Exposure of Cattle Breeding Herds to Naturally Co-Contaminated Zearalenone and Deoxynivalenol: The Relevance of a Urinary Mycotoxin Monitoring System for Herd Health and Food Safety.

The widespread presence of Fusarium mycotoxins in animal feed is a global issue, not only for the health of livestock but also for ensure the safety of food as an end product. High concentrations of zearalenone (ZEN) and deoxynivalenol (DON) have been detected in the diets of Japanese Black (JB) and Holstein Friesian (HF) breeding herds. Consequently, we monitored serum biochemical parameters over a long time in both herds, focusing on anti-Müllerian hormone (AMH) levels and acute-phase inflammation. Additionally, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and progesterone levels were measured in the HF herd. The JB herd, a ZEN-dominant model with low DON contamination, demonstrated ZEN levels that exceeded the Japanese limit in the purchased total mixed rations (TMR). Conversely, the HF herd, which primary consumes DON-dominant feed with low ZEN contamination, had high DON levels in the dent corn silage. Specifically, the JB herd's TMR contained 1.79 mg/kg ZEN and 0.58 mg/kg DON, whereas the HF herd's silage had 15.3 mg/kg DON (dried sample) and 0.1 mg/kg ZEN. Enzyme-linked immunoassay were used to measure urinary ZEN-DON levels following confirmation through liquid chromatography-tandem mass spectrometry. Urinary ZEN-DON levels measured were significantly correlated (p < 0.05, r > 0.6) in both herds. In the HF herd, AMH levels increased (p = 0.01) and serum amyloid A (SAA) levels decreased (p = 0.02) when contaminated and at the end of the monitoring period. Additionally, urinary ZEN and DON levels were significantly correlated with SAA levels (ZEN: p = 0.00, r = 0.46; DON: p = 0.03, r = 0.33), with an increase in ZEN and DON levels resulting in higher SAA levels. The JB herd showed no significant differences. Additionally, in the HF herd, 8-OHdG/Cre levels increased significantly during major contamination periods (p < 0.05). Clinical data from the HF herd indicated an increase in mastitis cases and treatment rates during periods of major contamination. Abortion rates in the HF herd decreased from 22.9% (before monitoring) to 8.9% (during the high contamination period) and finally to 1% (at the end of the monitoring period), with corresponding increases in progesterone levels. ZEN-DON contamination adversely affects breeding cattle's productivity, reproductive performance, and health. Therefore, monitoring urinary ZEN-DON is valuable for detecting contaminants and ensuring the safety of food products.

Aberrantly Expressed tRNA-Val Fragments Can Distinguish Canine Hepatocellular Carcinoma from Canine Hepatocellular Adenoma.

Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can be difficult to differentiate but must be diagnosed correctly as treatment and prognosis for these tumors differ markedly. Relevant diagnostic biomarkers are thus needed, and those identified in dogs may have utility in human medicine because of the similarities between human and canine HCA and HCC. A tRNA-derived fragment (tRF), tRNA-Val, is a promising potential biomarker for canine mammary gland tumors but has not previously been investigated in hepatic tumors. Accordingly, we aimed to elucidate the potential utility of tRNA-Val as a biomarker for canine HCA and HCC using clinical samples (tumor tissue and plasma extracellular vesicles [EVs]) and tumor cell lines with qRT-PCR assays. We also investigated relevant functions and signaling pathways with bioinformatic analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes). tRNA-Val was markedly downregulated in HCC tumor tissue versus HCA tumor tissue and normal liver tissue, and a similar trend was shown in plasma EVs and HCC cell lines versus healthy controls. Based on areas under the receiver operating characteristic curves (AUCs), tRNA-Val significantly distinguished HCC (AUC = 1.00, p = 0.001) from healthy controls in plasma EVs and HCC from HCA (AUC = 0.950, p = 0.01). Bioinformatics analysis revealed that tRNA-Val may be primarily involved in DNA repair, mRNA processing, and splicing and may be linked to the N-glycan and ubiquitin-mediated proteasome pathways. This is the first report on the expression of tRNA-Val in canine HCC and HCA and its possible functions and signaling pathways. We suggest that tRNA-Val could be a promising novel biomarker to distinguish canine HCC from HCA. This study provides evidence for a greater understanding of the role played by tRNA-Val in the development of canine HCC.

Novel Approach for Evaluating Pregnancy-Associated Glycoprotein and Inflammation Markers during the Postpartum Period in Holstein Friesian Cows.

We evaluated the relationship between decreased pregnancy-associated glycoprotein (PAG) levels, inflammatory parameters (serum amyloid A [SAA] and milk amyloid A [MAA]), postpartum inflammatory conditions (mastitis, ketosis, and follicular cysts), and the FOXP3 gene. Nineteen Holstein-Friesian cows were included in this study. Up to approximately eight weeks after delivery, weekly health examinations were performed for mastitis and ketosis, and reproductive organ ultrasonography was performed. The decreasing PAG rate was negatively correlated with SAA concentration (r = -0.493, p = 0.032). Cows with mastitis exhibited a slower trend of PAG decrease (p = 0.095), and a greater percentage of these cows had MAA concentrations above 12 µg/mL (p = 0.074) compared with those without mastitis. A negative correlation, although nonsignificant (r = -0.263, p = 0.385), was observed between the day-open period and decreased PAG rate. The day-open period was correlated with the presence or absence of follicular cysts (p = 0.046). Four cows that developed follicular cysts were homozygous for the G allele of the FOXP3 gene related to repeat breeders. These results indicate a relationship between a decreased PAG rate and inflammatory status during the postpartum period. Thus, suppressing inflammation during the perinatal period may improve reproductive efficiency in the dairy industry.

Transcript abundance of hepatic drug-metabolizing enzymes in two dog breeds compared with 14 species including humans.

Drug-metabolizing enzymes are important in drug development and therapy, but have not been fully identified and characterized in many species, lines, and breeds. Liver transcriptomic data were analyzed for phase I cytochromes P450, flavin-containing monooxygenases, and carboxylesterases and phase II UDP-glucuronosyltransferases, sulfotransferases, and glutathione S-transferases. Comparisons with a variety of species (humans, rhesus macaques, African green monkeys, baboons, common marmosets, cattle, sheep, pigs, cats, dogs, rabbits, tree shrews, rats, mice, and chickens) revealed both general similarities and differences in the transcript abundances of drug-metabolizing enzymes. Similarly, Beagle and Shiba dogs were examined by next-generation sequencing (RNA-seq). Consequently, no substantial differences in transcript abundance were noted in different breeds of pigs and dogs and in different lines of mice and rats. Therefore, the expression profiles of hepatic drug-metabolizing enzyme transcripts appear to be similar in Shiba and Beagle dogs and pig breeds and the rat and mouse lines analyzed, although some differences were found in other species.

YRNA and tRNA fragments can differentiate benign from malignant canine mammary gland tumors.

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA-derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio.

Severe glomerular fibrin thrombosis in a dog.

Here, we present a case of severe glomerular fibrin thrombosis in a dog with lymphoma. A 3-year-old neutered male Chihuahua presented with acute kidney injury, hypoalbuminemia, and transudate ascites. The dog showed symmetric enlargement of the spleen, which was diagnosed as B-cell lymphoma based on cytology and polymerase chain reaction tests. The dog died after intensive care, and the kidneys were removed for histopathological examination. Light microscopy, immunofluorescence, and electron microscopy analyses were performed for renal pathology; however, the findings did not support the evidence of protein-losing nephropathy. Instead, the endocapillary accumulation of fibrin thrombi was prominent in most glomeruli. A diagnosis of severe glomerular fibrin thrombosis was established, and hypoalbuminemia was considered the underlying cause of kidney damage.

Effects of dietary feed supplementation with heat-treated Lactobacillus sakei HS-1 on the health status, blood parameters, and fecal microbes of Japanese Black calves before weaning.

Background and Aim: Our previous research suggested that heat-killed Lactobacillus sakei HS-1 (HK-LS HS-1) is potentially beneficial for improving intestinal microbes and reducing the number of medical treatments. This study aimed to investigate the effect of HK-LS HS-1 as a supplement in milk replacers (MRs) on clinical health during the 1-month preweaning period. Materials and Methods: Eighteen female calves were randomly assigned to either a group receiving the HK-LS HS-1 supplement (n = 9) or a control group without it (n = 9). We then investigated the effect of including supplementary HK-LS HS-1; 0.2% in MRs twice daily at 09:00 and 16:00 on the health, serum biochemical parameters (measured using an automated biochemical analyzer), and fecal bacteriological changes of preweaning Japanese Black calves at the day of the start of supplementation (before HK-LS HS-1 supplementation; day 0), at weaning (day 30), and at 2 weeks (day 45) and 4 weeks (day 60) after weaning. Results: During the supplementation period (0-30 days), (1) an increase (p = 0.023) was observed in albumin, and there was a tendency of increase in total cholesterol level in the HK-LS HS-1 group but not in the control group; (2) substantial differences were obtained after the weaning period (30-60 days), although no differences were observed from 0-30 days in both groups. The anti-Müllerian hormone (AMH) level was substantially increased after weaning in the control group. No differences were observed in the amounts of Coliform spp. and Staphylococcaceae spp. between the two groups; thus, HK-LS HS-1 supplementation had similar antibacterial effects. A significant reduction was observed in the time to weaning of the HK-LS HS-1 group in the field trial. Conclusion: Supplementation with HK-LS HS-1 from an early stage after birth to weaning is a cost-effective treatment to improve the growth rate of preweaning calves. However, supplementation during only preweaning periods appears to have no beneficial effects on preventing weaning stress, especially in terms of AMH levels.

Therapeutic effect of aged garlic extract on gingivitis in dogs.

Periodontal disease is one of the most common dental health problems in dogs. Clinical studies in humans have shown that aged garlic extract (AGE), which contains stable and water-soluble sulfur-containing bioactive compounds, improves the symptoms of periodontal diseases. Our previous study demonstrated that oral administration of AGE in healthy Beagle dogs at 90 mg/kg/day for 12 weeks had no adverse effects such as hemolytic anemia, which is well known to occur as a result of ingestion of Allium species, including onions and garlic, in dogs. However, the therapeutic potential of AGE in canine periodontal disease remains unclear. Accordingly, we investigated the therapeutic effects of AGE in Beagle dogs with mild gingivitis. Feeding 18 mg/kg/day of AGE for 8 weeks resulted in the improvement of gingival index score, level of volatile sulfur compounds in exhaled air, and enzyme activity of periodontal pathogens without any adverse effects on clinical signs and hematological and serum biochemical parameters. Moreover, AGE increased the concentration of salivary cathelicidin, an antimicrobial peptide that contributes to the oral innate immune response. These results suggest that AGE could be a potential therapeutic agent for canine gingivitis.

Immunohistochemical analysis of renal oxidative damage in senior and geriatric cats with chronic kidney disease.

Oxidative stress is a well-known cause of chronic kidney disease (CKD). In this study, renal oxidative damage in azotaemic and non-azotaemic aged cats with naturally occurring CKD was investigated using immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxynonenal (4-HNE) as markers of oxidative tissue damage. Kidneys were obtained from aged (>10 years old) azotaemic (n = 13) and non-azotaemic (n = 7) cats. Immunoreactivity for 8-OHdG was found in the nuclei of glomeruli, proximal and distal tubules, loops of Henle and collecting ducts, whereas 4-HNE-positive signals were detected in the cytoplasm of distal nephrons in azotaemic and non-azotaemic cats. Quantitative analysis did not identify any significant differences between the azotaemic and non-azotaemic groups for any of the parameters examined. These results indicate that renal oxidative damage occurs in the kidneys of aged cats with CKD, regardless of whether they are azotaemic or non-azotaemic, emphasizing the importance of oxidative stress during early-stage CKD in senior and geriatric cats.

Usefulness of Measuring Serum Amyloid A Concentration in Japanese Black Cattle in Clinical Practice.

This study investigated the concentration of serum amyloid A (SAA), an acute phase protein, in Japanese Black cattle. Four practical trials were performed to evaluate the transition of SAA and sialic acid before and after dehorning, the relationship between the SAA concentration and other blood test parameters, the SAA dynamics in the diseased cattle, and the blood test results, including the SAA concentrations, of the two cases with a follow-up. The SAA concentration increased with dehorning but decreased 7 days after dehorning. The SAA concentration is positively correlated with the α-globulin, sialic acid, and fibrinogen concentrations and negatively correlated with the serum iron concentration. The SAA concentration in the deceased herd was significantly higher than that in the cured outcome herd. In addition, the SAA concentration in the cured group decreased significantly from the first test to retesting but increased significantly in the disuse group. Thus, SAA is a sensitive index of inflammation and a monitoring tool in Japanese Black cattle, and its measurement is considered useful in clinical practice.

Measurement of serum short-chain fatty acid concentrations in cattle after oral administration of difructose anhydride III.

Background and Aim: We previously reported the mitigation effects of difructose anhydride III (DFA III) on mycotoxins, such as zearalenon and sterigmatocystin, based on the urinary concentrations of these molecules in calves. This study was aimed at evaluating the effects of dietary supplementation of DFA III and the fermented status of DFA III in the intestine by comparing serum levels of short-chain fatty acid (SCFAs) in DFA III-supplemented cattle with those in non-supplemented control cattle. Materials and Methods: Serum SCFA concentrations were measured in 30 Japanese Black heifers, aged 9-10 months, from two herds, using gas chromatography on days 0 (before DFA III supplementation), 9, and 14 after DFA III supplementation. Results: A notably different trend was observed for isobutyric acid and enanthic acid, which may reflect the different fermentation status of supplementary DFA III in the intestine. Our results indicate the possibility that this trend observed in the intestinal tract following DFA III administration is associated with changes in the environment of intestinal bacterial flora, which may partially reflect the effects of DFA III supplementation on cattle. Conclusion: Difructose anhydride III supplementation for at least 2 weeks affects the trend of blood SCFA concentrations in cattle. Our results provide evidence supporting the effects of DFA III on the intestinal environment and intestinal barrier function.

Acupuncture Treatment Improves Transport Stress in Microminipigs Through the Acupoint in Ears.

BACKGROUND/AIM: This study aimed to investigate the effects of acupuncture treatment through the ear acupoints on transport stress in experimental microminipigs. MATERIALS AND METHODS: Experiment 1: Six animals were equally divided into two groups (Control and Treatment). In the treatment group, before transportation (6 h; vehicle and plane), short, ultrathin circular transdermal needles were applied to locations corresponding to the acupoints on the apical area of both ears. Peripheral blood samples were collected from the cranial vena cava 2 days before and immediately after transportation. Blood stress markers, biochemistry indicators, and oxidative stress levels were examined. Experiment 2 (follow-up study: diarrhea incidence after transportation): Diarrhea incidence after transportation in the control and treatment groups was investigated. RESULTS: Experiment 1: Transport stress induced an increase in blood cortisol, serum amyloid A (SAA), glucose, non-esterified fatty acid, and derivatives of reactive oxygen metabolites (d-ROMs) and decreased the biological antioxidant potential (BAP)/d-ROMs ratio yet did not affect BAP. Acupuncture suppressed the increases in SAA and d-ROMs values and the decrease in BAP/d-ROMs ratio. Experiment 2: The total diarrhea incidence was 25% in the control group, whereas diarrhea was not observed in the treatment group. CONCLUSION: Acupuncture treatment suppresses hypothalamic-pituitary-adrenal function and, as a result, reduces transport stress without affecting the suppression of the central catecholaminergic system. Acupuncture treatment for transport stress can improve animal welfare.

Pyruvate kinase deficiency mutant gene carriage in stray cats and rescued cats from animal hoarding in Hokkaido, Japan.

The mutant allele frequency of the Pyruvate kinase (PK) gene has been investigated mostly in pure breed cats. We investigated the PK mutant gene in stray and animal hoarding mongrel cats in Hokkaido, Japan. We also investigated the kinship of individuals carrying the mutant gene. Genotyping was conducted using the previously reported real-time PCR method. Fourteen microsatellite markers were used to identify the parents and offspring of cats carrying the PK mutant gene, and some kinship such as parent-offspring and siblings was observed. Some stray and animal hoarding cats carried the PK mutation gene and that consanguinity was confirmed among these cats indicated that the PK mutation gene was spread by unregulated interbreeding.

Novel Mutation in the Feline NPC2 Gene in Cats with Niemann-Pick Disease.

Niemann-Pick disease (NP) type C is an autosomal, recessive, and inherited neurovisceral genetic disorder characterized by the accumulation of unesterified cholesterol and glycolipids in cellular lysosomes and late endosomes, with a wide spectrum of clinical phenotypes. This study aimed to determine the molecular genetic alterations in two cases of felines with NP in Japan, a Siamese cat in 1989 and a Japanese domestic (JD) cat in 1998. Sanger sequencing was performed on 25 exons of the feline NPC1 gene and 4 exons of the feline NPC2 gene, using genomic DNA extracted from paraffin-embedded tissue specimens. The sequenced exons were compared with reference sequences retrieved from the GenBank database. The identified mutations and alterations were then analyzed using different prediction algorithms. No pathogenic mutations were found in feline NPC1; however, c.376G>A (p.V126M) was identified as a pathogenic mutation in the NPC2 gene. The Siamese cat was found to be homozygous for this mutation. The JD cat was heterozygous for the same mutation, but no other exonic NPC2 mutation was found. Furthermore, the JD cat had a homozygous splice variant (c.364-4C>T) in the NPC2 gene, which is not known to be associated with this disease. The NPC2:c.376G>A (p.V126M) mutation is the second reported pathogenic mutation in the feline NPC2 gene that may be present in the Japanese cat population.

Practical Application of a Urinary Zearalenone Monitoring System for Feed Hygiene Management of a Japanese Black Cattle Breeding Herd-Relevance to Anti-Müllerian Hormone and Serum Amyloid A Clarified from a Two-Year Survey.

In this study, a herd of Japanese Black (JB) breeding cattle with sporadic reproductive disorders was continuously monitored for an additional year to assess the effects of the urinary zearalenone (ZEN) concentration and changes in parameters (AMH and SAA) with time-lag variables and herd fertility (reproductive performance). This herd had high (exceeded the Japanese dietary feed regulations) urinary ZEN and rice straw ZEN concentrations (1.34 mg/kg). Long-term data of the herd with positive ZEN exposure revealed a decreasing ZEN concentration in urine and a gradual decrease in the AMH level with age. The AMH level was significantly affected by the ZEN value 2 months earlier and the AMH level in the previous month. The changes in ZEN and SAA values were significantly affected by the ZEN and SAA values in the previous month. Additionally, calving interval data between pre-monitoring and post-monitoring showed a significantly different pattern. Furthermore, the calving interval became significantly shorter between the time of contamination (2019) and the end of the monitoring period (2022). In conclusion, the urinary ZEN monitoring system may be a valuable practical tool for screening and detecting herd contamination in the field, and acute and/or chronic ZEN contamination in dietary feeds may affect herd productivity and the fertility of breeding cows.

Novel Mutation in the Feline GAA Gene in a Cat with Glycogen Storage Disease Type II (Pompe Disease).

Glycogen storage disease type II (Pompe disease: PD) is an autosomal recessively inherited fatal genetic disorder that results from the deficiency of a glycogen hydrolyzing enzyme, acid α-glucosidase encoded by the GAA gene. Here, we describe the molecular basis of genetic defects in an 8-month-old domestic short-haired cat with PD. The cat was previously diagnosed with PD based on the clinical and pathological findings of hypertrophic cardiomyopathy and excessive accumulation of glycogen in the cardiac muscles. Sanger sequencing was performed on 20 exons of the feline GAA gene using genomic DNA extracted from paraffin-embedded liver tissues. The affected cat was found to be homozygous for the GAA:c.1799G>A mutation resulting in an amino acid substitution (p.R600H) of acid α-glucosidase, a codon position of which is identical with three missense mutations (p.R600C, p.R600L, and p.R600H) causing human infantile-onset PD (IOPD). Several stability and pathogenicity predictors have also shown that the feline mutation is deleterious and severely decreases the stability of the GAA protein. The clinical, pathological, and molecular findings in the cat were similar to those of IOPD in humans. To our knowledge, this is the first report of a pathogenic mutation in a cat. Feline PD is an excellent model for human PD, especially IOPD.

Usefulness and Limitations of Cryopreservation for Immunocytochemical Staining of Canine Cytological Specimens for Detection of Cytokeratin and Vimentin.

Immunocytochemistry is an advanced diagnostic tool for identifying the origin of tumor cells. This study aimed to highlight the usefulness of cryopreserved, air-dried cytological samples in detecting cytokeratin and vimentin. Air-dried cytological smear samples were prepared from a total of 39 resected canine tumors and stored in a medical freezer without fixation. The duration of cryopreservation ranged from 2 to 56 months. The same tumors were processed for routine histopathological examination. Based on the morphological diagnosis, cryopreserved FNA smears from epithelial tumors were stained by enzymatic immunocytochemistry (ICC) for cytokeratin; those from mesenchymal and melanocytic tumors were stained by ICC for vimentin. To ascertain the positivity of tumor cells to the selected markers, tissue paraffin-embedded sections were also stained by immunohistochemistry (IHC) for the same markers. Immunoreactivity for cytokeratin was detected in cryopreserved cytological smears for a maximum of 46 months. Immunoreactivity for vimentin was clearly detected for 33 months. Smears stored at room temperature for 1 week did not show any signals under immunocytochemical examination. Thus, immunocytochemistry for cytokeratin and vimentin can be safely applied to air-dried smears cryopreserved in a freezer for at least 33 months.

Potential Biomarkers for Chronic Seasonal Heat Stress in Kagoshima Berkshire Pigs Reared in the Subtropical Region.

Introduction: Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region were investigated by comparing the biomarker changes in the summer (a period of chronic heat stress) and winter (a thermoneutral period) seasons. Material and Methods: Pigs were allocated to summer- and winter-finishing cohorts, 12 each. The evaluations included assessment of carcass traits and internal organs' normality carried out at the time of slaughter, and measurement of biomarkers in whole blood: derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential as markers of oxidative stress, and serum amyloid A and albumin/globulin (A/G) ratio as markers of acute and chronic inflammation, respectively. Results: The summer-finished pigs reared under subtropical field conditions showed lower carcass quality than the winter-finished pigs, indicating a potential adverse effect of summer temperatures on the swine industry. Marginal changes were observed in d-ROMs and the A/G ratio between the summer- and winter-finishing cohorts. Conclusion: The results demonstrate that d-ROMs and the A/G ratio could be used as sensitive markers for heat stress under field conditions.

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate.

Canine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease reported in multiple canine breeds, including the German Shepherd Dog (GSD). Clinical signs include progressive motor neuron paralysis, which begins in the pelvic limbs and eventually leads to respiratory distress, which may necessitate euthanasia. A common DM-associated mutation is a single nucleotide substitution that causes an amino acid substitution (c.118G>A, p.E40K) in the canine SOD1 gene. This SOD1 mutation and the clinical progression rate of A/A risk genotype in the Japanese GSD population have not been analyzed before. Therefore, the aim of this study was to determine the frequency of the mutated allele and analyze the clinical progression rate in the Japanese GSD population. We studied 541 GSDs registered with the Japanese German Shepherd Dog Registration Society between 2000 and 2019. Genotyping was performed using real-time PCR with DNA extracted from the hair roots of each dog. The study revealed 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%), indicating a frequency of the mutant allele of 0.220, which are in Hardy−Weinberg equilibrium. We analyzed the clinical signs in A/A dogs with an age limit of 10 years based on information obtained from the dogs' owners. Of the seven A/A dogs older than 10 years, owners reported DM-related clinical signs, indicating a clinical progression rate of 100%. These results, further genotyping, and thorough clinical examinations of SOD1 A/A risk genotype will help control and prevent DM in the Japanese GSD population.

Cytochrome P450 2J Genes Are Expressed in Dogs, Cats, and Pigs, and Encode Functional Drug-Metabolizing Enzymes.

Cytochrome P450s (P450s) have been identified and analyzed in dogs and pigs, species that are often used in preclinical drug studies. Moreover, P450s are clinically important for drug therapy not only in humans, but also in species under veterinary care, including dogs and cats. In the present study, seven P450s homologous to human CYP2J2, namely, dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J35, CYP2J91, and CYP2J93, were newly identified and characterized, along with pig CYP2J34 previously identified. The cDNAs of these CYP2Js contain open reading frames of 502 amino acids, except for CYP2J35 (498 amino acids), and share high sequence identity (77%-80%) with human CYP2J2. Phylogenetic analysis revealed that dog and cat CYP2J2 were closely related, whereas pig CYP2Js formed a cluster. All seven CYP2J genes contain nine coding exons and are located in corresponding genomic regions, with the pig CYP2J genes forming a gene cluster. These CYP2J2 mRNAs were predominantly expressed in the small intestine with additional expression in the kidney and brain for dog CYP2J2 and pig CYP2J91 mRNAs, respectively. All seven CYP2Js metabolized human CYP2J2 substrates terfenadine, ebastine, and astemizole, indicating that they are functional enzymes. Dog CYP2J2 and pig CYP2J34 and CYP2J35 efficiently catalyzed ebastine primary hydroxylation and secondary carebastine formation at low substrate concentrations, just as human CYP2J2 does. Velocity-versus-substate plots exhibited sigmoidal relationships for dog CYP2J2, cat CYP2J2, and pig CYP2J33, indicating allosteric interactions. These results suggest that dog, cat, and pig CYP2Js have similar functional characteristics to human CYP2J2, with slight differences in ebastine and astemizole oxidations. SIGNIFICANCE STATEMENT: Dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J34, CYP2J35, CYP2J91, and CYP2J93, homologous to human CYP2J2, were identified and characterized by sequence, phylogenetic, and genomic structure analyses. Intestinal expression patterns of CYP2J mRNAs were characteristic in dogs, cats, and pigs. Dog, cat, and pig CYP2Js likely play roles as drug-metabolizing enzymes in the small intestine, similar to human CYP2J2.