visit. Preoperative elevation of total bilirubin levels was observed in 16 dogs (21%). LC was successfully completed in 71 dogs (93%); the median operative time was 124 min. Although gall bladder ruptures were observed in 2 (2.6%) dogs, the operations were completed successfully. Three dogs (4.1%) had to be converted to open cholecystectomy and 2 (2.6%) underwent reoperation. Two dogs (2.6%) died intraoperatively and 2 (2.6%) died postoperatively. LC was a feasible, safe, and appropriate procedure considering the current operative indications for benign gall bladder diseases in dogs.

Tick-borne encephalitis (TBE) and severe fever with thrombocytopenia syndrome (SFTS) are both tick-borne zoonotic diseases caused by TBE virus (TBEV) and SFTS phlebovirus (SFTSV). In 2016, a second domestic TBE case was reported in Hokkaido, Japan, after an absence of 23 years. We conducted IgG ELISA for TBEV and SFTSV on 314 deer (Cervus nippon yesoensis) serum samples collected from 3 places in Hokkaido. There were 7 seropositive samples for TBEV but none for SFTSV by ELISA. The specificity of the 7 positive samples was confirmed by neutralization tests against TBEV, and 5 sera showed 320 to 640 of 50% focus reduction endpoint titers. Our results provide information about the infectious status of TBEV in wild deer in Hokkaido, Japan.

The composition of the intestinal microbiota is related to the health and immune function of the host. Administration of antibiotics affects the composition of the intestinal microbiota. However, the effects of immune function on the composition of the intestinal microbiota are still unclear. In this study, we investigated the lymphocyte composition and determined the relationships between lymphocyte function and the intestinal microbiota following antibiotic treatment in mice. To change the composition of the intestinal microbiota, mice were treated with or without antibiotics. Analysis of intestinal microbiota was performed by metagenomic analysis targeting 16S rRNA. Lymphocyte subsets of splenocytes were measured by flow cytometry. For functional analysis of T cells, splenocytes were stimulated with concanavalin (Con A), and cytokine gene expression was measured by real-time polymerase chain reaction. Firmicutes were predominant in the control group, whereas Bacteroidetes predominated in the antibiotic-treated group, as determined by metagenomic analysis. The diversity of the microbiota decreased in the antibiotic-treated group. Analysis of lymphocyte subsets showed that CD3 + cells decreased, whereas CD19 + cells increased in the antibiotic-treated group. All cytokine genes in splenocytes treated with Con A were downregulated in the antibiotic-treated group; in particular, genes encoding interferon-γ, interleukin (IL)-6, and IL-13 significantly decreased. Taken together, these results revealed that changes in the composition of the intestinal microbiota by antibiotic treatment influenced the population of lymphocytes in splenocytes and affected the immune response.

The intestinal microbiota of newborn calves changes during the early postnatal period and influences their health and immune function. We studied the compositional changes in the intestinal microbiome of newborn calves during the first week after birth by metagenomic analysis. In feces from newborn calves, we identified 4 bacterial phyla, namely, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The relative abundance of Lactobacillaceae significantly increased from day 1 to day 7. We evaluated Lactobacillus spp. colony numbers using selective agar plates and confirmed that the abundance of Lactobacillus spp. significantly increased during the first 7 days after birth. In conclusion, Lactobacillus spp. colonized the intestinal tract of calves during the first 7 days after birth.

Borna disease virus (BDV) is a virus that causes a neurological disease in domestic animals, including a variety of animal species in Japan. Few studies have examined the mode of transmission of this virus in cattle, and the exact mechanisms underlying the transmission of the virus need to be elucidated. This study aimed to examine the contribution of vertical transmission of the virus, which occurs when the virus is transmitted from the mother to offspring during gestation or birth. We used an epidemiological approach. The relative risk (RR) was calculated for cattle born to BDV sero-positive cows from farms with a higher within-herd prevalence of BDV (56.8%). We tested the sera of 1,122 dairy cattle from 24 dairy herds in Hokkaido Prefecture, Japan, for BDV infection using the ELISA and western blotting method. The overall level of BDV sero-prevalence was 22.1%. Seroprevalence was significantly higher in closed-breeding herds that do not have buying in cows (39.7%) than in farms that restock cattle by buying in cows (4.4%, P<0.01). The overall RR of BDV vertical transmission from infected mothers to their daughters was 1.86 (95% confidence interval (CI): 1.54-2.56). Our results show that vertical transmission contributes significantly to BDV transmission in the farms tested in this study.

We investigated the influence of Borna disease virus (BDV) infection on the clinical state of dairy cows. Sera from 149 cows were examined using enzyme-linked immunosorbent assay and western blotting detect antibodies to the BDV-nucleoprotein antigen. Among 149 investigated cows, 25 (16.8%) showed a positive reaction to BDV antigen. No significant difference existed in milk production or medical history between seropositive and seronegative cows. Although the estrus cycle appeared normal even in the seropositive cows, the frequency of artificial insemination and calving-to-conception intervals significantly increased in seropositive cows. Therefore, fertilization failure was recognized in the BDV-antibody positive cows.

This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α(1)-acid glycoprotein and IL-1β in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 µg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.

We have examined the seroprevalence of BDV in wild Raccoons (Procyon lotor) in Hokkaido, Japan. Serum samples from raccoons were examined using ELISA and Western blot assays to detect the presence of serum antibodies that react specifically to BDV antigens. Among 549 investigated individuals, eleven (2.0%) showed a positive reaction to BDV antigens. Brain tissue samples from five individuals were subjected to RT-PCR, which detected BDV sequences in three of them. Sequence analysis revealed a high degree of genetic conservation between BDV sequences derived from raccoons and previously published sequences derived from other animal species.

Reproductive diseases after parturition are a serious problem in dairy cattle. It is important to predict postpartum reproductive diseases early and to develop prophylaxis. The objectives of this study were to demonstrate changes in the peripheral blood concentration of interleukin-6 (IL-6) before parturition, which was mainly produced by T helper 2 type (Th2) cells, and to investigate a correlation between the IL-6 concentration and the occurrence of the postpartum retained placenta, endometritis and/or follicular cyst in dairy cattle. Twenty-seven Holstein-Friesian cows were used for this study. Thirteen had no clinical disease, 8 had retained placenta, 4 were diagnosed with endometritis by vaginal inspection, and 2 were diagnosed with follicular cyst by rectal palpation at 1 and 2 months after parturition. Blood samples were collected 60 days pre- and post-partum. They used for IL-6, progesterone (P(4)) and estradiol-17beta (E(2)) concentration determination. This study showed that the IL-6 concentration prepartum was higher than postpartum. Low levels of IL-6 and P(4) in peripheral blood prepartum tended to affect retained placenta and a high level of IL-6 prepartum tended to affect endometritis. These results indicate that measurement of change in the IL-6 concentration during pregnancy is one useful tool for predicting crisis in postpartum reproductive diseases in dairy cattle.

We previously reported that small wild rodents in Japan harbor two types of novel Babesia microti-like parasites (designated as Hobetsu and Kobe types), but not the type commonly found in the northeastern United States (U.S. type) where human babesiosis is endemic. To determine whether these new types of parasites are distributed in places surrounding Japan, an epizootiologic survey was undertaken in three geographically distant areas in northeastern Eurasia; South Korea, Vladivostok in Russia, and Xinjiang in China. Blood samples were collected from a total of 387 animals comprising 24 species. DNAs extracted from the samples were tested by nested PCR targeting babesial nuclear small-subunit rRNA gene (rDNA), which revealed that small rodents harboring B. microti exist in all three survey areas. Sequence analysis showed that all PCR-positive samples had rDNA sequences virtually identical to that of U.S.-type B. microti. However, when beta-tubulin gene sequences were compared, evident geographic variations were seen. By use of primers specific for each of the beta-tubulin genes of Kobe-, Hobetsu-, and U.S.-type parasites, a type-specific PCR was developed. Parasite with Hobetsu- or Kobe-type sequence was not detected from any of the three survey areas. These findings suggest that U.S.-type B. microti is widely distributed among small wild mammals in temperate zones of not only North America, but also Eurasia, whereas that Hobetsu- and Kobe-type parasites may be uniquely distributed in Japan.

To obtain basic information on the state of proinflammatory cytokines in newborn calves, we determined the kinetics of 5 cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist) in sera of newborns during the first 4 weeks of life. At birth, none of the 5 cytokines were detected in almost all serum samples, but the cytokines became detectable within 12 hr after being fed colostram. The mean concentrations of the cytokines reached peak levels by 24 hr and then gradually decreased and became undetectable by 4 weeks after birth. Cytokine mRNA expressions in peripheral blood mononuclear cells of newborns were observed without reference to the cytokine concentrations in sera. Serum cytokines detected in newborn calves are probably colostral origin.

Changes in interleukin (IL)-1beta, IL-6 and IL-8 in serum, and their mRNA expression on neutrophils from a 4.6-month old Holstein young calf with bovine leukocyte adhesion deficiency (BLAD) during the acute phase were evaluated. IL-1beta concentrations in the serum of the calf with BLAD at age 143-162 days ranged from 8.7 to 16.6 ng/ml, whereas the values were less than 2.7 ng/ml in control calves. Serum IL-6 (0.04 ng/ml) was only detected on the 1st day when the animal was diagnosed with the BLAD. IL-1beta and IL-8 mRNA expression on neutrophils from the affected calf appeared to be similar to those of controls. Serum cytokine levels and their mRNA expression on neutrophils from the calf with BLAD appeared to be little affected by the deficient expression of beta(2)-integrin on leukocytes, and are considered to be modulated by the inflammatory stimuli.

Serum cytokine levels and their expression of mRNA on neutrophils from a bone marrow (BM) transplanted heifer with leukocyte adhesion deficiency were evaluated. The clinical condition of the affected heifer was relatively stable after BM-transplantation. Persistent hyper gamma-globulinemia and increased serum immunoglobulin G (IgG) concentrations were monitored longitudinally. The concentration of interleukin (IL)-1beta in serum from the affected heifer ranged from 15.8 to 321.7 ng/ml, and maximum concentration occurred at the time which coincided with peak IL-6. Serum levels of IL-6 ranged from 0.32 to 27.9 ng/m l, and they appeared to be associated with the increment of serum IgG in the affected heifer. mRNAs for IL-1beta, IL-6, IL-8 and granulocyte and macrophage colony stimulating factor (GM-CSF) were increased in neutrophils from the affected heifer compared to controls. Persistent hyper gamma-globulinemia of the affected heifer appeared to be associated with enhanced mRNA expression for IL-6 and its serum levels. These findings suggest that humoral immunity of the affected heifer is activated and the production of neutrophils appears to be enhanced under the incapability of beta(2) integrin-mediated functions of phagocytic cells.

Concentrations of interferon-gamma (lFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-gamma and TNF-alpha-specific ELISA. In serum and whey samples from healthy cows. IFN-gamma was almost undetectable and TNF-alpha was detected at low levels. At the onset of illness, INF-gamma in sera and whey and TNF-alpha in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-gamma and TNF-alpha in whey from mastitic cattle decreased significantly as the cows recovered.

The coexistence of interleukin (IL)-1beta with IL-1 receptor antagonist (ra) in bovine colostrum and the possibility of simultaneous transfer of these cytokines to neonates via colostrum have been demonstrated. In the present study, we investigated the effect of IL-1ra on the mitogenic response of calf peripheral blood mononuclear cells (PBMC) stimulated by concanavalin A (ConA), which was mediated by IL-1. Pretreatment of PBMC with recombinant bovine (rb) IL-1ra alone significantly suppressed the proliferation of ConA-stimulated cells. However, in the presence of rbIL-1beta, the suppressive activity of rbIL-1ra was counteracted. These results suggest that coexistence of IL-1ra with IL-1 in colostrum may have no effect on the activation of the neonatal immune system by IL-1beta.

A sandwich ELISA for the bovine IL-1 receptor antagonist (bIL-1ra) was developed using recombinant (r) bIL-1ra produced by Escherichia coli, anti-rbIL-1ra rabbit IgG, its biotinylated one and avidin-peroxidase. This ELISA system enabled detection of rbIL-1ra at a concentration of more than 2 ng/ml. This ELISA was applied to quantitation of bIL-1ra in sera and whey of mastitic and healthy cows. The results indicate that although IL-1ra levels in healthy and mastitic sera and whey were comparable, serum IL-1ra/IL-1beta ratio of euthanized cows was significantly lower than that of the recovered.

We tried to develop an experimental system for quantifying piroplasm transmission in Theileria sergenti infection using a SCID mouse model, of which red blood cells (RBC) had been substituted with bovine (Bo). A mouse hybridoma cell producing antibody against Holstein RBC was established for distinguishing Holstein RBC from Japanese Black RBC by a immuno-staining method. T. sergenti piroplasms in RBC were stained by 4',6-diamidino-2-phenylindole. With the aid of these techniques the piroplasm transmission from one to another race of Bo-RBCs could successfully be quantified in SCID mice.

We have previously demonstrated that the red blood cells (RBCs) in the blood circulation of SCID mice could be almost completely substituted with bovine RBCs by means of repeated transfusions. In the present study, transfusion experiments were carried out with RBCs from various animal species to investigate the ability of SCID mice to accept xenogeneic RBCs. Bovine (Bo), equine (Eq), human (Hu) and murine (Mu) RBCs were labeled with a fluorescent dye to trace their trafficking in the blood stream. Following the intravenous injection these RBCs were cleared from the circulation at various rates; the 1/100 reduction time in the labeled cell counts was 2 to 7 days, 4 to 7 hr and 1 to 2 hr with Bo-, Hu- and Eq-RBCs, respectively. In contrast, labeled Mu-RBCs from not only syngeneic but also allogeneic mouse strain were able to stay over 50 days. The difference in clearance rates was attributable to the difference in uptake of the RBCs by the splenic macrophages. The clearance rates of labeled RBCs were significantly decreased by co-transfusion with unlabeled RBCs of the same species, indicating that xenogeneic RBCs were recognized by the reticulo-endothelial system of SCID mice in a species-specific fashion. Furthermore, at least in the case of Hu-RBC, the complement component 3 may play a role in the Hu-RBC clearance in SCID mice since C3 deposition was observed on Hu-RBCs but not on Bo- and Eq-RBCs.

Two calves (5 and 9 months old) affected with pneumonia and gingivitis were also diagnosed as having bovine leukocyte adhesion deficiency (BLAD). The gene of leukocyte adhesion molecule CD18 in these BLAD calves and their dams (carrier) were examined by means of polymerase chain reaction (PCR) and digestion of restriction endonuclease. The splicing in mRNA coded CD18 reported in human LAD was not recognized in BLAD on the basis of the results of PCR amplification. The region including the portion of point mutation, which corresponded to the region reported in the human patient, was amplified by PCR, and the PCR product was then digested with Taql. An obvious difference was recognized in the pattern of digestion among healthy calves, BLAD calves and their dams. In BLAD, therefore, the point mutation reported in human patients was strongly suggested. Moreover it may be a method able to be used in detecting the carrier.