Analytical validation of five diagnostic tests for the detection of polymorphonuclear cells in stallion semen.

The objectives of this study were to evaluate the ability of five diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 × 106 purified sperm were incubated, in triplicate, with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 × 106 PMN/ml, 3) 0.5 × 106 PMN/ml, 4) 2.5 × 106 PMN/ml, 5) 5 × 106 PMN/ml, 6) 10 × 106 PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD13 immunolabeling. Sperm motility was evaluated after 4 h at 38 °C. The number of leucocytes detected with the LEDT differed among treatments (P<0.0001), from negative results in control samples to moderate or large numbers in the samples with the highest PMN concentration. The hemacytometer count and CD13 immunostaining detected differences with the control treatment at the lowest PMN concentration (2.5 × 106 PMN/ml; P<0.001). Sperm motion was lower in samples with ≥5 × 106 PMN/ml (P<0.0001). Thus, a sample was considered leucospermic if it contained ≥5 × 106 PMN/ml. The LEDT had the best sensitivity (100%), followed by cytology (78%), peroxidase test (60%), CD13 immunostaining (56%) and hemacytometer count (47%). The LEDT had the lowest specificity (65%), which was 95% for all other tests. In conclusion, the LEDT was a simple, economic and sensitive stall-side test to screen semen for presence of PMNs. Because of the lower specificity, positive LEDT results should be confirmed with the identification of peroxidase-positive cells or CD13-positive cells.

Effect of prophylactic use of tulathromycin on gut bacterial populations, inflammatory profile and diarrhea in newborn Holstein calves.

This objective of this study was to evaluate the use of tulathromycin on the timing of appearance and number of four indicator organisms representing the gastrointestinal microbial community, the incidence of diarrhea and a measure of the systemic inflammatory profile in Holstein heifers. Twenty-six Holstein heifer calves were distributed between receiving (ATB+) or not receiving (ATB-) tulathromycin at a dose of 2.5 mg/kg by 12 h of age. Samples from the calves were collected at six times during the neonatal period. Stool samples were used to determine the dry matter content and quantitative analysis of specific indicator bacterial populations. Samples of whole blood and serum were collected to determine the total number of neutrophils, the number of CD62L+ neutrophils, quantity of haptoglobin, and to allow for ex vivo measurement of reactive oxygen species. A higher frequency of diarrhea was detected in the ATB+ calves (84.6%) than ATB- (53.8%) on days 13-15 (P = 0.084). ATB- calves had a greater number of Bifidobacterium in stool on day 3-5 (P = 0.002), and on days 7-9 (P = 0.018). The ATB+ calves tended to have a higher number of Escherichia coli in stool on days 20-23 and days 27-30 (P = 0.052 and P = 0.072). Both the total number of neutrophils (P = 0.013) and the capacity for ROS production was higher in ATB- (P = 0.038) than ATB+ calves at all points tested. ATB+ calves had higher levels of haptoglobin (P = 0.032) on days 13-15. Administration of tulathromycin appeared to negatively impact the establishment of a normal microbiome and to modulate the development of innate immune function.

Influence of feeding fresh colostrum from the dam or frozen colostrum from a pool on indicator gut microbes and the inflammatory response in neonatal calves.

The aim of this study was to evaluate the capacity of cells from colostrum to modulate the intestinal microbial colonization, the activity of the inflammatory response, and for their influence on the development of diarrheal disease in calves. Twenty calves were distributed into two groups: COL+ (n = 10) receiving fresh whole colostrum; COL- (n = 10) receiving pooled frozen colostrum, containing no viable cells. All assessments were made before colostrum intake (D0), the next day (D2), and weekly on the 7th (D7), 14th (D14), 21st (D21) and 28th (D28) day of age. Diarrhea was assessed using a fecal score, and the systemic inflammatory status was assessed using a combination of temperature, anemia, total serum iron level, total haptoglobin concentration and the need for systemic antimicrobial treatment. The number of indicator bacteria present in the fecal population was estimated using qPCR. However, COL- calves presented more frequent signs of systemic inflammatory response including, fever at D7 (P = 0.011); indicator haptoglobin levels on D7 and D14, and lower levels of iron on D7, D14. Anemia was detected more often in the COL- calves on D21 (P = 0.043) and D28 (P = 0.016). COL- calves had a 1.66 greater chance of having elevated haptoglobin and a 1.8 greater chance of needing treatment with antimicrobials than COL+. A lower number of DNA copies of Clostridium perfringens were detected in COL+ calves on D2 (P = 0.088) and D7 (P = 0.040). Similarly, a low number of DNA copies was observed for Escherichia coli and Lactobacillus spp. (P = 0.012) in the fecal samples of COL+ calves on D7.

An Assessment of Secondary Clinical Disease, Milk Production and Quality, and the Impact on Reproduction in Holstein Heifers and Cows from a Single Large Commercial Herd Persistently Infected with Bovine Viral Diarrhea Virus Type 2.

The aim of this study was to evaluate secondary clinical disease, milk production efficiency and reproductive performance of heifers and cows persistently infected (PI) with bovine viral diarrhea virus type 2 (BVDV type 2). PI animals (n = 25) were identified using an antigen capture ELISA of ear notch samples. They were distributed into three age groups: ≤ 12 (n = 8), 13 to 24 (n = 6) and 25 to 34 (n = 11) months old. A control group of BVDV antigen ELISA negative female cattle that were age matched to the PI animals was utilized from the same herd. The PI group had a 1.29 higher odds ratio for diarrhea than controls (p = 0.001, IC95% = 1.032-1.623) and 1.615 greater chance of developing bovine respiratory disease (BRD) (p = 0.012, IC95% = 1.155-2.259). The age at first insemination (p = 0.012) and number of insemination attempts required to establish the first pregnancy (p = 0.016) were both higher for PI than controls. Milk production was higher for control cows than PI cows during most of the sampling periods. Somatic cell counts (SCC) were higher in PI cows than the controls at all sampling points across lactation (p ≤ 0.042). PI cattle had a higher incidence of disease, produced less milk, a higher SCC, and poorer reproductive performance than control cattle in this study.

Effect of maternal cells transferred with colostrum on the health of neonate calves.

The objective of this research was to evaluate the influence of cells from colostrum on the health of neonate calves. Animals were distributed in 2 groups: COL+ (n=9) which received fresh colostrum from their own damns; and COL- (n=10) which received frozen colostrums from donors. Heifers were assessed before colostrum intake - D0; D2; D7; D14; D21 and D28. Heifers were monitored by clinical examination, hematological profile and serum iron. COL- had a higher diarrhea intensity score (typically 3) on D7. Moreover, a single case each of bronchopneumonia and navel inflammation were observed in COL- calves. COL- had fewer red blood cells (RBC) (6.5±0.8×106/µL) and less hemoglobin (Hgb) (8.3±1.4g/dL) than COL+ (RBC=7.2±0.8×106/µL; Hgb=9.6±1.3g/dL) at D14 (P≤0.05). COL- had more anemia on D21 (P=0.03) and on D28 (P=0.02). Iron was lower in COL- (5.6±2.7µM/L) than COL+ (10.7±6.2µM/L) (P=0.03) on D7. Lymphocytes was lower in COL- than COL+ on D7 (3.8±1.0×103/µL COL+ and 5.4±2.2×103/µL COL-, P=0.02). COL- calves had more anemia and lower serum iron concomitant with diarrhea on D7. The number of leukocytes was relatively consistent in the COL+ calves, while COL- calves showed an increasing number of of lymphocytes starting on D7.

Innate immune response in neonate Holstein heifer calves fed fresh or frozen colostrum.

The aim of this research was to evaluate the influence of maternal cells from colostrum on the development and function of the innate immune response in Holstein calves. Calves were divided into 2 groups: COL+ (n=10) received fresh colostrum; and COL- (n=10) which received frozen colostrum containing no viable cells. The calves were assessed before colostrum intake (D0), 48h of age (D2), and weekly from D7 up to D28. Blood samples were collected for analysis of the distribution of leukocytes, cellular phenotype and in vitro granulocyte function. COL+ calves tended to have a high number of neutrophils on D7 (p=0.073). COL- calves took up significantly more Escherichia coli (measured as MFI) on D7 (p=0.034). Endogenous production of radicals (as percentage of cells) tended to be higher in COL- calves on D14 (p=0.061). The intensity of endogenous reactive oxygen species (ROS) produced by granulocytes tended to be higher in COL+ calves on D21 (p=0.094). Overall, ROS production (percent of cells, and MFI) induced by Staphylococcus aureus and Escherichia coli were higher in COL+ calves than COL- calves. It was our observation that COL+ calves developed an innate immune response more quickly and efficiently after natural exposure to pathogens after birth. In contrast, COL- calves mounted an innate response more slowly that yielded a persistent inflammatory response after natural exposure to these bacteria agents. This research provides evidence of an advantage to the calf of receiving fresh colostrum on the development and function of the innate immune system.