Comprehensive Analyses of the Bacterial Population in Non-Healing Claw Lesions of Dairy Cattle.

Non-healing claw lesions (NHCLs) are a newly characterized disorder affecting the deep dermis of the hoof in dairy cattle. Although NHCLs are thought to be associated with bovine digital dermatitis (BDD), their precise etiology is not yet understood. To investigate the bacterial populations present in each type of NHCL (toe necrosis: TN, non-healing white line disease: nhWLD, and a non-healing sole ulcer: nhSU), and the newly added entity non-healing verrucous-like lesions (nhVLL), 16S rRNA-based metagenomic analysis with next-generation sequencing (NGS) was employed. Twelve cases of NHCLs (3 TN, 3 nhWLD, 4 nhSU, and 2 nhVLL) were collected from five dairy farms in two prefectures in Japan. Three samples of healthy hoof dermis collected from two farms and a slaughterhouse were used as controls. Furthermore, culture-dependent and -independent approaches were conducted for detecting Treponema species and Fusobacterium necrophorum. As reported in BDD, Treponema species and F. necrophorum were detected frequently from NHCLs by PCR and immunohistochemistry, but NGS showed that these bacterial genera were not predominant in NHCLs. The predominant bacterial genera in NHCLs differed among the lesions examined, suggesting that Treponema species present predominantly in BDD were not predominant in NHCLs and that the bacterial population in NHCLs may vary among individual cattle and/or farms.

Validation of the bovine blood calcium checker as a rapid and simple measuring tool for the ionized calcium concentration in cattle.

Point-of-care (POC) devices that veterinary practitioners can use to easily and rapidly measure blood ionized calcium (iCa) levels in cows immediately after withdrawing a blood sample on the dairy farm are needed. Aims of present studies was to compare the commercially available ion-selective electrode handheld iCa meter (bovine blood iCa checker) with the benchtop blood gas analyzer GEM premier 3500 and handheld analyzer i-STAT 1. Sixty-two paired-point whole blood samples were obtained from three cows with hypocalcemia experimentally induced by Na2-EDTA infusion. Whole blood samples were also obtained from the 36 cows kept on a farm in field conditions. The results using the bovine blood iCa checker correlated with those using the GEM premier 3500 and i-STAT 1. Bovine blood iCa checker was "compatible" with the GEM premier 3500 and i-STAT 1 because the frequency of differences between the measurements within ± 20% of the mean were 100% (65/65, >75%) and 90.8% (59/65, >75%), respectively. In the field trial, the blood iCa concentration measured by the bovine blood Ca checker was significantly positively correlated with that measured by the i-STAT 1 portable analyzer. Bovine blood iCa checker was "compatible" with the i-STAT 1 because the frequency of differences between the measurements within ± 20% of the mean was 100% (36/36, >75%). Results from these findings, the bovine blood iCa checker may be applied as a simplified system to measure the iCa concentration in bovine whole blood.

Combination of procedure for ultra rapid extraction (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Mycoplasma bovis in milk.

Polymerase chain reaction (PCR) is typically used for the early detection of mycoplasma in bovine milk; it requires 3 days to obtain results because of the necessary enrichment process. A more rapid, simple, and accurate detection method is required to directly detect the Mycoplasma bovis (M. bovis) gene in milk. In this study, we assess the utility of combining the following two methods to achieve this goal: the loop-mediated isothermal amplification (LAMP), which is more sensitive than PCR, and the procedure for ultra rapid extraction (PURE), which adsorbs and filters components that inhibit DNA amplification/detection. LAMP was examined using DNA extracts obtained by four methods. This showed that PURE had the highest sensitivity and specificity and that the combination of PURE and LAMP was able to detect M. bovis in milk. We then showed that the detection limit of M. bovis was 102 colony-forming units per milliliter of milk using the PURE-LAMP. Finally, the respective sensitivities of the PURE-LAMP and PCR were 57% and 86% for bulk tank milk, 89% and 74% for mature milk, 85% and 92% for colostrum/transitional milk, and 97% and 95% for mastitis milk. The specificity was 100% for all milk samples in both LAMP and PCR. We conclude that PCR was suitable for detecting mycoplasma in bulk tank milk and that the PURE-LAMP could detect mycoplasma within 2 hr and was also effective for mature and mastitis milk.

Availability of oral swab sample for the detection of bovine viral diarrhea virus (BVDV) gene from the cattle persistently infected with BVDV.

Bovine nasal and oral discharges were used as samples for bovine viral diarrhea virus (BVDV) gene detection. Viral genes in serum (S), nasal discharge (N) and oral discharge (O) were quantified with real-time polymerase chain reaction using SYBR Green by the relative quantification method, and findings were compared among samples. Although the quantity of the BVDV gene in S was greater than those in N and O, all samples were available to identify persistently infected (PI) cattle with BVDV by reverse transcription polymerase chain reaction (RT-PCR). The swab samples were able to be stored for a few days at 4 degrees C with a little decrease of amplification signal in RT-PCR. Oral swab sampling was easier than nasal swab sampling, and was also less uncomfortable for the cattle than other sampling methods without pain or unnecessary retention. This sampling method can be performed without any special technique and equipment. Therefore, the oral swab sampling method is useful for screening to detect BVDV PI cattle by RT-PCR.

Relationship of bovine viral diarrhea virus persistent infection to incidence of diseases on dairy farms based on bulk tank milk test by RT-PCR.

The prevalence of bovine viral diarrhea virus (BVDV) in dairy herds in Hokkaido, Japan, was estimated by reverse transcription polymerase chain reaction (RT-PCR) using bulk tank milk samples. Sixteen out of 265 dairy herds were identified as BVDV positive, and at least one persistently infected (PI) cattle was recognized in each of the positive herds except for two herds of which, owners did not agree to examine individual cows. The proportion of positive herds with a history of BVDV PI was significantly higher than that with no history of BVDV PI (odds ratio (OR) 4.25, 95% confidence interval (CI) 1.471-12.278, p = 0.004). The herds examined for BVDV were divided into two groups, high and low disease incidence groups based on the occurrence of diseases such as diarrhea, pneumonia or abortion in the past 1 year. The BVDV positive herds in the high disease incidence group were significantly more than that in the low disease incidence group (OR 2.92, CI 1.110-7.683, p = 0.024). It was observed that there were significantly (p = 0.008) more PI calves or heifers in farms of high disease incidence group than in farms of low disease incidence group. These results suggested that bulk tank milk test was available method for the detection of PI animals in dairy herds, and the existence of PI non-lactating cows in herd correlated with the incidence of diseases of the diarrhea or respiratory disorders.