We previously developed a dual-target detection qPCR assay called the BLV-CoCoMo Dual qPCR assay (Daul-CoCoMo assay) for the quantification of bovine leukemia virus (BLV) proviral load (PVL) by improving the original BLV-CoCoMo-qPCR-2 assay (Single-CoCoMo assay). Compared to the Single-CoCoMo assay, the Dual-CoCoMo assay is a simple, user-friendly and cost-effective tool that detects two target genes simultaneously. However, the Dual-CoCoMo assay may have a lower diagnostic sensitivity than the Sinlgle-CoCoMo assay. Here, we aimed to enhance the sensitivity of the Dual-CoCoMo assay by optimizing DNA extraction methods. First the performance of DNA extraction was compared between the conventional Wizard method and a newly selected Pure SPIN method. The average total DNA yields of the Pure SPIN method were higher than the Wizard method. Therefore, PVL was quantified using these DNA samples. In all six samples that were negative by the Wizard+Single-CoCoMo assay, which is the most sensitive conventional method, PVL was quantified by the Pure SPIN+Dual-CoCoMo assay, demonstrating the method is highly effective for quantifying BLV PVL.

The JPN/1/2018 strain of classical swine fever virus (CSFV) currently circulating in Japan is classified as having moderate pathogenicity. Within the last five years, six distinct CSF outbreaks have been documented on pig farms in Tochigi Prefecture. Histopathological analysis was conducted on 23 pigs diagnosed with CSF, and lesion severity was systematically scored to assess pathological features and explore possible underlying factors. Notably, hemorrhagic and lymphocyte-depleted lesions were frequently identified in the spleen, kidneys, and superficial lymph nodes. Nonetheless, the analysis did not reveal any definitive factors influencing the lesion scores. When employed alongside immunohistochemistry (IHC), in situ hybridization (ISH) exhibited sensitivity superior to IHC. Furthermore, ISH alone detected positive signals indicative of encephalitis, thereby establishing a link between CSFV infection and encephalitic lesions.

In Aichi Prefecture, Japan, vaccination against classical swine fever (CSF) was started in October 2019, but low rates of the immunization of animals have been observed on some farms. This study evaluated the relationship between porcine reproductive and respiratory syndrome virus (PRRSV) infection and the proportion of animals immunized via CSF vaccination. A survey was conducted on 69 farms to collect various information and classify them into five stages of infection based on their PRRSV infection status. Univariable and multivariable analyses were performed using generalized linear models, with the PRRS infection stage and other survey items as independent variables. The analysis revealed that farms in PRRS infection stage 2, in which vertical transmission is progressing on the farm (difference in logit −1.303, p＜0.001), and farms that vaccinated against PRRSV (difference in logit −0.525, p＜0.001) had lower a proportion of animals immunized via CSF vaccination. The percentage of PRRS infection stage 2 farms in the region was 7.2%. It may be possible to increase the proportion of animals immunized via CSF vaccination on these farms in the region by improving their PRRS stage.

In October 2022, a 9-month-old beef calf died from pneumonia, and bovine herpesvirus type-4 (BoHV-4) was isolated from the lung. Immunohistochemically BoHV-4 antigen was detected in the lung, suggesting its potential involvement in the pathogenesis of pneumonia. The result of a retrospective antibody survey using 166 serum samples collected from cattle in the Tokachi district between 2018 and 2021 revealed a seropositivity rate of 51.8％ in the region. Additionally, using polymerase chain reaction (PCR) testing, the BoHV-4 genome was detected in the 98 formalin-fixed paraffin-embedded lung tissue of four cattle that had shown signs of pneumonia during the same period. In these four cases, BoHV-4 antigen was immunohistochemically detected in one case. These findings suggest that BoHV-4 is more widespread in the Tokachi district than previously recognized and may play a role in the onset or progression of pneumonia in cattle.

Since 2021, Japan has maintained a surveillance program to preserve its officially tuberculosis-free status in cattle. In this program, intradermal tuberculin testing followed by an interferon-gamma (IFN-γ) assay is used to determine cell-mediated immune responses and identify potentially infected cattle. Nevertheless, in recent years, several cases have been confirmed in which nontuberculous mycobacteria (NTM) other than Mycobacterium bovis were isolated from cattle classified as suspected cases. The data has raised concerns regarding the specificity of the current diagnostic test, particularly in tuberculosis-free countries. In this study, we conducted genomic analysis of five NTM isolates obtained from three cattle suspected of bovine tuberculosis in Japan. Three of these strains were identified as belonging to the same mycobacterial species closely related to the M. terrae complex, which is generally found in environmental sources such as soil. To determine the impact of these isolates on the diagnosis of bovine tuberculosis, we conducted a sensitization experiment using guinea pigs and cattle. Results showed that sensitization with these NTM isolates induced nonspecific immune responses that cross-reacted with antigens derived from M. bovis, suggesting that environmental exposure to NTM may affect test outcomes. However, when the tuberculosis-specific peptide antigen cocktail PC-EC was incorporated into the IFN-γ assay, animals that demonstrated nonspecific positive responses to bovine purified protein derivative (B-PPD) were reclassified as negative. These results suggest that the combined use of PC-EC with conventional B-PPD in IFN-γ assays could improve the diagnostic specificity of bovine tuberculosis surveillance without compromising sensitivity, particularly in tuberculosis-free countries such as Japan.

H9N2 subtype avian influenza virus (AIV) is widely distributed in poultry in Asia, the Middle East, and Africa. If they were introduced into poultry in Japan, the rapid diagnosis of AIVs would be difficult because the current high pathogenicity avian influenza (HPAI) diagnosis system in Japan primarily targets H5 and H7 subtypes and could not identify H9 subtype AIVs. In this study, we compared five real-time RT-PCR methods under the same conditions using in vitro-transcribed RNAs from H9N2 AIV strains (n＝24) belonging to the Y439, G1, and Y280 lineages. The Saito method showed the highest detection efficiency, while reduced sensitivity was observed for one strain in the G1 lineage, possibly due to sequence mismatches within the forward primer binding region. To increase G1 strain detection efficiency, we redesigned the forward primers incorporating degenerate bases. The method using the redesigned primers successfully restored high sensitivity across all strains in three lineages and improved the overall robustness of detection. We also confirmed there was no cross-reactivity of the method using the redesigned primer with AIVs of other subtypes and live vaccine strains of other pathogens. Taken together, these results demonstrated that the method developed in this study provides a way to specifically and sensitively detect H9N2 AIVs, and it is expected to complement the AIV diagnosis system in Japan.

A basic study was conducted on the clinical use of ultrasound biomicroscopy (UBM) with contact-type soft membranes (SM-UBM) in the evaluation of canine iridocorneal angle (cICA). Unclear cICA images were observed when the soft membrane (SM) was used according to the equipment instructions, but it was possible to obtain clear cICA images using SM-UBM with the SM mounting raised, suggesting that the SM mounting should be investigated for the use of SM-UBM in veterinary medicine. The appropriate amount to raise the SM mounting within the range of 1-3 mm and inter- and intra-examiner reliability were examined regarding the opening distance and area of the ciliary cleft-like structures of a thawed porcine eye. The results indicated that the optimal position of the SM was one that was raised 3 mm. The cICA images obtained from SM-UBM with the SM raised 3 mm were comparable to or clearer than those obtained with conventional UBM. These results indicated that an SM in a position that is raised 3 mm was suitable for using SM-UBM to evaluate cICA in the veterinary field. These findings suggest that the cICA can be stably evaluated using SM-UBM under these conditions.

This retrospective study evaluated the efficacy and safety of ropinirole ophthalmic solution compared with intravenous tranexamic acid (TXA) for inducing emesis in dogs. Medical records of 1,161 dogs that underwent emetic induction at Japan Animal Medical Center between 2021 and 2023 were reviewed. Dogs were assigned to a TXA group (n＝581), a ropinirole group (ROP; n＝580), or a combined ropinirole + TXA group (R&T; n＝74). The initial emesis success rate was 25.0% in the TXA group and 70.7% in the ROP group. Adverse events, mostly prolonged vomiting, occurred in 1.9% and 3.1% of dogs in the TXA and ROP groups. The R&T group achieved a 91.9% success rate without adverse events. These findings indicate that ropinirole provides a markedly higher initial success rate than TXA for inducing emesis. In addition, the add-on administration of the alternate drug in cases unresponsive to monotherapy was safely performed. Together, these results suggest that ropinirole is a valuable option for clinical use, while combination therapy may serve as a safe secondary approach when initial treatment fails.

A dog that experienced severe systemic circulatory failure during anesthesia developed abnormal behavior and neurological abnormalities upon awakening. In this case, an additional injection of propofol was administered during inhalation anesthesia, and immediately afterward, a drop in blood pressure and heart rate led to asystole and spontaneous respiratory arrest. Chest compressions, artificial respiration, and an adrenaline injection were performed, and heart rate and breathing resumed within two minutes. Blood pressure also returned to normal within 30 minutes with a continuous infusion of noradrenaline. However, the patient subsequently exhibited violent uncoordinated movements and vocalizations, leading to a suspicion of delirium upon awakening and various treatments. Treatments such as medetomidine and benzodiazepines were ineffective, but the patient was stabilized by Alfaxan administered approximately 20 hours later. It was believed that circulatory failure during anesthesia caused the severe and prolonged abnormal behavior and neurological abnormalities.

The use of protamine in open-heart surgery involving a cardiopulmonary bypass may induce hypotension after administration, although the mechanism for this remains unclear. This study investigated possible factors associated with protamine-induced hypotension (PIH) in dogs. In Study 1, the dose dependency of PIH was evaluated by comparing a group of dogs that received 4 mg/kg (n＝6) and a group that received 2 mg/kg (n＝6). In Study 2, the same 12 dogs received 1 mg/kg of protamine to assess potential allergic involvement. Mean arterial pressure, the number of PIH events, and the time to PIH onset were measured. Although no significant difference was detected (P＝0.221), PIH occurred more frequently in the higher-dose group (3/6 vs. 1/6), and most cases (77.8%) developed within 10 minutes of administration. No significant difference in anti-heparin activity was observed between the 4 mg/kg and 2 mg/kg groups (P＝0.552). These findings suggest a there was higher incidence of PIH in the higher-dose group, but the association between dose and PIH remains unclear. Allergic involvement could neither be confirmed nor excluded (P＝0.558).

A 45-day old broiler chicken with a gastrointestinal tract duplication cyst was subjected to pathological investigation. A spherical cyst, measuring 30 mm in diameter, was closely attached near the center of the pancreas, with separated ventral and dorsal lobes in the adhesion area. The cyst was unilocular, showed a nearly smooth inner surface, and the lumen was filled with pale mucus. There was no communication between the cyst and the pancreatic duct, common bile duct or hepatoenteric duct. Histopathologically, the cystic wall consisted of lining epithelia similar to intestinal mucosal epithelia, lamina propria, smooth muscular layers and capsules. In some areas of the pancreas that adhered to the cyst, acinar cell degeneration with dilatated acinar lumina and perivascular lymphocyte infiltration in the stroma were observed. The positional relationship, with the pancreas tissue encompassing the cyst, might suggest that an embryological abnormality developing into the formation of the cyst had initiated before the pancreatic anlages separated from the foregut. This is the first report of a gastrointestinal tract duplication cyst of the pancreas in a chicken.

From August 2023 to August 2024, we collected cecal feces from horses and rectal feces from cows brought into the slaughterhouse under our jurisdiction. After enrichment culturing, we performed genetic screenings for Shiga toxin-producing Escherichia coli (STEC) and attempted to isolate bacteria from positive samples. As a result, a non-major serogroup STEC was isolated from one horse, STEC Og157:Hg7 from one cow, STEC Og26 from one cow, and STEC from non-major serogroups were isolated from 22 cows. Furthermore, there was no significant seasonal differences in the STEC carrier rate for cows. The STEC carrier rate for horses was significantly lower than that for cows, suggesting that the risk of STEC contamination of horse carcasses originating from the contents of horse digestive tracts during horse slaughter seem to be low. In contrast, the results suggested that cows may be carrying STEC to a certain degree regardless of the season and slaughterhouses must consistently strive to thoroughly manage sanitation, including measures to prevent secondary contamination.