TCR signaling and cellular metabolism regulate the capacity of murine epidermal γδ T cells to rapidly produce IL-13 but not IFN-γ.

Resident epidermal T cells of murine skin, called dendritic epidermal T cells (DETCs), express an invariant γδ TCR that recognizes an unidentified self-ligand expressed on epidermal keratinocytes. Although their fetal thymic precursors are preprogrammed to produce IFN-γ, DETCs in the adult epidermis rapidly produce IL-13 but not IFN-γ early after activation. Here, we show that preprogrammed IFN-γ-producing DETC precursors differentiate into rapid IL-13 producers in the perinatal epidermis. The addition of various inhibitors of signaling pathways downstream of TCR to the in vitro differentiation model of neonatal DETCs revealed that TCR signaling through the p38 MAPK pathway is essential for the functional differentiation of neonatal DETCs. Constitutive TCR signaling at steady state was also shown to be needed for the maintenance of the rapid IL-13-producing capacity of adult DETCs because in vivo treatment with the p38 MAPK inhibitor decreased adult DETCs with the rapid IL-13-producing capacity. Adult DETCs under steady-state conditions had lower glycolytic capacity than proliferating neonatal DETCs. TCR stimulation of adult DETCs induced high glycolytic capacity and IFN-γ production during the late phase of activation. Inhibition of glycolysis decreased IFN-γ but not IL-13 production by adult DETCs during the late phase of activation. These results demonstrate that TCR signaling promotes the differentiation of IL-13-producing DETCs in the perinatal epidermis and is needed for maintaining the rapid IL-13-producing capacity of adult DETCs. The low glycolytic capacity of adult DETCs at steady state also regulates the rapid IL-13 response and delayed IFN-γ production after activation.

Predicting an increased risk of severe clinical mastitis and economic loss using a threshold value of bovine leukemia virus proviral load.

OBJECTIVE: To establish a threshold value of bovine leukemia virus (BLV) proviral load (PVL) to identify increased risk of severe clinical mastitis, and to examine the prognosis and economic loss of clinical mastitis based on the newly established PVL cut-off value. ANIMALS: 97 lactating Holstein cows with clinical mastitis. METHODS: Blood and milk samples were collected aseptically from each cow. Youden index was used for receiver-operating characteristic curve analysis with the severity rate of clinical mastitis as the dependent variable and PVL as an independent variable. PVL cut-off value was used as a criterion to compare the severity rate of clinical mastitis, percentage of cows with and without systemic treatments, number of treatments, cost of treatment, and prognosis. RESULTS: PVL cut-off value was 17.8 copies/10 ng DNA for the dependent variable MILD vs SEVERE. The severity rate of clinical mastitis, percentage of cows given systemic treatments, and technical fees for medical treatment were significantly higher in the group above the PVL cut-off value than in the group below the PVL cut-off value and the negative group. Number of treatments was significantly higher in the group above the cut-off value than in the group below the cut-off value. There was no significant difference in prognosis after mastitis among the 3 groups. CLINICAL RELEVANCE: These results suggested that PVL cut-off value of 17.8 copies/10 ng DNA was a useful threshold for increased economic losses in BLV-infected cows; it may also serve as a new standard value for the detection and culling of BLV-infected cows in Japan.

Antemortem diagnosis of external iliac arterial thromboembolism in a calf using contrast-enhanced computed tomography: a case report.

A 28-day-old Holstein calf with astasia (i.e., right hind limb flaccid paralysis) was referred to Large Animal Veterinary Educational Center. Thoracic radiographs showed an alveolar pattern and air bronchograms indicating pneumonia, whereas skeletal radiography showed muscle atrophy of the right hind limb but no abnormalities in the coxofemoral and stifle joints. Contrast-enhanced computed tomography revealed the pulmonary abscess in the right cranial pulmonary lobe and a thrombus (5 × 5 × 20 mm) in the right external iliac artery distal to the bifurcation of the abdominal aorta. These findings were confirmed via pathological examination. Streptococcus bovis/equinus complex colonies were isolated from the thrombus and pulmonary abscess. Thus, the pulmonary abscess was considered the infectious source of the thromboembolism in the right external iliac artery. Arterial thromboembolism should be included in the differential diagnoses in calves with astasia but without radiographic skeletal abnormalities.

Effects of Intramammary Antimicrobial Treatment on the Milk Microbiota Composition in Mild Clinical Bovine Mastitis Caused by Gram-Positive Bacteria.

The purpose of this study was to clarify the effects of antimicrobial treatment for mild mastitis caused by Gram-positive bacteria on the milk microbiota in dairy cattle. Sixteen quarters of sixteen cows with mild clinical mastitis from the same herd were included in the study. On the day of onset (day 0), the cows were randomly allocated to a no-treatment (NT; n = 10) group or an intramammary antimicrobial treatment (AMT) group that received AMT starting on day 0 (AMT-AMT group; n = 6). The next day (day 1), the cows in the NT group were randomly allocated into an NT group (NT-NT group; n = 3) that received no treatment or an AMT group that received AMT starting on day 1 (NT-AMT group; n = 7). Milk samples were collected on days 0, 1, 3 and 7, and the milk microbiota of each sample was comprehensively analyzed via 16S rRNA gene amplicon sequencing of the milk DNA. During the treatment period, the milk microbiota of the NT-NT group did not change, but those of the NT-AMT and AMT-AMT groups changed significantly on days three and seven. Thus, the use of antimicrobials for mild mastitis caused by Gram-positive bacteria changes the milk microbiota composition.

Antimicrobial susceptibility of bovine clinical mastitis pathogens in Japan and development of a simplified agar disk diffusion method for clinical practice.

This study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.

Effects of ozonized glycerin on inflammation of mammary glands induced by intramammary lipopolysaccharide infusion in goats.

Although ozone shows antimicrobial activity against mastitis-causing pathogens in ruminants, its anti-inflammatory effect on mammary glands remains unclear. The aim of this study was to examine the anti-inflammatory effects of ozonized glycerin (OG) on experimentally induced inflammation in the mammary glands of six Shiba and two Tokara lactating goats. We infused 1 µg lipopolysaccharide (LPS) into all udders on day -1. On day 0, post LPS infusion, OG (ozone group), and glycerin (control group) were infused into the right and left sides of the udders, respectively. Milk samples were collected once daily from days -1 to 7. The somatic cell count and lactoperoxidase (LPO) activity, along with the interleukin-1ß (IL-1ß), tumor necrosis factor-α, IL-8, IL-10, lactoferrin, and sodium ion concentrations in milk were measured. IL-8, IL-10, and lactoferrin levels after LPS infusion in the ozone group were significantly lower than those in the control group, and the LPO activity tended to be lower than that observed in the control group. This study showed that OG has anti-inflammatory potential against LPS-induced inflammation in the mammary glands.

Differences in antimicrobial components between bacterial culture-positive and culture-negative bovine clinical mastitis milk.

A bacterial culture of milk is the most common test to determine the presence of mastitis-causing pathogens, which informs appropriate treatment. However, a certain proportion of clinical mastitis milk shows no growth of any mastitis-causing pathogens. We hypothesized that bacterial culture-negative clinical mastitis milk is associated with the activity of antimicrobial components contained in the milk. In this study, the differences in antimicrobial components (lactoferrin, transferrin, lysozyme, lactoperoxidase, and lingual antimicrobial peptide [LAP]) between bacterial culture-positive and culture-negative bovine clinical mastitis milk were investigated using Holstein cows. Our results showed that 37 out of 71 samples of clinical mastitis milk had negative bacterial cultures. The LAP concentration in bacterial culture-negative milk was lower than that in positive milk (31.95 ± 1.64 nM vs. 42.85 ± 4.01 nM). In contrast, the lysozyme concentration in bacterial culture-negative milk was higher than that in positive milk (0.76 ± 0.15 µg/ml vs. 0.42 ± 0.06 µg/ml). In conclusion, the concentration of antimicrobial components was different between bacterial culture-positive and culture-negative bovine clinical mastitis milk, which suggests that antimicrobial components are related to bacterial culture results.

Effect of glycyrrhizin administration followed by symptom-based antimicrobial selection therapy on antimicrobial use in clinical mastitis without systemic symptoms.

In bovine mastitis, antimicrobial treatment is often initiated before the causative organism is identified a problem in the prudent use of antimicrobials. In this study, we aimed to reduce the total amount of antimicrobials used in mastitis treatment by administering glycyrrhizin, an anti-inflammatory drug, instead of antimicrobials at the onset of clinical mastitis without systemic symptoms, followed by symptom-based antimicrobial selection therapy (ST), to examine the effect of this treatment strategy on treatment outcomes and antimicrobial use. Comparisons between cases that received antimicrobial treatment starting from the day of diagnosis (blanket antibiotic therapy [BT] group: 33 cases) and cases that received ST starting from the day after the diagnosis (ST group: 57 cases) revealed no difference in the cure rate, milk withholding period, or recurrence rate. However, the ST group had a significantly lower amount of antimicrobials than the BT group. Additionally, a single administration of glycyrrhizin before ST significantly relieved the udder symptoms and reduced the antimicrobial amount when compared with cases without glycyrrhizin administration. Thus, a single administration of glycyrrhizin followed by ST can reduce the total antimicrobial use.

Regression tree analysis of the relationship between the concentrations of antimicrobial components and the microbiota of normal milk from dairy cows.

The purpose of this study was to determine the concentrations of antimicrobial components (immunoglobulin A (IgA), lactoferrin (LF), lingual antimicrobial peptide (LAP), and S100A7) in normal milk and their relation to host factors (Age, somatic cell count (SCC), days in milk, richness, and alpha diversity of the milk microbiota) in dairy cows using multivariate regression tree analyses, and to clarify how the milk microbiota is related to the obtained results. Thirty normal milk samples were collected from a commercial dairy farm in June 2020. The thresholds that predicted the concentration of each antimicrobial component in milk were obtained by regression tree analysis, and the beta-diversity of the milk microbiota composition between groups divided according to each threshold was compared by an analysis of similarities test. The IgA and LF concentrations were mainly predicted by the SCC (177,500 and 70,000 cells/ml, respectively), and the LAP and S100A7 concentrations were predicted by Age (29.667 and 40.3 months, respectively). No relationship was observed between the concentration of IgA, LAP, or S100A7 and the milk microbiota composition between the groups divided by the threshold for prediction, but the milk microbiota composition was significantly different between the groups divided by the threshold for predicting the LF concentration. Our results indicated that the LF concentration in normal milk may be associated with the milk microbiota composition.

Examination of the microbiota of normal cow milk using MinIONTM nanopore sequencing.

The aim of this study was to evaluate the microbiota of normal milk in dairy cows and their relationship with host factors, such as the age of the cow (Age), somatic cell counts in milk (SCCs), and days in milk (DIM). We investigated 48 milk samples from 22 cows with no systemic or local clinical signs using MinIONTM nanopore sequencing for a 16S rRNA gene amplicon. Bacterial richness was positively correlated with the DIM (P=0.043), and both the Shannon-Wiener Index and Simpson's Index, which are metrics of alpha-diversity, were also significantly positively correlated with the SCC (P<0.001). The composition ratios of both Actinobacteria at the phylum level and Kocuria spp. at the genus level in the milk microbiota were significantly correlated with the SCC (P<0.001 and P<0.001, respectively). In the beta-diversity test, the one-way analysis of similarities test showed a significant difference (P=0.0051) between the low- and high-SCC groups. This study clarified that the composition of the normal milk microbiota in this herd was related to the SCC. It also raised the possibility of variations in bacterial genera in the normal milk microbiota between the low- and high-SCC groups. However, to clarify the actual condition of the milk microbiota and to elucidate the relationship with the SCC, it is necessary to perform further analyses taking into account not only the relative abundance, but also the absolute abundance of microbes.

Immune response during the onset of coliform mastitis in dairy cows vaccinated with STARTVAC®.

The immune response during the onset of coliform mastitis in vaccinated cows was investigated by measuring lactoferrin (LF), interleukin-8 (IL-8), and interleukin-1ß (IL-1ß) concentrations and somatic cell counts in 28 milk samples at the onset of acute coliform mastitis (ACM) and 73 milk samples at the onset of peracute coliform mastitis (PCM). Vaccinated ACM, unvaccinated ACM, and vaccinated PCM showed significantly higher values for LF and IL-1ß levels than unvaccinated PCM (p < .01). The IL-8 concentration was lower in vaccinated PCM than in unvaccinated PCM (p < .05). There was no significant difference in somatic cell counts for each parameter. There were no significant differences in the parameters between vaccinated and unvaccinated ACM cows, or vaccinated ACM and PCM cows. From the above results, it is suggested that mastitis vaccination improved the early immune response, particularly at the onset of PCM, and played a large role in host defense against the initial infection.

Outlines of the Japanese guidelines for the management of primary cutaneous lymphomas 2020.

Since the publication of the Japanese "Guidelines for the management of cutaneous lymphomas" in 2011, the World Health Organization (WHO) classification of hematolymphoid neoplasms and the WHO-European Organisation for Research and Treatment of Cancer classification for primary cutaneous lymphomas were updated and a number of novel systemic drugs for cutaneous T-cell lymphoma had been approved in Japan. In 2020, we revised the Japanese guidelines for the management of cutaneous lymphomas with consideration of the recent advances in the understanding of the pathophysiology and classification of cutaneous lymphomas together with the update of treatment strategies reflecting the advent of novel drugs. In addition to a brief explanation of epidemiology, diagnosis, staging system, prognosis and management of each subtype of cutaneous lymphomas, the recommendations for nine clinical questions regarding treatment options that can vary even among experts are also described. A systematic review process and determination of recommendations in answer to each clinical question have been performed in accordance with the Grading of Recommendations, Assessment, Development and Evaluation scheme by a multidisciplinary expert panel consisting of dermatologists, a hematologist and a radiation oncologist. In this article, we present the outlines of the revised Japanese "Guidelines for the management of cutaneous lymphomas".

Metagenomic identification, sequencing, and genome analysis of porcine hepe-astroviruses (bastroviruses) in porcine feces in Japan.

Recently, hepe-astrovirus-like RNA viruses named bastroviruses (BastVs), have been found in human, pig, bat, and rat fecal samples. In this study, we determined nearly complete genome sequences of four BastVs in the feces of healthy pigs. Genetic characterization revealed that these porcine BastVs (PBastVs) and BastVs from other animals including humans, had the same genome organization, that is, they contained three predicted conserved domains of viral methyltransferase, RNA helicase, and RdRp in the nonstructural ORF1 and the astrovirus capsid domain in the structural ORF2. Phylogenetic analyses using RNA-dependent RNA polymerase and the capsid region revealed that PBastVs branched with bat and rat BastVs; however, the groups formed by each host were distantly related to human BastVs. Pairwise amino acid sequence comparison demonstrated that PBastVs shared 95.2-98.6% and 76.1-95.5% sequence identity among each other in the ORF1 and ORF2 regions, respectively; the sequence identities between PBastVs and BastVs from other animals were 21.4-42.5% and 9.1-20.6% in the ORF1 and ORF2 regions, respectively. This suggested that BastVs were derived from a common ancestor but evolved independently in each host population during a prolonged period. Putative recombination events were identified in the PBastV genome, suggesting that PBastVs gain sequence diversity and flexibility through recombination events. In an analysis of previously obtained metagenomic data, PBastV sequence reads were detected in 7.3% (23/315) of fecal samples from pigs indicating that PBastVs are distributed among pig populations in Japan.

Diagnosis of a sublaryngeal abscess in a Japanese Black calf using computed tomography.

A 76-day-old Japanese Black calf presented with severe stridor, resenting palpation of the laryngeal region. Endoscopic examination revealed an expansile process restricting the esophageal and tracheal lumina caudal to the arytenoid cartilage, hyperemia and edema of the pharyngeal mucosa, right arytenoid cartilage swelling and displacement, and marked airway obstruction. The absence of an endotracheal wall abnormality impeded a definitive diagnosis. Computed tomography (CT) revealed a mass (CT value: 40-45 HU) caudal to the arytenoid cartilage, causing tracheal stenosis and esophageal displacement. The presence of gas in the mass suggested the presence of an abscess. Diagnosis of deep retropharyngeal lesions by conventional endoscopic and ultrasonographic examinations may be challenging; CT can then provide more comprehensive diagnostic information on a lesion.

A case study of improving milking cow performance and milking system performance with using a flow simulator.

Efforts to improve dairy performance have been focused on increasing milk productivity of cows through improved feeding systems and genetic potential. However, methods for evaluating milking system performance based on milk productivity have not yet been established. Milking system performance was evaluated by measuring the claw vacuum at five flow rates (1.9-8.7 kg/min) produced using a flow simulator for a single eight-swing milking parlor with a high-line system. Based on these results, a double eight-parallel milking parlor with a low-line system was installed and tested. Farmers can take data obtained from evaluations of milking system performance into account for future management decisions, such as renewing the milking system. By renewing the milking system, average milking productivity, somatic cell linear score (LS) of bulk milk, and LS of each cow were significantly improved in the year after installing the new system (p < .01). In addition to checking conventional milking systems, this novel diagnostic method using a flow simulator can be used for checking new installations and also for proposing renovations.