Quantitative analysis of viremia and viral shedding in pigs infected experimentally with classical swine fever virus isolates obtained from recent outbreaks in Japan.

Although classical swine fever occurred in September 2018 for the first time in 26 years, its virulence is thought to be moderate based on field observations by veterinary authorities and our previous experimental infections. We quantified viremia and viral shedding in pigs infected with recent Japanese classical swine fever virus isolates, as well as a highly virulent strain. The results show that pigs infected with the Japanese strains exhibited lower viremia and viral shedding than those infected with the highly virulent strain. However, horizontal transmission occurred in pigs infected with the Japanese strains, similar to those infected with the highly virulent strain. Additionally, viremia and neuralization antibodies coexisted in pigs infected with the Japanese strains, presenting challenges for control measures.

Risk of transmission of foot-and-mouth disease by wild animals: infection dynamics in Japanese wild boar following direct inoculation or contact exposure.

Understanding of disease dynamics and viral shedding in wild boar and of the potential for disease spreading within wild boar and domestic pig populations is critical for developing effective control and eradication measures for foot-and-mouth disease (FMD). Accordingly, we infected experimentally wild boar and domestic pigs with FMD virus (FMDV) strains O/TAI/315/2016 and A/MOG/2013, and studied their susceptibility and viral transmissibility in both populations. Similar to FMDV-infected pigs, wild boar inoculated with both viruses exhibited vesicular lesions on their feet, snout, tongue and lip, although they did not show lameness. Further, inoculated wild boar were equally capable of transmitting the virus to all of their contact animals. While all contact pigs developed vesicular lesions after contact with inoculated animals, in contrast, no wild boar when exposed to the same infected animals showed obvious clinical signs. These results will be useful for further understanding of the critical roles in occurring and sustaining an FMD outbreak, and will be useful for establishing epidemiological surveillance programs and effective countermeasures for wild boar.

Toward better control of classical swine fever in wild boars: susceptibility of boar-pig hybrids to a recent Japanese isolate and effectiveness of a bait vaccine.

We analyzed the pathogenicity of a recent Japanese classical swine fever virus (CSFV) to wild boars via an experimental infection using boar-pig hybrids as an alternative to wild boars. We also investigated the effectiveness of a bait vaccine against the CSFV. Naïve boar-pig hybrids and pigs showed clinical signs such as fever, leucopenia, anorexia and conjunctivitis following the experimental infection. In contrast, the boar-pig hybrids administered the bait vaccine did not show any clinical signs. Our data indicated that boar-pig hybrids and domestic pigs have similar susceptibility to the recent Japanese CSFV. Additionally, the bait vaccine is effective against the CSFV.

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.

Microminipigs as a new experimental animal model for toxicological studies: comparative pharmacokinetics of perfluoroalkyl acids.

In this study, we evaluated the efficacy of a novel minipig strain, the Microminipig (MMPig), as an animal model for studying the pharmacokinetics of a mixture of 10 perfluoroalkyl acids (PFAAs). After a single oral dose was given, we found that the blood depuration of PFAAs (blood t1/2), which we calculated using first-order elimination curves, ranged from 1.6 to 86.6 days. Among the five body compartments analyzed, the liver was the greatest site of accumulation of perfluorooctanesulfonate and longer chain perfluorinated carboxylates such as perfluorodecanoic acid, perfluoroundecanoic acid and perfluorododecanoic acid. We observed an increasing accumulation trend of perfluorinated carboxylates in the organs associated with the fluorinated carbon chain length. The perfluorononanoic acid burden was the highest among the treated compounds 21 days after a single exposure, as 29% of the given perfluorononanoic acid dose was accumulated in the tissues. The persistence of PFAAs in edible pig tissues even after 21 days post-exposure raises concerns about the safety of swine products. This was the first study to use MMPigs to elucidate the pharmacokinetics of a group of environmental pollutants. We found that MMPigs could be excellent experimental animals for toxicological studies due to their easy handling, cost efficacy for target compounds and ease of waste treatment.

Effect of doubled dose administration of foot-and-mouth disease vaccine against heterologous virus infection in cattle.

Vaccination is a feasible approach for controlling foot-and-mouth disease (FMD). In FMD-free countries, vaccines are stored as a precautionary measure to control potential outbreaks. However, the challenge lies in pre-stocking optimal vaccines against the newly emerging strains. This study examined the potency of pre-stocked vaccines administered at elevated doses during emergencies. We vaccinated the cows with either a single or double trivalent vaccine dose containing two serotype O and one serotype A strains. Subsequently, vaccinated and unvaccinated cows were exposed to virulent strains of serotype O (O/JPN/2010; topotype Southeast Asia/Mya-98 lineage) or A (A/IRN/2016; topotype ASIA/G-VII lineage), which were genetically and antigenically distinct from the vaccine strains. Following challenge infections, all cows that received a single dose vaccination exhibited vesicular lesions with excreted viruses in the oral and nasal discharges. However, a substantial reduction was observed in the total clinical scores and virus titers in the sera and nasal discharges compared to those in the unvaccinated group. Cows receiving a doubled dose vaccination were completely protected from infection with O/JPN/2010 or demonstrated a significant decrease in viral shedding and clinical scores against A/IRN/2016. To note, vesicular lesions harbor significant amounts of viruses; thus, by mitigating their formation, viral transmission can be impeded, thereby slowing viral spread in the field. Furthermore, increasing the vaccine dose induced higher neutralizing antibody titers against heterologous strains. These findings suggest an alternative strategy for the effective management of future epidemics using pre-stocked vaccines.

Development of a Novel Indirect ELISA for the Serological Diagnosis of African Swine Fever Using p11.5 Protein as a Target Antigen.

African swine fever is a hemorrhagic viral disease with a mortality rate of nearly 100% in pigs. Hence, it is classified as a notifiable disease by the World Organization for Animal Health. Because no field-available vaccine exists, African swine fever virus (ASFV) control and eradication solely depend on good farm biosecurity management and rapid and accurate diagnosis. In this study, we developed a new indirect serological enzyme-linked immunosorbent assay (ELISA) using recombinant p11.5 protein from ASFV as a solid-phase target antigen. The cutoffs were determined by receiver operating curve analysis performed with serum samples obtained from naïve and infected pigs. Based on the results of a commercially available serological ELISA, the relative sensitivity and specificity of our assay were 93.4% and 94.4% (N = 166; area under the curve = 0.991; 95% confidence interval = 0.982-0.999), respectively. Furthermore, to compare the performance of the serological ELISAs, we conducted the assays on a panel of sera collected from pigs and boars experimentally infected with different ASFV isolates. The results indicated the greater sensitivity of the newly developed assay and its ability to detect anti-ASFV antibodies earlier after virus inoculation.

A Spontaneously Occurring African Swine Fever Virus with 11 Gene Deletions Partially Protects Pigs Challenged with the Parental Strain.

African swine fever (ASF) is an infectious Suidae disease caused by the ASF virus (ASFV). Adaptation to less susceptible, non-target host cells is one of the most common techniques used to attenuate virulent viruses. However, this may induce many mutations and large-scale rearrangements in the viral genome, resulting in immunostimulatory potential loss of the virus in vivo. This study continuously maintained the virulent ASFV strain, Armenia2007 (Arm07), to establish an attenuated ASFV strain with minimum genetic alteration in a susceptible host cell line, immortalized porcine kidney macrophage (IPKM). A mutant strain was successfully isolated via repeated plaque purification in combination with next-generation sequencing analysis. The isolated strain, Arm07ΔMGF, which was obtained from a viral fluid at a passage level of 20, lacked 11 genes in total in the MGF300 and MGF360 regions and showed marked reduction in virulence against pigs. Moreover, all the pigs survived the challenge with the parental strain when pigs were immunized twice with 105 TCID50 of Arm07ΔMGF, although viremia and fever were not completely prevented after the challenge infection. These findings suggest that this naturally attenuated, spontaneously occurring ASFV strain may provide a novel platform for ASF vaccine development.

Pathology of spontaneous tumour lesions in pullets and adult chickens in commercial farms - Short communication.

Twenty pullets and adult chickens, aged 100 to 403 days, from several commercial chicken farms were examined by gross and histopathology. Grossly, all chickens had white-greyish masses in the visceral organs with or without enlargement of the peripheral nerves. Histopathological examination revealed Marek's disease (MD) lymphoma, lymphoid leukosis (LL) and myeloid leukosis (ML) in 14/20, 5/20 and 1/20 of the chickens, respectively. Lesions of the sciatic nerves in chickens diagnosed as having MD lymphoma were various. No neoplastic and/or inflammatory cells were noted in the peripheral nerves of chickens diagnosed as having LL and ML. These results indicated that MD lymphoma could also develop in older chickens; thus, microscopic examination is needed to identify MD in older chickens showing lymphocyte-derived tumours.

Relationship between tumour development and detection of Marek's disease virus in the feather follicular epithelium of older chickens.

To demonstrate the relationship between tumour development and virus replication, eight specific-pathogen-free pullets of line P2 (Group P; 14 weeks old) and five adult chickens (Group A; 96 weeks old) were inoculated with virulent Marek's disease virus (vMDV). Five chickens of Group P died or were euthanised due to moribund condition following the development of neoplastic lesions between days 53 and 91. On histopathological examination, these lesions were characterised by the proliferation of lymphoid cells of variable size. On analysis by polymerase chain reaction (PCR), the MDV meq gene was detected in Group P from day 21, and it was continuously identified in five chickens until they died or were euthanised. Abnormal signs and histopathological changes were not observed in chickens of Group A. The MDV meq gene was temporarily detected in some chickens of Group A, but it remained almost undetectable throughout the experimental period. In older chickens inoculated with vMDV, the onset of MD lymphoma development tended to be delayed as compared with the young chicks. The relationship between MD lymphoma development and virus replication in older chickens has been suggested. Our data might indicate the underlying existence of an age-related resistance to vMDV challenge.

Immunohistochemical analysis of the distribution of classical swine fever (CSF) viral antigen in boar-pig hybrids and pigs four weeks after infection.

We detected the classical swine fever virus (CSFV) antigen in three boar-pig hybrids (hybrids) and three pigs. All animals were experimentally infected with CSFV strain JPN/27/2019 to optimize diagnostic sampling and risk assessment of virus dissemination. Two hybrids died 17- and 19-days post-inoculation (dpi). The other animals were euthanized at 28 dpi. The detection of CSFV antigen at 28 dpi in epithelial cells of the apocrine sweat and sebaceous glands in the skin, salivary glands, mucosal epithelial cells in the rectum, and epithelial cells in the kidney and urinary bladder, suggests that CSFV persists in these tissues and spreads via sweat, saliva, feces, and urine for at least 4 weeks. These findings reveal that hybrids and pigs represent a high risk of virus dissemination four weeks after infection with CSFV strain JPN/27/2019. Prominent CSFV antigens were also detected in hair follicles of the skin. These results suggest that postmortem sampling of animal skin may be effective for CSF diagnosis and can be used to develop a rapid and easy diagnostic method using hair follicles.

Usability of Immortalized Porcine Kidney Macrophage Cultures for the Isolation of ASFV without Affecting Virulence.

Immortalized porcine kidney macrophage (IPKM) cells are highly susceptible to major African swine fever virus (ASFV) isolates. To clarify the compatibility of this cell line for ASFV isolation from biomaterials, animal experiments and in vitro isolation were performed. Pork products seized at international airports were subjected to virus inoculation in pigs (in vivo) and IPKM cell cultures (in vitro) to examine the viability and virulence of the contaminating viruses. Moreover, the viruses isolated using IPKM cells were inoculated into pigs to assess the virulence shift from the original materials. All pigs that were inoculated with either homogenate samples of seized pork product or IPKM-isolated ASFVs developed typical symptoms of ASF and died (or were euthanized) within the term of the animal experiments. The success rate of virus isolation in IPKM cells was comparable to that observed in porcine primary alveolar macrophage (PAM) cells. The IPKM cell line would be an ideal tool for the isolation and propagation of live ASFVs with high efficiency and enhanced usability, such as immortal, proliferative, and adhesive properties. The isolated viruses retained biologically similar characteristics to those of the original ones during isolation in vitro.

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.

Administration of the antiviral agent T-1105 fully protects pigs from foot-and-mouth disease infection.

Foot-and-mouth disease (FMD) is a contagious disease affecting cloven-hoofed animals. Its transmissibility and antigenic variety make this disease difficult to control. Antiviral agents are expected to have an immediate effect that is independent of viral antigenicity; thus, they can serve as effective tools for inhibiting the spread of the causative agent, the FMD virus (FMDV), from infected animals. In this study, we investigated the antiviral activity of a pyrazinecarboxamide derivative, T-1105, against FMDV. Cytopathic effect inhibition assays revealed that T-1105 strongly inhibited the replication of 28 reference strains of all seven FMDV serotypes at non-cytotoxic concentrations. The antiviral effect of T-1105 against FMDV was also evaluated by experimental infection of domestic pigs. T-1105 was administered orally to pigs starting 1 h before or 6 h after the inoculation of a porcinophilic FMDV serotype O, topotype CATHAY. None of the pigs administered with T-1105 showed clinical signs of FMD. Moreover, no infectious FMDVs or FMDV-specific genes were detected in their sera, oral and nasal discharges, or tissues collected 48 h after virus inoculation. These findings strongly suggest that administration of T-1105 is effective in controlling the spread of FMDV in pigs.

Histopathological and Immunohistochemical Studies of Pineoblastoma in a Cow.

We report the first case of pineoblastoma in a cow. At necropsy, a soft, gelatinous greyish yellow, 4 × 3 × 2 cm mass was found midsagittally on the dorsal surface of the midbrain. The mass enveloped the brainstem anterior to the cerebellum, extended to the pituitary fossa and was adherent to the ventromedial aspect of the occipital lobes. Histologically, the mass was unencapsulated, with extramedullary proliferation into the lobes and meninges, but was demarcated from the adjacent brain tissue. Histological findings were consistent with a pineoblastoma, as reported in other species, and the neoplasm was strongly immunopositive for synaptophysin. Glial fibrillary acidic protein-positive spindloid astrocytic processes surrounded blood vessels and extended around neoplastic cells.

Quantitative analysis of infection dynamics of foot-and-mouth disease virus strain O/CATHAY in pigs and cattle.

Foot-and-mouth disease virus (FMDV) serotype O, topotype CATHAY is a known porcinophilic virus that has caused devastating damage to the pig industry. However, the minimum infectious dose via a natural infection route in pigs, the infection dynamics in cattle, and risk of viral transmission from infected cattle to pigs have not been quantitatively analyzed. The FMDV strain O/HKN/1/2015 was serially diluted and inoculated into pigs via an intraoral route to determine the infectious dose. We found that a 104.0 tissue culture infectious dose (TCID50) of the virus was insufficient, but 105.5 TCID50 was sufficient to infect pigs via the oral route. While cows inoculated with the strain showed increased temperature in their feet, typical clinical signs including vesicular development were not observed. The cows showed short-term and low levels of viremia and virus excretion only before the detection of virus neutralizing antibodies. FMDV genes were not detected in esophageal-pharyngeal fluid from cows after 14 days post inoculation. No genetic insertions that could be associated with host adaptation were observed in viruses isolated from infected cows. These findings indicate that cows infected with FMDV of O/CATHAY have a low risk of viral transmission or persistence. Information on the dynamics of virus infection is essential for ensuring the rapid and accurate diagnosis of this disease, and its surveillance.

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact.

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD50)/ml, 101 HAD50/ml and 106 HAD50/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD50/ml and 101 HAD50/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD50/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 103 HAD50/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.

Late Marek's disease in adult chickens inoculated with virulent Marek's disease virus.

Recently, excessive losses from Marek's disease (MD) have been noted in adult laying flocks over the age of 40 weeks. We defined these late outbreaks in adult chickens as "late MD", and experimentally reproduced the disease in adult SPF P2 line (50-week-old) or commercial line (74-week-old) chickens inoculated with a virulent strain of Marek's disease virus (MDV). Commercial line chickens were given MDV vaccines (HVT and CVI 988) at hatch. The occurrence of MD was evaluated periodically by the evidence of neurologic signs such as paralysis, torticollis, ataxia, and/or nervous tics, as well as histopathological examination. In P2 line chickens, neurologic signs and MD lymphoma were observed from day 21 onward, and they tended to increase in a time-dependent manner. Meanwhile, in commercial line chickens, only one chicken exhibited MD lymphoma on day 70 post inoculation, but its pathogenesis was questionable. No regression of MD lymphoma was noted in either case. The lesions in the visceral organs, thymus, peripheral nerves, and feather pulps of P2 line chickens were characterized by proliferation of variably sized lymphoid cells. In the feather follicle epithelium, numerous inclusion bodies were noted on day 21 post-inoculation, which tended to decrease afterwards. The morphological findings obtained resembled late MD in field cases. In conclusion, our results demonstrate that adult SPF P2 line chickens are susceptible to virulent MDV, and would be useful for investigation of late MD.

Changes in hepato-renal gene expression in microminipigs following a single exposure to a mixture of perfluoroalkyl acids.

It is evident that some perfluoroalkyl acids (PFAAs), a group of globally dispersed pollutants, have long biological half-lives in humans and farm animals. However, the effects of PFAAs in domestic animals have not been fully elucidated. The present study investigated how exposure to a single dose of a mixture of 10 PFAAs influenced hepatic and renal gene expression and histopathology, as well as plasma clinical biochemistry, in microminipigs (MMPigs) over 21 days. In animals treated with PFAAs, the mRNA expression of twelve genes related to fatty acid metabolism was upregulated in the kidney, while only few of these genes were induced in the liver. The expression of several kidney injury-associated genes such as, IGFBP1, IGFBP6, GCLC X2, GCLC X3, MSGT1, OLR1 was upregulated in the kidney. Interestingly, the expression of IGFBP-genes was differentially altered in the liver and kidney. Our findings thus identified hepato-renal gene expression changes in MMPigs that were associated with various molecular pathways including peroxisome proliferation, lipid metabolism, kidney injury, and apoptosis. Furthermore, serum HDL levels were significantly decreased following exposure to PFAAs, whereas no significant histopathological changes were detected, as compared to the vehicle group. Taken together, the present study provided the first indication that a single exposure to a mixture of PFAAs can produce changes in MMPig renal gene expression that were observed three weeks post exposure, suggesting that more attention should be paid to the kidney as a primary target organ of PFAAs.