ABSTRACTBacterial isolates from frogs were phenotypically identified asOchrobactrum anthropi, but 16S rRNA sequencing showed up to 100% identity withBrucella inopinata. Further analysis ofrecA,omp2a,omp2b,bcsp31, and IS711and multilocus sequence analysis (MLSA) verified a close relationship withBrucella, suggesting the isolates may actually represent novel members of this growing genus of zoonotic pathogens.

Somatic cell counts (SCC) are generally used as an indicator of udder health. In Germany, a cutoff value of 100,000 cells/mL is currently used to differentiate between healthy and diseased mammary glands. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed evaluation of the udder health status. The aim of this study was to differentiate immune cells in milk of udder quarters classified as healthy based on SCC values of <100,000 cells/mL. Twenty cows were selected and 65 healthy udder quarters were compared with a control group of 15 diseased udder quarters (SCC>100,000 cells/mL). Cells were isolated from milk of all quarters to measure simultaneously percentages of lymphocytes, macrophages, and polymorphonuclear neutrophilic leukocytes (PMNL) by flow cytometric analysis. The bacteriological status of all 80 quarters was also determined. Differential cell count patterns of milk samples (n = 15) with extreme low SCC values of ≤ 6,250 cells/mL revealed high lymphocyte proportions of up to 88%. Milk cell populations in samples (n = 42) with SCC values from >6,250 to ≤ 25,000 cells/mL were also dominated by lymphocytes, whereas DCC patterns of 6 out of 41 milk samples with SCC values from ≥ 9,000 to ≤ 46,000 cells/mL indicated already inflammatory reactions based on the predominance of PMNL (56-75%). In 13 of 15 milk samples of the diseased udder quarters (SCC >100,000 cells/mL), PMNL were categorically found as dominant cell population with proportions of ≥ 49%. Macrophages were the second predominant cell population in almost all samples tested in relation to lymphocytes and PMNL. Further analysis of the data demonstrated significant differences of the cellular components between udder quarters infected by major pathogens (e.g., Staphylococcus aureus; n = 5) and culture-negative udder quarters (n = 56). Even the percentages of immune cells in milk from quarters infected by minor pathogens (e.g., coagulase-negative staphylococci; n = 19) differed significantly from those in milk of culture-negative quarters. Our flow cytometric analysis of immune cells in milk of udder quarters classified as healthy by SCC <100,000 cells/mL revealed inflammatory reactions based on DCC.

Somatic cell count (SCC) is generally regarded as an indicator of udder health. A cut-off value of 100×10(3) cells/ml is currently used in Germany to differentiate between normal and abnormal secretion of quarters. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed analysis of the udder health status. The aim of this study was to differentiate somatic cells in foremilk samples of udder quarters classified as normal secreting by SCC <100×10(3) cells/ml. Twenty cows were selected and 72 normal secreting udder quarters were compared with a control group of six diseased quarters (SCC >100×10(3) cells/ml). In two severely diseased quarters of the control group (SCC of 967×10(3) cells/ml and 1824×10(3) cells/ml) Escherichia coli and Staphylococcus aureus were detected. DCC patterns of milk samples (n = 25) with very low SCC values of ≤6·25×10(3)cells/ml revealed high lymphocyte proportions of up to 92%. Milk cell populations in samples (n = 41) with SCC values of (>6·25 to ≤25)×10(3) cells/ml were also dominated by lymphocytes (mean value 47%), whereas DCC patterns of milk from udder quarters (n = 6) with SCC values (>25 to ≤100)×10(3)cells/ml changed. While in samples (n = 3) with SCC values of (27-33)×10(3) cells/ml macrophages were predominant (35-40%), three milk samples with (43-45)×10(3) cells/ml indicated already inflammatory reactions based on the predominance of polymorphonuclear leucocytes (PMN) (54-63%). In milk samples of diseased quarters PMN were categorically found as dominant cell population with proportions of ≥65%. Macrophages were the second predominant cell population in almost all samples tested in relationship to lymphocytes and PMN. To our knowledge, this is the first study evaluating cell populations in low SCC milk in detail. Udder quarters classified as normal secreting by SCC <100×10(3) cells/ml revealed already inflammatory processes based on DCC.

The control of Johne's disease requires the identification of Mycobacterium avium ssp. paratuberculosis (MAP)-positive herds. Boot swabs and liquid manure samples have been suggested as an easy-to-use alternative to sampling individual animals in order to diagnose subclinical Johne's disease at the herd level, but there is a need to evaluate performance of this approach in the field. Using a logistic regression model, this study aimed to calculate the threshold level of the apparent within-herd prevalence as determined by individual faecal culture, thus allowing the detection of whether a herd is MAP positive. A total of 77 boot swabs and 75 liquid manure samples were taken from 19 certified negative and 58 positive dairy herds. Faecal culture, three different polymerase chain reaction (PCR) methods and the combination of faecal culture with PCR were applied in order to detect MAP. For 50% probability of detection, a within-herd prevalence threshold of 1·5% was calculated for testing both matrices simultaneously by faecal culture and PCR, with the threshold increased to 4·0% for 90% probability of detection. The results encourage the use of boot swabs or liquid manure samples, or a combination both, for identifying MAP-positive herds and, to a certain extent, for monitoring certified Johne's disease-negative cattle herds.

The aim of the present study was the examination of the boot swab sampling technique for the collection of environmental material in order to identify Mycobacterium avium ssp. paratuberculosis (MAP)-infected herds. Eight dairy herds were included into the study. Four of them had a well-known history of MAP-infection from a herd surveillance programme conducted since 2006. Cows in these herds were repeatedly tested positive in Pourquier® MAP-ELISA (Pourquier, Montepellier, France); in some MAP could be isolated in individual faecal culture despite that symptoms of paratuberculosis were never reported. In four presumably negative herds nearly all cows were repeatedly tested serologically negative for MAP. The pathogen was never isolated from faecal samples of cows by culture. The study was initiated with the aim of standardising environmental samples as a herd diagnostics, in which overall 130 pairs of boot swab samples from the cows' surroundings were taken In 58 of 64 swab samples (90·6%) from confirmed MAP-infected herds the organism could be isolated by mycobacterial culture of the boot swab. Contrarily, in 66 samples from presumably MAP-negative herds only one swab was positive (1·5%). The utilisation of boot swabs as a standardised technique for environmental sampling offers an effective and inexpensive tool for identifying herds infected with MAP. This is the first report of using boot swabs for the collection of environmental samples for MAP- detection in cattle herds. This easy to perform technique enables the economical detection of MAP herd status.