Detection of mastitis in the bovine mammary gland by flow cytometry at early stages

Subclinical mastitis is a costly disease and its diagnosis is difficult. Besides the somatic cell count (SCC) and bacteriology, the differential inflammatory cell count (DICC) is a meaningful tool for mastitis detection. As microscopy is very subjective because of the low number of events to be counted, flow cytometry has often been proposed for the differentiation of milk cells. The objective of this study was to determine whether it is possible to identify subclinical mastitis in cattle at an early stage by a simple and fast flow cytometric method. The aim was to identify the main leucocyte populations in flow cytometric dotplots (polymorphonuclear neutrophils (PMN), lymphocytes and macrophages) and, with these, to elaborate a method of mastitis prognostics. Milk from 15 German Holstein cows was sampled in cross-sectional studies and SCC determined. After preparation, the milk cells were incubated with different specific antibodies that bind to different cell types and also to propidium iodide (PI), which differs between viable and non-viable cells. This procedure made it possible to localize cell types in a flow cytometric dot plot and to differentiate between viable and non-viable PMN. Percentages of viable PMN can be determined by a procedure consisting of a simple centrifugation, incubation with PI, and flow cytometric measurement. So it is possible to quickly determine the stage of the inflammation even in quarters with a low SCC.     

Lymphocyte blastogenesis inhibition by milk whey as an indicator of mastitis

Sixty milk whey samples prepared from quarters of five cows with a history of mastitis were tested for their ability to inhibit DNA synthesis in mitogen-stimulated lymphocytes. The inhibitory activity was compared with milk SCC, electrical conductivity, pH, and the number of colony-forming bacteria in the milk. Milk whey contained factors that inhibited DNA synthesis in cultured lymphocytes. Inhibition of mitogen-induced DNA synthesis increased with the clinical severity of mastitis and with increased values of indirect indicators of mastitis. The increases in inhibition and electrical conductivity were delayed past the increases in SCC. Milk whey (10 microliters) from quarters with clinical mastitis and from quarters with SCC greater than 900,000 inhibited 96 to 100%, 84 to 100%, and 69 to 100% of DNA synthesis in 3-d cultures of lymphocytes stimulated with Concanavalin A, phytohemagglutinin P, and pokeweed mitogen, respectively. The numbers of colony-forming bacteria correlated least with the inhibitory activity.     

Detection of multiple virulence-associated genes in Listeria monocytogenes isolated from bovine mastitis cases

Clinical samples (n=725) were collected from bovines (n=243) which were positive for mastitis using the California mastitis test (CMT) and somatic cell count (SCC). The clinical samples comprising blood (n=239), milk (n=243), and faecal swabs (n=243) were examined for the presence of pathogenic Listeria spp. Isolation of the pathogen was done using selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Confirmation of the isolates was based on biochemical tests and Christie, Atkins, Munch-Petersen (CAMP) test followed by pathogenicity testing. Pathogenicity of the isolates was tested by phosphatidylinositol-specific phospholipase C (PI-PLC) assay as well as in vivo tests namely, chick embryo and mice inoculation tests. The isolates were subjected to PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. Listeria spp. were isolated from 12 (1.66%) samples. Of these 4 (0.55%) and 1 (0.14%) were confirmed as Listeria monocytogenes and Listeria ivanovii, respectively. L. monocytogenes and L. ivanovii were recovered from milk samples (2) and faecal (3) of mastitic cattle (3) and buffaloes (2). L. monocytogenes recovered from the milk of mastitic cattle and L. ivanovii from the faecal swab of buffalo turned out to be pathogenic. However, the remaining three hemolytic isolates exhibiting positive CAMP test turned out to be negative in PI-PLC assay, chick embryo and mice inoculation. L. monocytogenes and L. ivanovii isolates characterized as pathogenic by PI-PLC assay and in vivo pathogenicity tests were found to possess all the five virulence-associated genes and three genes, plcA, prfA and actA respectively. The remaining three hemolytic but non-pathogenic L. monocytogenes isolates were negative for plcA by PCR. It seems that the plcA gene and its expression (in the PI-PLC assay) have an important role as virulence determinants in pathogenic Listeria spp. In conclusion, the PI-PLC assay and virulence genes targeted PCR (plcA, prfA and hlyA genes for L. monocytogenes and plcA, prfA and actA genes for L. ivanovii) hold a good promise as rapid and reliable in vitro alternatives to in vivo pathogenicity tests.     

Potentiation of antibiotic therapy for bovine mastitis by recombinant bovine interleukin-2.

Adjunct therapy with recombinant bovine interleukin-2 and antibiotics for Staphylococcus aureus IMI was investigated in an attempt to improve the therapy of antibiotics alone. Treatment of established S. aureus IMI with Na-cephapirin or Cefa-Lak produced average cures of 32.0 and 41.8%, respectively. When Na-cephapirin treatment was combined with recombinant bovine interleukin-2 at either 3.3 or 10 mg, the therapeutic efficacy was improved by an average of 20 to 30%. When Cefa-Lak treatment was combined with recombinant bovine interleukin-2 at 10 mg, the therapeutic efficacy was improved on average by 20%. Recombinant bovine interleukin-2, formulated in the excipient of the commercial Cefa-Lak, also improved the therapeutic efficacy by 16% compared with Cefa-Lak alone. Recombinant bovine interleukin-2, formulated in Cefa-Lak, maintained biological activity at room temperature for at least 21 d. After intramammary infusion of recombinant bovine interleukin-2, no biologically active interleukin-2 was detected in milk 48 h (four milkings) after administration. These data suggest that cytokines may be used as adjunct therapy with existing mastitis antibiotics or formulations of existing commercial products to improve the therapeutic efficacy.     

Specific agglutination of Streptococcus agalactiae from bovine mastitis by casein components of bovine milk

Streptococcus agalactiae strains freshly isolated from bovine mastitis cases clumped within 15 min of addition of small amounts of bovine milk to a broth culture. This reaction was not observed with isolates from human infections or bovine strains that had been maintained in the laboratory for extensive periods. Intensity of the clumping response as measured by a microtiter dilution assay was highly variable. Analysis of several single colony isolates derived from one strain indicated that the clumping phenotype was genetically unstable. The clumping reaction took place in the presence of rifampicin or chloramphenicol. Milk components that caused aggregation were heat stable, relatively insensitive to proteases, and were larger than 10,000 daltons. Purified casein also induced clumping in these strains.     

Detection of mastitis pathogens by analysis of volatile bacterial metabolites

The ability to detect mastitis pathogens based on their volatile metabolites was studied. Milk samples from cows with clinical mastitis, caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli were collected. In addition, samples from cows without clinical mastitis and with low somatic cell count (SCC) were collected for comparison. All mastitis samples were examined by using classical microbiological methods, followed by headspace analysis for volatile metabolites. Milk from culture-negative samples contained a lower number and amount of volatile components compared with cows with clinical mastitis. Because of variability between samples within a group, comparisons between pathogens were not sufficient for classification of the samples by univariate statistics. Therefore, an artificial neural network was trained to classify the pathogen in the milk samples based on the bacterial metabolites. The trained network differentiated milk from uninfected and infected quarters very well. When comparing pathogens, Staph. aureus produced a very different pattern of volatile metabolites compared with the other samples. Samples with coagulase-negative staphylococci and E. coli had enough dissimilarity with the other pathogens, making it possible to separate these 2 pathogens from each other and from the other samples. The 2 streptococcus species did not show significant differences between each other but could be identified as a different group from the other pathogens. Five groups can thus be identified based on the volatile bacterial metabolites: Staph. aureus, coagulase-negative staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae as one group), E. coli, and uninfected quarters.     

Preventing bovine mastitis by a postmilking teat disinfectant containing acidified sodium chlorite

A split-herd study was performed to determine if an acidified, sodium chlorite teat disinfectant, UDDERgold Platinum Germicidal Barrier Teat Dip (UG Pt, Ecolab Inc., Redmond, WA), was effective in preventing new intramammary infections (IMI) in lactating dairy cows compared with a licensed, iodophor teat disinfectant (Iosan, Novartis Animal Health, Ltd., Whittlesford, UK), and to show that the test product was tolerated equally well by teat skin. The study lasted 114 d and covered all weather conditions. The teats of 176 cows were dipped after each milking in UG Pt and the teats of 172 cows were dipped in Iosan, the positive-control product. Routine milk samples were taken from each quarter of every cow every 4 wk. Additional samples were taken from newly calved cows joining the trial and from cows with clinical signs of mastitis. Milk samples were cultured for the presence of bacteria and the cause of clinical mastitis. Each quarter was eligible for only 1 infection during the trial. The number of clinical cases was identical in each group (n = 13) and the number of subclinical infections was slightly lower in the UG Pt group than in the Iosan group (n = 27 and 31, respectively). These rates of infection suggest that the products did not differ in their ability to prevent a new IMI. At least 203 cows were assessed for skin integrity before the start of the trial and every 28 d throughout. The UG Pt teat dip had no adverse effects on teat condition. The prevalence of hyperkeratosis did not change with time for both groups (0.90 ± 1.08 and 0.95 ± 1.06 at wk 0 vs. 0.65 ± 0.87 and 0.49 ± 0.74 at wk 16 for fore and hind teats, respectively, for UG Pt and 1.02 ± 1.25 and 1.16 ± 1.11 at wk 0 vs. 0.51 ± 0.71 and 0.45 ± 0.65 at wk 16, respectively, for Iosan); no redness of the skin was observed in either group. Application of recommended statistical methods to demonstrate noninferiority was problematic.     

Deacylation of endotoxin during natural cases of bovine mastitis

Acyloxyacyl hydrolase, a lysosomal enzyme that deacylates and thus detoxifies lipopolysaccharide (endotoxin) has been identified in bovine peripheral blood and milk neutrophils. Enzymatic activity increases on a per neutrophil basis during cases of experimental Escherichia coli mastitis. The objective of this study was to quantify acyloxyacyl hydrolase activity from milk neutrophils collected from mammary glands naturally infected with a variety of bacteria. Acyloxyacyl hydrolase activity was detectable in milk neutrophils isolated from cases of both Gram-negative and Gram-positive bacterial infections, with highest activities found in milk neutrophils from glands infected with organisms known to cause the most severe forms of mastitis. In addition, acyloxyacyl hydrolase activity was inhibited to varying degrees in mastitic milk by a nonprotein inhibitory substance. Nonenzymatic deacylation of endotoxin also occurred in mastitic milk, but to a lesser degree than enzymatic deacylation. Nonenzymatic deacylation of endotoxin was not found to occur in clinically normal milk. Severity of coliform mastitis in individual cows may be dependent in part on the interaction of endotoxin with milk neutrophil acyloxyacyl hydrolase activity, inhibition of acyloxyacyl hydrolase activity by an inhibitory substance, and the inherent ability of milk to deacylate endotoxin nonenzymatically.     

Naturally occurring mastitis disrupts developmental competence of bovine oocytes

We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n = 50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n = 7), (2) medium SCC (n = 16), or (3) high SCC (n = 27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8 d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the germinal vesicle stage). The disruption was associated with elevation of SCC rather than bacterial type. The results may provide a partial explanation for the low fertility of cows that have contracted mastitic pathogens before insemination.     

Effect of mastitis on proteolytic activity in bovine milk

Proteolytic activity of milk was studied before, during, and after experimental-induced mastitis. An inoculum of Streptococcus agalactiae was infused into one quarter of each udder of six cows to elicit an infection. Bacteriological cultures and SCC of milk were used to monitor infection status. Sodium dodecyl sulfate-PAGE was used to measure proteolytic activity of milk. Inhibitor 6-amino-n-hexanoic acid was used to determine the relative proportion of plasmin and nonplasmin proteolytic activity of milk. Somatic cell count, total milk proteolytic activity, and nonplasmin proteolytic activity were higher in infected quarters than in quarters preinfection. After elimination of infections, SCC and nonplasmin proteolytic activity decreased to preinfection amounts. Total proteolytic activity of milk decreased after infections were cured but remained significantly higher than preinfection activity. This postinfection proteolytic activity in milk may be due to an increase in milk plasmin activity. Our data suggest that detrimental effects of mastitis on milk quality can continue after infection has been eliminated and milk SCC have returned to low values.     

Host-pathogen interactions in bovine mammary epithelial cells and HeLa cells by Staphylococcus aureus isolated from subclinical bovine mastitis

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap⁺icaA⁺icaD⁺ isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay.     

Assessment of beta-glucuronidase levels in goat's milk as an indicator of mastitis: comparison with other mastitis detection methods

The use of somatic cell counts (SCCs) for the diagnosis of mastitis is not a well-established procedure for the caprine species, because nonleucocytic cell-like particles are normally observed as a result of the apocrine secretion process of the goat mammary gland. The infection levels of 124 goats were measured by the beta-glucuronidase test, which was compared with the SCC method and the California mastitis test (CMT). Seventy-nine of 124 samples (63.7%) showed SCCs lower than 1.3 x 10(3) cells per ml. Of these samples, 93% showed low levels of beta-glucuronidase activity (< 15 U/ml). In the remaining 36.3% of the samples, SCCs were higher than 1.3 x 10(3) cells per ml. Of these samples, 88% showed high levels of beta-glucuronidase activity (15 to 100 U/ml). The CMT gave similar results. In this study, the beta-glucuronidase test was standardized for goat milk and shown to be reliable, enabling one to count only the somatic enzyme cells in milk and avoiding the interference encountered with the SCC method.     

Viability of milk neutrophils and severity of bovine coliform mastitis

To study the host-pathogen interactions during Escherichia coli mastitis, we first determined whether E. coli infection would change blood and milk polymorphonuclear neutrophil (PMN) chemiluminescence (CL) and viability. We then hypothesized that when E. coli invade the mammary gland, the viable PMN in milk would efficiently phagocytose and destroy E. coli before establishment of infection. We observed that the phagocytosis-dependent and independent CL were closely linked to PMN viability and were crucial to the outcome of mastitis. Maximal PMN influx and colony-forming units in infected quarters appeared at postinfection hours (PIH) 6 to 24. This further boosted PMN recruitment through bone marrow-blood barrier as well as blood-milk barrier. The survival of recruited PMN in the E. coli-infected quarters was much higher than that of noninfected quarters. Chemiluminescence activity of PMN from the infected quarters significantly increased following E. coli infection, even exceeding that of blood at PIH 6, 12, and 18 to 24; no such increase was observed in noninfected quarters, suggesting that the various responses of milk PMN to stimuli resulted largely from PMN viability. The highest CL intensity and durability was observed in milk PMN from infected quarters at PIH 12. Whereas an increased viability of PMN in the noninfected quarters was only significant at PIH 6, the viability of PMN in infected quarters was long lasting and significantly higher at PIH 6 to 72. Importantly, higher preinfection milk PMN viability correlated with bacterial clearance, which was accompanied by faster recovery. Our study strongly supports the hypothesis that boosting milk PMN viability could be a strategy with which to prevent or reduce the severity of coliform mastitis in dairy cows. This strategy might be achieved through strengthening bone marrow functionality.     

Phage typing of Staphylococcus aureus associated with subclinical bovine mastitis

Six hundred and seventeen isolates of Staphylococcus aureus from subclinical clinical mastitis cases in 63 dairy herds in Northern Ireland were typed using a set of 25 phages. Ninety-four per cent of the isolates were typable, with nine phages, predominantly from groups I and III, being responsible for almost all of the lysis. Although 68 phage patterns were found, six of them typed 47.2% of the isolates. One strain accounted for 14.7% of the isolates, but the largest number of strains (44) was restricted to individual farms. The epidemiological significance of these findings for on-farm mastitis control is discussed.     

A coagulase-negative variant of Staphylococcus aureus from bovine mastitis milk

Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates of Staphylococcus aureus which gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex? test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically 'coagulase-negative variant of Staph. aureus'. The fact that coagulase-negative Staph. aureus variants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis 'coagulase-negative staphylococci (CNS)' in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such as coa is necessary in these cases.     

Cost benefit analysis of bovine mastitis in the UK.

Bovine mastitis reduces the yield and quality of milk and increases the rate of culling and veterinary costs. This reduces the profitability of farm milk production but the calculation of the extent of this economic loss is complex because of the many factors involved and deficiencies in the evidence on the relationship between the disease and various production factors. This paper examines the available evidence for the UK and provides a consistent analytical framework within which the benefits arising from reduced mastitis in dairy herds constrained by quota can be considered. It is estimated that since 1970 the farms that have followed the recommended control procedures have reduced the average annual number of cases of clinical mastitis from 135 to 40 cases/100 cows each year, while the quarters remaining uninfected for a whole year has increased from 65 to 80% of the total quarters. The costs of the main control procedures (e.g. 8.60 pounds/cow for dry-cow therapy and teat dipping or spraying) are broadly covered by the reduction in clinical mastitis, leaving the benefits of reduced subclinical infection (e.g. 3810 pounds for a 100 cow herd unconstrained by quota and achieving the average reduction in infection) as a substantial bonus. The imposition of quotas reduces the financial benefit of mastitis control but it still remains a worthwhile investment. The results of this analysis can be used to suggest maximum costs of additional new control measures produced by research. It also indicates that there is considerable value in production research which gives more precise knowledge of production systems, thus allowing producers to respond optimally to quota cuts.     

Influence of bovine mastitis on lipolysis ond proteolysis in milk

Lipolysis and proteolysis in milk were determined before, during, and after experimentally induced mastitis. Streptococcus agalactiae was infused into one quarter of five cows to elicit an infection. Milk protease activity was higher during infection, but milk lipase activity was unchanged. Lipolytic damage to milk fat and proteolytic damage to milk casein occurred in the udder prior to milking during an infection. Lipolysis increased due to increased susceptibility of the milk fat to lipase action during infection. The mechanism of the increased susceptibility of the fat to lipolysis was not determined. After infections were eliminated, SCC, initial and stored FFA concentrations, and initial tryosine values returned to preinfection levels. However, after infections were eliminated, milk protease activity as determined by an increase in tryosine values remained elevated as milk SCC returned to preinfection levels. Protease activity returned to preinfection levels within 10 d after SCC returned to preinfection levels.     

乳酶作为奶牛隐性乳房炎诊断指标的研究进展

概述了牛髓过氧化物酶(MPO)、N-乙酰基-β-D-氨基葡萄糖苷酶(NAGase)、乳过氧化物酶(LP)、碱性磷酸酶(ALP)、乳酸且脱氢酶(LDH)、过氧化氢酶(CAT)、脂酶、谷胱甘肽过氧化物酶(GPx)在奶牛隐性乳房炎诊断应用中的研究进展.     

放射免疫法快速检测牛血清中磺胺类药物

目的应用Charm Ⅱ放射免疫分析方法检测牛血清中磺胺类药物残留,以便能大大提高检测速度,快速高效地完成磺胺类药物检测。方法在活体家畜上采集血清样品进行检测。确定控制点设定的方法,采集6个牛血清样品进行添加检测,设定控制点,样品结果与控制点比较。评价免疫反应体系的灵敏度和特异性。测得数值与控制点比较,测的数值大于控制点即为阴性,反之测得数值小于控制点即为阳性样品。结果磺胺类检测限可达20μg/kg,短时间可出检测结果,此方法特异性强,与除磺胺类药物外的其他药物无交叉反应。结论此方法快速,简便。虽然此方法只是初筛方法,测得阳性结果时则需采用其他检测方式进一步确证。但已经大大提高了检测效率,既保持了家畜的鲜活状态,又能快速得到检测结果,大大加快通关速度,促进了我国的活畜出口量。     

Effect of chronic staphylococcal mastitis on mitogenic responses of bovine lymphocytes

We compared mitogenic responses of milk and peripheral blood lymphocytes from nonmastitic (control) cows and cows with experimentally induced staphylococcal mastitis in one gland. Milk lymphocytes from infected glands were essentially unresponsive to Concanavalin A, phytohemagglutinin-P, and pokeweed mitogen. Proliferative responses of milk lymphocytes from uninfected glands of infected cows were not as depressed as those from infected glands but were significantly less than those of milk lymphocytes from control cows. Proliferative responses of peripheral blood lymphocytes from control and infected cows to Concanavalin A and phytohemagglutinin-P were similar; however, peripheral blood lymphocytes from infected cows responded in reduced fashion to pokeweed mitogen compared with peripheral blood lymphocytes from control animals. These observations demonstrate that in vitro lymphocyte blastogenesis is markedly depressed during infection, suggesting that in vivo lymphocyte function is compromised, possibly contributing to the chronicity of staphylococcal mastitis.