In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Expression profile analysis of the transient receptor potential (TRPM) channel, a possible target of praziquantel in Schistosoma japonicum.

The WHO considers schistosomiasis, which is controlled by the mass administration of the drug praziquantel (PZQ), to be a neglected tropical disease. Despite its clinical use for over four decades, PZQ remains the only choice of chemotherapy against this disease. Regarding the previous studies that demonstrated that PZQ activates the transient receptor potential (TRP) channel in Schistosoma mansoni (Sm.TRPMPZQ), the expression profile of the ortholog of this channel gene (Smp_246790.5) in S. japonicum (EWB00_008853) (Sj.TRPMPZQ) was analyzed. The relative expression of this gene in various stages of the parasite lifecycle was analyzed by quantitative real-time reverse transcription-PCR (qRT-PCR), and the expression of Sj.TRPMPZQ was observed by immunohistochemical staining using anti-serum against the recombinant Sj.TRPMPZQ protein. qRT-PCR revealed the significantly lower mRNA expression in the snail stage in comparison to other stages (p < 0.01). The relative quantity of the Sj.TRPMPZQ expression for paired females, unpaired males, and eggs was 60%, 56%, and 68%, respectively, in comparison to paired males that showed the highest expression (p < 0.05). Interestingly, immunostaining demonstrated that Sj.TRPMPZQ is expressed in the parenchyma which contains muscle cells, neuronal cells and tegument cells in adult worms. This may support the two major effects of PZQ-worm paralysis and tegument disruption-induced by channel activation. Moreover, the channel was expressed in both the eggshell and the miracidia inside, but could not be observed in sporocyst. These results suggest that the expression of Sj.TRPMPQZ corresponds to the known sensitivity of S. japonicum to PZQ.

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice.

Introduction: NcSAG1 is one of most widely investigated antigens of Neospora caninum in various research fields. Such studies demonstrated the proficiency of NcSAG1 in the regulatory process of parasite adhesion and invasion of host cells. Accordingly, the contribution of NcSAG1 to the pathogenesis of neosporosis can undoubtedly be extrapolated, but direct evidence is lacking. Herein, we provide the first successful attempt at the gene disruption of NcSAG1 and novel data on the invasion and virulence potentials of N. caninum in vitro and in vivo. Methods: The disruption of the NcSAG1 gene was applied using the CRISPR/Cas9 system and confirmed by PCR, western blot and indirect fluorescent antibody tests as NcSAG1 knockout parasites (NcSAG1KO). Then, we investigated the role of NcSAG1 in the growth kinetics of the parasite in vitro. Results and discussion: The deletion of the NcSAG1 gene significantly decreased the infection rate and reduced the egress rate of the parasite. An in vivo study using nonpregnant female and male BALB/c mice revealed a significantly higher survival rate and lower body weight change in the group infected with the NcSAG1KO parasite than in the parental strain (Nc-1)-infected group. Regarding the vertical transmission model of BALB/c mice, the absence of the NcSAG1 gene significantly enhanced the survival of pups and greatly lowered the parasite burden in the brains of pups. In conclusion, our study suggested NcSAG1 as a key molecule in the pathogenesis of N. caninum.

Involvement of chemokine receptor CXCR3 in the defense mechanism against Neospora caninum infection in C57BL/6 mice.

C-X-C motif chemokine receptor 3 (CXCR3) is an important receptor controlling the migration of leukocytes, although there is no report regarding its role in Neospora caninum infection. Herein, we investigated the relevance of CXCR3 in the resistance mechanism to N. caninum infection in mice. Wild-type (WT) C57BL/6 mice and CXCR3-knockout (CXCR3KO) mice were used in all experiments. WT mice displayed a high survival rate (100%), while 80% of CXCR3KO mice succumbed to N. caninum infection within 50 days. Compared with WT mice, CXCR3KO mice exhibited significantly lower body weights and higher clinical scores at the subacute stage of infection. Flow cytometric analysis revealed CXCR3KO mice as having significantly increased proportions and numbers of CD11c-positive cells compared with WT mice at 5 days post infection (dpi). However, levels of interleukin-6 and interferon-γ in serum and ascites were similar in all groups at 5 dpi. Furthermore, no differences in parasite load were detected in brain, spleen, lungs or liver tissue of CXCR3KO and WT mice at 5 and 21 dpi. mRNA analysis of brain tissue collected from infected mice at 30 dpi revealed no changes in expression levels of inflammatory response genes. Nevertheless, the brain tissue of infected CXCR3KO mice displayed significant necrosis and microglial activation compared with that of WT mice at 21 dpi. Interestingly, the brain tissue of CXCR3KO mice displayed significantly lower numbers of FoxP3+ cells compared with the brain tissue of WT mice at 30 dpi. Accordingly, our study suggests that the lack of active regulatory T cells in brain tissue of infected CXCR3KO mice is the main cause of these mice having severe necrosis and lower survival compared with WT mice. Thus, CXCR3+ regulatory T cells may play a crucial role in control of neosporosis.

Transcriptomic Analysis of the Effects of Chemokine Receptor CXCR3 Deficiency on Immune Responses in the Mouse Brain during Toxoplasma gondii Infection.

The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals, including humans. We previously revealed through a whole-brain transcriptome analysis that infection with T. gondii in mice causes immune response-associated genes to be upregulated, for instance, chemokines and chemokine receptors such as CXC chemokine receptor 3 (CXCR3) and its ligand CXC chemokine ligand 10 (CXCL10). Here, we describe the effect of CXCR3 on responses against T. gondii infection in the mouse brain. In vivo assays using CXCR3-deficient mice showed that the absence of CXCR3 delayed the normal recovery of body weight and increased the brain parasite burden, suggesting that CXCR3 plays a role in the control of pathology in the brain, the site where chronic infection occurs. Therefore, to further analyze the function of CXCR3 in the brain, we profiled the gene expression patterns of primary astrocytes and microglia by RNA sequencing and subsequent analyses. CXCR3 deficiency impaired the normal upregulation of immune-related genes during T. gondii infection, in astrocytes and microglia alike. Collectively, our results suggest that the immune-related genes upregulated by CXCR3 perform a particular role in controlling pathology when the host is chronically infected with T. gondii in the brain.

Toll-Like Receptor 2 is Involved in Abnormal Pregnancy in Mice Infected with Toxoplasma gondii During Late Pregnancy.

Infection with Toxoplasma gondii during pregnancy causes failure of pregnancy maintenance, resulting in fetal death, abortion, stillbirth, or premature birth, but the mechanism of disease onset remains unclear. Although Toll-like receptor 2 (TLR2) is expressed on antigen-presenting cells and trophoblasts, the role of TLR2 in T. gondii infection during pregnancy is unknown. In this study, we investigated the role of TLR2 in congenital toxoplasmosis using TLR2-deficient (TLR2-/-) mice. T. gondii infection on gestational day 12.5 (Gd12.5) induced more abnormal pregnancy, including premature birth and stillbirth, in wild-type mice than in TLR2-/- mice. Multiple calcifications were observed in the placentas of the infected wild-type mice. At Gd18.5 (6days postinfection), the parasite numbers in the placenta and uterus and the histological changes did not differ significantly between the wild-type and TLR2-/- mice. However, T. gondii infection reduced the mRNA expression of interleukin-12p40 (IL-12p40) and increased IL-4 and IL-10 mRNAs in the placentas of the wild-type mice. In contrast, the placentas of the TLR2-/- mice showed no changes in the expression of these cytokines, including IL-6 and tumor necrosis factor α, in response to T. gondii infection. Serum interferon-γ levels were significantly lower in the infected TLR2-/- mice than in the infected wild-type mice on Gd18.5. Thus, the TLR2-/- mice were less susceptible to the induction of immune responses by T. gondii infection during late pregnancy. Therefore, TLR2 signaling may play a role in the development of disease states during pregnancy, specifically placental hypofunction.

Age-Related Arteriolar Changes With Lipid and Amyloid Deposits in the Gonads of Dogs.

Arteriolar lesions with lipid and/or amyloid deposits are frequently detected in canine gonads by routine histopathologic examination; however, they have never been examined in detail. In the present study, a total of 139 testes/epididymides and 200 ovaries from 72 male (4 months to 14 years old) and 105 female (7 months to 16 years old) dogs were examined for arteriolar lesions. Arteriolar lesions were detected in 21 of 72 male dogs (29%) and 54 of 105 female dogs (51%). These lesions were histologically classified into 4 types: "fibromuscular hypertrophy," characterized by thickening of the tunica intima; "focal vasculitis," characterized by mononuclear cell infiltration; "vacuolar change," consisting of lipid accumulation and infiltration of foamy cells; and "hyalinosis," characterized by irregular thickening with amyloid deposits. In the lesions of vacuolar change and hyalinosis, lipid deposition and infiltration of α-SMA-positive cells and Iba-1-positive cells were also observed. Foamy cells and amyloid deposits were immunopositive for apolipoproteins and oxidized low-density lipoprotein-related proteins. These results indicate that vacuolar change is possibly an early stage of atherosclerosis, and that amyloid may deposit as a consequence of the microenvironment associated with atherogenesis. Logistic regression analysis revealed that arteriolar lesions with lipid deposits were associated with age and interstitial cell tumors in male dogs, and with age in female dogs. Aging is likely an important risk factor of arteriolar lesions with lipid deposits of the canine gonads.

Chronic Inflammatory and Proliferative Lesions of the Gallbladder in Aged Pigs.

Primary epithelial tumors of the gallbladder are rarely reported in animals. In this study, 9 aged pigs (6-12 years old) were histopathologically examined for gallbladder proliferative lesions. At necropsy, a large gallstone occupied the lumen of the gallbladder of 3 pigs. Histopathological examination revealed chronic cholecystitis in all 9 pigs, mucosal hyperplasia in 2 pigs, adenoma in 1 pig, and adenocarcinoma in 2 pigs. Bacilli were detected in the gallbladder lumen of 6 pigs by Warthin-Starry stain. Mucosal hyperplasia, adenoma, and adenocarcinoma were characterized by papillary projections of the mucosa with occasional acinar structures. Tumor invasion of the surrounding tissue was observed in the cases of adenocarcinoma. On Alcian blue and periodic acid-Schiff double-stained sections, the acinar structure of gallbladder mucosa in chronic cholecystitis and mucosal hyperplasia was stained in a mosaic pattern, indicating pyloric gland metaplasia. The results of immunohistochemistry revealed a CD10-positive epithelial brush border and mucin (MUC) 2-positive goblet cells in chronic cholecystitis, adenoma, and adenocarcinomas, indicating intestinal metaplasia. Immunoreactivity of MUC5 AC and cytokeratin 19 was weaker in adenoma and adenocarcinomas compared with the normal and hyperplastic gallbladder mucosa. The number of p53-positive nuclei and the Ki-67 index were higher in adenocarcinomas compared with benign lesions. These results suggest that chronic cholecystitis associated with gallstones and/or bacterial infections may contribute to metaplastic changes and development of gallbladder tumors in aged pigs. Alteration of mucin, cytokeratin, and p53 profiles in gallbladder proliferative lesions in pigs were similar to that in humans, suggesting a common pathogenesis in tumor development.

A retrospective survey on canine intracranial tumors between 2007 and 2017.

To clarify the prevalence of canine intracranial tumors in Japan, a retrospective study was performed using data on 186 canine intracranial tumors. Of 186 cases, 159 cases (85.5%) were primary and 27 cases (14.5%) were secondary intracranial tumors. Among primary intracranial tumors, meningioma (50.9%) was the most common, followed by glial tumors (21.4%) and primary intracranial histiocytic sarcoma (12.6%). These 3 tumors were most frequently found in middle-aged to elderly dogs without any sex predilection. Regarding glial tumors, the incidence of oligodendroglial tumors (79.4%) was higher than that of astrocytic tumors (17.6%). A significant breed predisposition (P<0.05) was observed for meningioma in Rough Collie, Golden Retriever, Miniature Schnauzer, and Scottish Terrier; for glial tumors in Bouvier de Flandres, French Bulldog, Newfoundland, Bulldog, and Boxer; for primary intracranial histiocytic sarcoma in Pembroke Welsh Corgi, Siberian Husky, and Miniature Schnauzer. The high incidence of oligodendroglial tumors in dogs and the breed predisposition for primary intracranial histiocytic sarcoma in Pembroke Welsh Corgi have not been reported in previous epidemiological studies on canine tumors. Since the incidence of intracranial tumors was vary among dog breeds, the present results demonstrate the uniqueness of the canine breed population in Japan.

Generation of a GFP Reporter Akabane Virus with Enhanced Fluorescence Intensity by Modification of Artificial Ambisense S Genome.

We previously generated a recombinant reporter Akabane virus expressing enhanced green fluorescence protein (eGFP-AKAV), with an artificial S genome encoding eGFP in the ambisense RNA. Although the eGFP-AKAV was able to detect infected cells in in vivo histopathological study, its fluorescent signal was too weak to apply to in vivo imaging study. Here, we successfully generated a modified reporter, eGFP/38-AKAV, with 38-nucleotide deletion of the internal region of the 5' untranslated region of S RNA. The eGFP/38-AKAV expressed higher intensity of eGFP fluorescence both in vitro and in vivo than the original eGFP-AKAV did. In addition, eGFP/38-AKAV was pathogenic in mice at a comparable level to that in wild-type AKAV. In the mice infected with eGFP/38-AKAV, the fluorescent signals, i.e., the virus-infected cells, were detected in the central nervous system using the whole-organ imaging. Our findings indicate that eGFP/38-AKAV could be used as a powerful tool to help elucidate the dynamics of AKAV in vivo.

A cutaneous mixed tumor in a dog.

The atypical cutaneous tumor of a 9-year-old mixed breed female dog was examined. The tumor was well-demarcated and histologically composed of a trichoblastic area, tricholemmal area and apocrine glandular area. Neoplastic cells in trichoblastic area and tricholemmal area had PAS-positive granules in the cytoplasm and were positive for pan-cytokeratin, cytokeratin 5/6, 14 and 19 and p63. Neoplastic cells in trichoblastic area were also positive for cytokeratin 15 and CD34. Neoplastic cells in apocrine glandular area were positive for pan-cytokeratin and cytokeratin 7, 18 and 19. Myoepithelial cell proliferation with osteocartilaginous metaplasia was observed in this area. Since neoplastic cells showed multiphenotypic differentiation for hair follicles and apocrine glands, the present case was diagnosed as a cutaneous mixed tumor.

Abdominal hamartoma with pancreatic and hepatic differentiation in a sow.

A 7-year-old Duroc sow exhibited emaciation, loss of appetite and rapid breathing, and was euthanized. Histopathological examination revealed mild to moderate fibrosis of the heart, cystic kidneys and ulcerative enteritis associated with Balantidium infection. Additionally, a small nodule was incidentally found in the peripancreatic fat tissue. The nodule consisted of disarranged cellular components: pancreatic islet cells (either insulin-, glucagon- or somatostatin-positive), pancreatic acinar cells, hepatocytes (human hepatocyte-positive) and ductal cells (cytokeratin 19-positive). Some of the human hepatocyte-positive cells were also positive for chromogranin A and cytokeratin 7, indicating that they were hepatic progenitor cells. The nodule was therefore diagnosed as hamartoma, probably originating from a fragment of the caudal verge of the liver bud, which contains hepatic and pancreatic progenitors.

Sarcocystis calchasi encephalitis in a rock pigeon.

A rock pigeon (Columba livia) caught in Akihabara, Tokyo, showed neurological symptoms, such as head tilt and circling. Pathological examinations revealed abundant Sarcocystic cysts in the skeletal muscle and myocardium with mild myositis, and numerous schizonts and sarcocysts with severe multifocal granulomatous T-lymphocytic infiltration in the central nervous system. A Sarcocystis calchasi-specific gene was detected in the muscle and brain. This case indicates S. calchasi was distributed in Japan and caused severe encephalitis to rock pigeons.