A method for preparation of chromosomes from bovine zygotes and blastocysts

A simple technique for making chromosome preparations from zygotes and early blastocysts is described. The morphological features of blastocysts and total number of cells greatly influence the quality of the preparation.     

A simple, rapid method for differential cell counts in porcine mammary secretions.

The use of the fluorescent dye acridine orange for making differential cell counts in mammary secretions from sows was investigated, and the variations in cell type during the lactation period were also studied. In untreated samples of mammary secretions polymorphonuclear leucocytes, mononuclear phagocytes, lymphocytes and epithelial cells could be identified with certainty, and the methodological error was small (1.80 to 2.22 per cent). The mammary secretions could be stored at 4 degrees C for six hours without any effect on the differential count. Washing the secretions decreased the percentage of polymorphonuclear leucocytes and increased the percentage of epithelial cells. The polymorphonuclear leucocytes predominated in colostrum (58.0 to 65.5 per cent) and epithelial cells predominated in milk (60 to 89 per cent). Polymorphonuclear leucocytes were the predominant phagocytes in all mammary secretions (7.6 to 65.5 per cent).     

Cytokine activity in bovine mammary gland secretions during the periparturient period.

The presence of cytokine activity in periparturient bovine mammary secretions was evaluated. Mammary secretions were modified for use in biological assays for interleukin-2 (IL-2) like and antiviral activity. The level of IL-2 like activity in mammary gland secretions was lower during the last week of gestation when compared to levels detected approximately two weeks prepartum. Antiviral titers gradually increased as parturition approached. Results from Western blots indicated that the antiviral activity observed in prepartum secretions may be due to tumor necrosis factor (TNF). Interferons (IFN) were not detected in the colostrum samples.     

A study of bovine T-cell subsets in the blood and mammary gland secretions during the dry period

Blood and mammary secretions were obtained from cows throughout the dry period. Quantitative and qualitative assays were performed to determine the cell types and cell distributions at weekly intervals from day of dry off until parturition. The total cell counts in secretions increased during involution and remained at high levels until a few weeks prepartum. The macrophages were the predominant cell type in mammary secretions whereas the numbers of lymphocytes were always less than neutrophils or macrophages. Enriched mononuclear cell populations derived from blood and mammary secretions were also evaluated using "T-cell rosette" assays. Changes observed in the relative distribution of three T-cell subsets in secretions did not reflect the dynamics of the cells in the peripheral blood. T-cell subsets that predominated in mammary secretions were the EN+ EAET+ and EN-EAET+ phenotypes. Distinct patterns of migration or differentiation of T-cell subsets were suggested by the changes of subsets observed in mammary secretions collected throughout the dry period.     

Immunofluorescent immunoglobulin differentiation of vibrio fetus antibodies from bovine cervico-vaginal secretions

Reports on discrepancies between local and systemic immunity started to appear about 50 years ago (cf. Tomasi & Bienenstock 1968). Protection against infections has been shown in many cases to be closely related to the antibody content of external secretions and more or less independent from the serum antibody level.     

Restriction endonuclease patterns of bovine herpesvirus type 1 isolated from bovine mammary glands

Restriction endonuclease analysis was used, in conjunction with viral neutralization and growth-curve experiments, to compare a bovine herpesvirus type 1 (BHV-1) isolate, originally obtained from bovine mammary gland lesions, with a standard BHV-1 strain, infectious bovine rhinotracheitis virus. Although differences were not detected by viral neutralization or growth-curve experiments, restriction fragment patterns generated by Bam HI, Eco RI, Hind III, and Hpa I, revealed definite differences between the isolate and the prototype strain. Additionally, Eco RI, Hind III, and Hpa I patterns revealed that the mammary gland isolate had DNA-fragment patterns characteristic of infectious pustular vulvovaginitis strain of BHV-1, type 2b. Seemingly, type-2b isolates, similar to types 1 and 2a, may be capable of causing divergent types of infection of variable severity in cattle.     

A modified method for complete bovine fetotomy

A modification of the "Utrecht technique" for complete bovine fetotomy was developed. In cranial presentation, the first step is decapitation, then oblique indirect section through the neck and thorax to remove 1 forelimb with a small part of the thoracic wall. This is followed by thoracic and abdominal evisceration. The final step is direct oblique section of the fetal pelvis or, alternatively, deep detruncation followed by bisection of the pelvis. In caudal presentation, the first step is removal of a hindlimb, then abdominal and thoracic evisceration, followed by thoracic detruncation and oblique section for removal of 1 forelimb and most of the remaining thorax. The neck and remaining forelimb are removed together to complete the procedure.     

An evaluation of the mononuclear cells derived from bovine mammary gland dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties and non-specific esterase staining.

The distribution of mononuclear cells isolated from the bovine mammary gland during the nonlactating (dry) period was examined using monoclonal antibodies against leukocyte cell surface antigens, cellular light scattering properties, and the presence of nonspecific esterase. Most of the mononuclear cells isolated during the dry period were lymphocytes. T cells predominated until about 1 week prior to parturition. During the week prior to calving, the percentage of B cells increased until it approximated T cells. The ratio of CD4:CD8 cells was 2-3:1 for mammary gland T cells. This was similar to the ratio found in peripheral blood. At dry-off, about 12% of mammary mononuclear cells were macrophages. The macrophage percentage increased (to about 30%) at mid-dry and remained at this levels until parturition. PMN's were isolated with the mononuclear cells during the first 2 weeks dry and the week prior to calving. Three methods were used to identify mammary macrophages. Esterase staining (as an enzymatic method), forward angle/90 degrees light scatter (based on size and internal complexity), and MHC class II/forward angle light scatter (based on size and surface markers) were compared. Each method yielded similar specificity for macrophage identification. Non-adherent cell fractions, obtained by passage of the cells over Sephadex G-10 columns, were enriched in CD4 positive T cells, somewhat depleted of B cells, and depleted of macrophages and PMN's. Cells eluted from G-10 columns, with lidocaine, were mostly lymphocytes, but reflected the cells loaded onto the column.     

A method for separation of bovine blood leukocytes for in vitro studies.

Various methods have been developed for separation of the cellular components of blood. Size and density differences in cellular blood elements of various animal species necessitate the use of specific separative procedures. This study describes a method which combines isopycnic density-gradient centrifugation with erythrocyte aggregation as a means of isolating leukocytes from bovine blood. The procedure yields a viable population of mixed leukocytes which has proven useful for cell culture and in vitro tests of cell-mediated immunity.     

Tetracycline resistance determinants in streptococcal species isolated from the bovine mammary gland.

Seventy-one streptococci isolated from dairy cows with clinical mastitis were tested for tetracycline resistance. Twenty-one (30%) isolates were tetracycline resistant (Tcr), and eight hybridized with the Tet O, one hybridized with the Tet L, and one hybridized with both the Tet L and Tet K determinants. The remaining Tcr isolates did not hybridize with any of the 5 Gram-positive Tet determinants tested. The Tet O determinants were plasmid-mediated, and four selected strains transferred the Tet O determinant at frequencies of 10(-6) to 10(-8). Strains which did not hybridize with known probes were tested for resistance to minocycline. All of the Streptococcus dysgalactiae had low minimum inhibitory concentrations (MICs) for minocycline, while the S. agalactiae and the one S. uberis showed high MICs to minocycline. This suggests that at least two different uncharacterized Tet determinants exist in these isolates, one conferring high resistance to both tetracycline and minocycline and one conferring only tetracycline resistance.     

Antimicrobial resistance in streptococcal species isolated from bovine mammary glands

Streptococcal species isolated from dairy cows with clinical mastitis were obtained from mastitis research workers in Florida, Louisiana, New York, Vermont, Washington, and West Virginia. Seventy-one streptococcal isolates were tested, including 39 strains of Streptococcus agalactiae, 21 strains of S dysgalactiae, and 11 strains of S uberis. The minimal inhibitory concentration of erythromycin, lincomycin, oxytetracycline, penicillin, spectinomycin, streptomycin, and tetracycline was determined for each isolate. Differences were not detected among strains with respect to geographic origin. None of the strains was resistant to penicillin. Lincomycin was the next most effective antimicrobial, with only 2 resistant strains of each streptococcal species. There were no differences among the streptococcal species with respect to resistance to either penicillin or lincomycin. Streptococcus uberis was more likely to be resistant to erythromycin than were S agalactiae and S dysgalactiae (P less than 0.02). Streptococcus agalactiae and S uberis had similar distributions for resistance to oxytetracycline, tetracycline, spectinomycin, and streptomycin. Strains of S dysgalactiae were more likely to have intermediate resistance to oxytetracycline and streptomycin than were strains of S agalactiae and S uberis, which were highly resistant to oxytetracycline and streptomycin (P less than 0.001). Differences were not detected among the streptococcal species with respect to resistance to spectinomycin. Resistance to multiple antimicrobials was observed in all streptococcal species tested. Although S dysgalactiae appeared to have a greater percentage of strains (73%) that were resistant to multiple antimicrobials than did S agalactiae (31%) or S uberis (45%), differences were not statistically significant.     

A simple method for determination of pepsinogen in bovine serum and plasma.

Calf serum pepsinogen can be determined by radial diffusion in a casein-containiing agarose gel prepared at pH 8.2 and afterwards immersed in a BaGla-containing buffer, pH 2.4, whereby the casein is precipitated homogeneously. Serum samples are placed in wells in the gel, and at the low pH pepsinogen present in the samples will be activated and the casein digested, whereby transparent, circular zones are formed in the otherwise opaque gel. The diameter of these zones can be measured directly and used as an expression of the serum pepsinogen level. On analysis of a number of sera from calves with or without ostertagiasis a positive correlation was found between the results obtained by this method and conventional absorption photometry (r = 0.87, n = 55).     

牛源胎儿弯曲菌PCR检测方法的建立

根据胎儿弯曲菌的16S rRNA基因序列设计引物,以微需氧培养的胎儿弯曲菌标准株菌体裂解物为模板,进行PCR扩增目的片段。同法检测了胎儿弯曲菌的10个参考菌株,结果均为阳性。采用异硫氰酸胍快速提取流产牛阴道分泌物中胎儿弯曲菌的DNA,然后用PCR方法检测,5份样品阳性,该试验可在24 h内完成,比病原分离法快5~7 d;而检测空肠弯曲菌、大肠杆菌、布氏杆菌、葡萄球菌、链球菌等相关病原时,均无特异性扩增产物,表明该检测方法具有快速、特异、实用性强的特点。     

Preliminary studies of mammary secretions in the mare to assess foetal readiness for birth

The status of the mare and foetus in relation to readiness for birth was assessed by measurement of the electrolytes sodium, potassium and calcium in mammary secretions pre-partum. Sixteen Thoroughbred mares were allowed to foal spontaneously and the ionic status of their mammary secretions was measured over three to five weeks pre-partum. From these measurements, a scoring system was developed where an ionic score of 35 points or more suggested that the mare was within 24 h of foaling. On the basis of this ionic score, 10 pony mares were induced with either oxytocin or fluprostenol and assessment of foal maturity was made by physical, behavioural and physiological criteria. Eight pony mares, induced when the ionic score was 35 points or more, delivered full term foals; two mares were induced when their scores were 30 and 20 points and delivered a full term and slightly immature foal respectively. These results suggest that foetal maturity may be related to electrolyte concentrations in mammary secretions and that an ionic score of 35 points or more may indicate that induction would be successful in terms of maturity of the newborn foal.     

Shedding of porcine reproductive and respiratory syndrome virus in mammary gland secretions of sows.

OBJECTIVE: To document shedding of porcine reproductive and respiratory syndrome (PRRS) virus in mammary gland secretions of experimentally inoculated sows, to evaluate effects of vaccination during gestation on virus shedding during the subsequent lactation, and to evaluate shedding of PRRS virus in milk of sows in commercial herds. ANIMALS: 6 sows seronegative for PRRS virus were used for experiment 1, and 2 sows were retained for experiment 2. For experiment 3, 202 sows in commercial herds were used. PROCEDURE: In experiment 1, 2 sows were inoculated with PRRS virus, 2 sows were vaccinated with modified-live PRRS virus vaccine, and 2 sows served as control pigs. Mammary gland secretions were assayed for PRRS virus. In experiment 2, pregnant vaccinated sows from experiment 1 were vaccinated with another modified-live PRRS virus vaccine. Mammary gland secretions were assayed in the same manner as for experiment 1. For experiment 3, milk collected from 202 sows in commercial herds was assayed for PRRS virus. RESULTS: In experiment 1, PRRS virus was detected in mammary gland secretions of both vaccinated and 1 of 2 virus-inoculated sows. In experiment 2, virus was not detected in samples from either vaccinated sow. In experiment 3, all samples yielded negative results. CONCLUSIONS AND CLINICAL RELEVANCE: Na?ve sows inoculated late in gestation shed PRRS virus in mammary secretions. Previous vaccination appeared to prevent shedding during the subsequent lactation. Results for samples obtained from sows in commercial herds suggested that virus shedding in mammary gland secretions of such sows is uncommon.     

Effect of mammary secretions on functions of polymorphonuclear leukocytes in pigs

OBJECTIVE: To determine the effects of porcine mammary secretions on polymorphonuclear (PMN) leukocyte function and to relate concentrations of estradiol-17beta and cortisol in mammary secretions to PMN cell function. SAMPLE POPULATION: Mammary secretions from 10 healthy sows and blood PMN leukocytes from 27 healthy sows. PROCEDURE: Mammary secretions were collected within 24 hours after parturition (colostrum) and 12 to 13 days later (milk). Chemoattractant properties were assessed by use of a cell migration assay. Phagocytic capacity of PMN cells in colostrum and milk was assessed by recording chemiluminescence following phagocytosis of Escherichia coli or zymosan. Estradiol-17beta and cortisol concentrations were determined by use of radioimmunoassays. RESULTS: Chemoattractant properties of colostrum and milk were significantly greater than that of zymosan-activated serum. However, chemoattractant properties did not differ significantly between the 2 types of secretions. The capacity of PMN cells in colostrum to phagocytose either zymosan or E. coli was less, compared with cells in milk, and the ability of cells in either type of mammary secretion to phagocytose E. coli was greater than the ability to phagocytose zymosan. Concentrations of estradiol-17beta and cortisol were greater in colostrum, compared with milk. No clear relation was evident between PMN cell activity and hormone concentrations in mammary secretions. CONCLUSIONS AND CLINICAL RELEVANCE: Although chemoattractant properties of colostrum and milk did not differ, the phagocytic capacity of PMN cells in colostrum was significantly less than that of cells in milk. This may predispose sows to coliform mastitis during the early postparturient period.     

A bovine herpesvirus isolated from sheep.

A viral agent was isolated from the trachea of a lamb that was suffering from a respiratory disorder. The physical and chemical properties of the isolates are characteristic of the herpesvirus group. It contains DNA in its virion, is ether sensitive, acid labile at pH 3.0 and heat labile at 56 degrees C after five minutes. The cytopathology observed provided further evidence of a herpesvirus isolate. The neutralization of the infectivity of the isolate with antiserum to bovine herpesvirus 1 is evidence that it should be considered an isolate of bovine herpesvirus 1. It is concluded that this is a report of a bovine herpesvirus infection in sheep.