Prevalence of contagious mastitis pathogens in bulk tank milk in Prince Edward Island

The purpose of this study was to 1) estimate the herd prevalence of contagious mastitis pathogens in bulk milk from Prince Edward Island (PEI) dairy farms, 2) determine the association between bulk milk culture results and mean bulk milk somatic cell count (BMSCC), and 3) investigate the agreement of repeated bulk milk cultures. Three consecutive bulk milk samples were obtained at weekly intervals from all 258 PEI dairy herds and were cultured using routine laboratory methods. Cumulative prevalence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma spp. (M. bovis and M. alkalescens) was 74%, 1.6%, and 1.9%, respectively. Bulk milk somatic cell count of Staph. aureus-positive herds was higher than that of negative herds. Agreement for Staph. aureus isolation between 3 consecutive tests was moderate (kappa = 0.46). Mycoplasma bovis and M. alkalescens in bulk milk are being reported for the 1st time in PEI ever and in Canada since 1972.     

Culture of bulk tank milk as a mastitis screening test: A brief review

The culture of a sample of bulk tank milk may be a useful technique by which to screen herds for major mastitis pathogens. Staphylococcus aureus and Streptococcus agalactiae, if identified on a culture of a sample of bulk milk, reliably indicate infection of the udder. Environmental bacteria, such as the other streptococci and coliforms, are unlikely to be indicative of the proportion of cows infected with these organisms.Samples of bulk milk are readily obtainable and can be rapidly and inexpensively cultured to screen large numbers of herds for mastitis-causing bacteria, yet the performance of the test has only recently been formally assessed for its ability to correctly classify herds according to infection status.A single culture of bulk tank milk has been found to be a test with low sensitivity and high specificity for determining the presence of S. agalactiae or S. aureus in the herd. This means that many infected herds will be called negative, but few uninfected herds will be classified as positive.The literature assessing the performance of bulk tank milk culture in comparison with other mastitis screening tests, the use of bulk milk culture for prevalence surveys, and factors affecting these results is discussed.     

Between-herd prevalence of Mycoplasma bovis in bulk milk in Flanders, Belgium

Mycoplasma bovis (M. bovis) is a highly infectious pathogen of cattle causing pneumonia, polyarthritis, otitis, and less frequently, subcutaneous abscesses, abortions and meningitis. Ineffective drugs treatments, culling of infected cows and loss of milk production can lead to significant economic loss on dairy farms. The early detection of cows excreting M. bovis bacteria to prevent mastitis outbreaks is warranted. Reports suggest that the risk of M. bovis mastitis is higher in larger dairy herds. The objective of this study is to estimate the herd-level prevalence of M. bovis in Flanders, Belgium by culturing bulk tank milk samples taken from dairy farms. Three bulk tank milk samples per dairy herd were taken over four weeks, with collection intervals of two weeks. Culturing was done after pre-incubation using modified Hayflicks media to increase the chances of recovery of bacteria. For the identification of M. bovis, tDNA intergenic spacer PCR was used. In three herds (1.5%) of the 200 herds sampled, M. bovis was isolated from one of the three consecutive bulk tank milk samples. We conclude that in Flanders in 2009 at least 1.5% of the dairy herds had one or more cows excreting M. bovis in the milk. The frequent monitoring of bulk tank milk to detect the presence of M. bovis, especially in expanding herds on farms that often purchase replacement animals, should be encouraged in order to detect the presence of M. bovis and to monitor the success of control procedures following an outbreak of mycoplasmal mastitis in the herd.     

Prevalence of udder infections and mastitis in 50 California dairy herds

The California mastitis test (CMT) and bacteriologic culture were performed on samples of bulk-tank milk and cow-composite milk (n = 23,138 cows) from 50 California dairies, 19 of the 50 with known mastitis problems. Thirty-eight (76.0%) bulk-tank milk samples and 12,334 (53.3%) cows were positive by results of the CMT. Potential mastitis agents were isolated from 5,085 (22%) cows. Staphylococcus aureus was isolated from all 50 herds, Streptococcus agalactiae was isolated from 47 herds, and Mycoplasma sp was isolated from 24 herds. For cow-composite milk samples, the prevalences were 9.3% for Str agalactiae, 9.1% for S aureus, 0.9% for Mycoplasma sp, 1.2% for coliform bacteria, 0.9% for other streptococci, 0.8% for coagulase-negative staphylococci, and 1.3% for other organisms. The relative sensitivity and the relative specificity of the CMT performed on cow-composite milk samples were 83.4% and 55.2%, respectively, and the predictive value of positive CMT results was 34.2%.     

Sensitivity to various antibiotics of coagulase-negative staphylococci isolated from samples of milk from Dutch dairy cattle

During recent years the prevalence of coagulase-negative staphylococci in milk samples from Dutch dairy cows has increased. In 1999 16.2% of the bacteria isolated from milk collected from cows with subclinical mastitis were coagulase-negative staphylococci. In 2004 this proportion was 42.2%. The proportion of coagulase-negative staphylococci of the bacteria isolated from milk samples from cows with clinical mastitis was 7.3% in 1999 and 14.1% in 2004. In this study, the susceptibility of 108 coagulase-negative staphylococci to oxacillin, cefquinome, streptomycin, neomycin, penicillin, and the combination of nafcillin, penicillin, and streptomycin was tested. The isolates were cultured from milk collected from cows with mastitis and typed using the Api-Staph system. Eight species were identified. Staphylococcus chromogenes was the predominant species (41.7%), followed by Staphylococcus xylosus (15.7%) and Staphylococcus simulans (10.2%). With the agar dilution method all strains proved to be sensitive to cefquinome and 90% to oxacillin. Three isolates (2.8%) were mecA-positive. Despite the agar dilution results, these three isolates should be considered resistant to all beta-lactam antibiotics (penicillins, penicillins combined with a beta-lactamase inhibitor and all generations of cephalosporins). In the agar diffusion test, all isolates proved to be sensitive to the combination of nafcillin-penicillin-streptomycin, 99% were sensitive to neomycin and 1% intermediate sensitive, and 95% were sensitive to streptomycin, 4% resistant, and 1% intermediate sensitive. The coagulase-negative staphylococci were highly resistant to penicillin (37.4%), although the level of resistance varied between species, from 0% for Staphylococcus simulans to 100% for Staphylococcus saprophyticus. Because coagulase-negative staphylococci are resistant to several antibiotics, sensitivity testing is important for targeted treatment of mastitis.     

Effect of preculture freezing and incubation on bacteriological isolation from subclinical mastitis samples

In this study the sensitivity of three methods of isolation of udder pathogens from milk samples from subclinical mastitis cases was compared. For analysis 1827 quarter milk samples were selected. Milk was cultured using a standard culture technique (0.01 ml of fresh milk streaked on a sheep blood agar plate and on Edward's medium). In addition, an inoculum of 0.01 ml of the original milk sample was incubated for 24h at 37 degrees C in broth, followed by culture using the standard culture technique. In the third method, the whole milk sample was frozen for 24h, and then incubated for 24h at 37 degrees C, followed by culture using standard culture technique. The isolation percentage of Staphylococcus aureus was 4.7% for standard culture technique, 14.2% for incubation in broth, and 21.5% for the combination of freezing plus incubation. Isolation percentage of Streptococcus dysgalactiae and Streptococcus agalactiae was highest using the standard culture technique, while isolation rate of Streptococcus uberis was not different among the three methods used. With increasing somatic cell count, the likelihood of S. aureus, S. dysgalactiae and S. uberis isolation increased.Based on the relative sensitivity, defined as the isolation rate using a single technique compared to the isolation rate of the three techniques together, a combination of standard culture technique and freezing plus incubation was most attractive for achieving a high isolation rate of S. agalactiae and S. dysgalactiae. Relative sensitivity of S. uberis isolation was highest using the standard culture technique and incubation in broth, while S. aureus was most often isolated using a combination of incubation in broth and freezing plus incubation. A combination of the three methods increased the isolation rate for S. dysgalactiae, S. uberis and S. aureus. The standard culture technique, together with the combination of freezing plus incubation, can be recommended for isolating major udder pathogens. If S. aureus is the pathogen of main interest, using incubation in broth together with the combination of freezing plus incubation performed best.     

Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.     

The suitability of ELISA for the detection of antibodies against Mycobacterium avium ssp. paratuberculosis in bulk milk samples from Rhineland-Palatinate

Paratuberculosis or Johne's Disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a notifiable disease in Germany which produces enormous economical losses in dairy farms. At present,there is no confirmed data about the actual number of infected livestock herds in Germany. A countrywide monitoring program to evaluate the prevalence in dairy herds would only be economically feasible on the basis of bulk milk testing. In this study, we evaluated two ELISA test kits (SVANOVIR Ptb-ELISA, IDEXX-M.pt. Milk test kit) for the detection of antibodies against MAP in bulk milk. First, the Paratuberculosis-status of the herd derived from the history of the farm was used as a gold standard. Paratuberculosis-negative farms were tested negative with each test, but paratuberculosis-positive or Paratuberculosis-serologically-positive farms were detected only in one case (Svanovir) or three cases (IDEXX), respectively. Even if inconclusive results are counted as positive, 82.9 % (Svanovir) or 80 % (IDEXX) of the paratuberculosis-positive or serologically paratuberculosis positive farms were not detected. Nevertheless, a re-validation of both ELISAs by means of ROC and TG-ROC analyses was attempted by searching for ideal cut-offs, optimised for bulk milk. If a high specificity was selected, no acceptable sensitivity could be reached.The best results were obtained using a sensitivity of 32.3 % at a specificity of 100 % (Svanovir). With a small change of the cut-off value, the sensitivity increased to still 57 %, but this reduced the specificity to 67 %. Similar results were obtained with the IDEXX-ELISA. We then evaluated the Svanovir-ELISA for the detection of bulk milk samples on the basis of the current paratuberculosis prevalence within 69 dairy herds from Rhineland-Palatinate using individual milk samples.When the bulk milk samples were tested in two different laboratories using the same ELISA, considerable differences in the results became evident. Nearly all samples were tested with a higher relative test result in one laboratory, which often led to differences in the classification of the prevalence levels.The estimated within-herd seroprevalences ranged between 0 % and 37 %.There was little agreement between the historical paratuberculosis herd status and the within-herd prevalence in milk serum, as reflected in a kappa-index of 0.146.To determine the sensitivity and specificity of the bulk milk ELISA by ROC and TG-ROC analysis, 116 bulk milk samples were used that had been obtained from the 69 dairy herds participating in the study. The optimal ratio of sensitivity (81 %) and specificity (77 %) relative to a "gold standard" was obtained when the cut-off was set at the 10 % level. These values for sensitivity and specificity were better than those obtained in an evaluation of the same ELISA in which the historical Paratuberculosis herd-status was used as a "gold standard." The results of this study question the suitability of the available ELISAs for bulk milk testing.Taking into account that the Svanovir-ELISA for individual milk samples has a sensitivity of 60 96% relative to the blood serum variant of the test, and that the latter has also a limited sensitivity due to the pathogenesis of paratuberculosis, the available test systems examined in this Study do not seem to be suitable for herd diagnosis by using bulk milk samples.     

Bulk tank milk analysis: factors associated with appearance of Mycoplasma sp. in milk

Factors associated with the presence of Mycoplasma sp. in bulk tank milk samples were evaluated from 664 herds during 2.25 years. Milk quality components were not strongly related to the presence of Mycoplasma sp. in bulk tank milk. The presence of other contagious mastitis pathogens, Staphylococcus aureus and Streptococcus agalactiae, was also not related to the presence of mycoplasma, suggesting that the aetiology and transmission of mycoplasma mastitis were different from transmission of other contagious mastitis pathogens. The occurrence of the first isolation of mycoplasma from a bulk tank was not correlated to season of the year. Mycoplasma in bulk tank milk samples were more likely to be found in herds shipping more milk, an indirect measure of herd size. This suggests that larger herds are more likely to have mycoplasma mastitis. However, the first appearance of mycoplasma mastitis in a bulk tank sample was followed by a sample without this pathogen in more than 60% of herds. Mycoplasma sp. was not detected in any herd a year after first isolation. These findings suggest that this pathogen could be controlled and eliminated from herds.     

Diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in individual milk and bulk milk samples of dairy herds

The objective of the study was to determine the diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Map) in individual milk samples and in bulk milk samples. For individual milk samples the specificity of the Pourquier ELISA was estimated by testing a panel of individual milk samples from certified Map-free herds. The relative sensitivity of the assay in individual milk samples and agreement of the results with those of serum samples was estimated by testing panels of paired serum-milk samples from seropositive cattle, whole-herd investigations, and moderate or heavy shedders. The specificity of the ELISA for individual milk samples was still 99.8% at a cut-off of 20% sample to positive (S/P) value, clearly lower than the cut-off defined by the manufacturer (30% S/P). The relative sensitivity for individual milk samples as compared with positive serum samples was 87% for a cut-off of 20% S/P, and 80% for a cut-off of 30% S/P. The sensitivity of this ELISA for detection of high shedders was >90% both for individual milk and serum samples, also agreement was very good (kappa=0.91 for all paired samples). The specificity of the Pourquier ELISA in bulk milk samples was investigated by testing bulk milk samples from certified Map-free herds. Feasibility of bulk milk testing was investigated by titrating ELISA positive individual milk samples in negative milk. In addition, 383 bulk milk samples from herds with a known within-herd seroprevalence were tested. The specificity of the ELISA for bulk milk samples was 100% at a cut-off of 12.5% S/P. At the cut-off recommended by the manufacturer (30% S/P) performance of the bulk milk ELISA related to herd status (> or =2 seropositive cows) was rather poor, corresponding with a sensitivity of 24% and a specificity of 99% relative to serology. However, at the revised cut-off for bulk milk of 12.5% S/P and a within-herd seroprevalence of > or =3%, sensitivity and specificity relative to serology were 85% and 96%, respectively. Given the current herd-level seroprevalence in The Netherlands, these test characteristics corresponded with positive and negative predictive values for bulk milk of 67% and 94%, respectively. In conclusion, the diagnostic performance of the Pourquier ELISA for individual milk samples creates opportunities for a cheaper and more feasible testing scheme, while the diagnostic performance for bulk milk samples warrants further consideration.     

Acute coliform mastitis in buffaloes (Bubalus bubalis): Clinical findings and treatment outcomes

This report was delineated to study the clinical, bacteriological and therapeutic aspects concerned with acute coliform mastitis in buffaloes. Bacteriological examination of 80 quarter milk samples obtained aseptically from 56 buffaloes with acute mastitis revealed that coliform bacteria was the most common pathogen (45 cases) followed by Staphylococcus aureus (seven cases) then Streptococcus uberis (three cases), and Streptococcus agalactiae (one case). Clinically, hotness, swelling and painful reaction with serous excretion containing clots was recorded in buffaloes with coliform mastitis. The efficacy of ceftiofur was evaluated in the treatment of buffaloes with acute coliform mastitis. Parenteral ceftiofur neither improved clinical signs nor returned milk to pre-infection production level, whereas intramammary ceftiofur and combination of intramammary with parenteral ceftiofur improved the clinical signs in 10/15 and 12/15 buffaloes, respectively. On quarter level, 3/17, 12/17 and 15/21 quarters recovered in groups received parenteral, intramammary and combination therapy, respectively. This study demonstrates that systemic ceftofur is not effective in the treatment of clinical coliform mastitis in buffaloes.     

Evaluation of a rapid coliform detection kit from clinical mastitis milk using colloidal gold nanoparticle–based immunochromatographic strips

The accurate identification of mastitis‐causing bacteria assists in effective management by both dairy farmers and veterinarians and can be used to implement the selective use of antimicrobials for treatment. The purpose of this study was to evaluate the ability of our developed anti–ribosomal protein-L7/L12 antibody–coated immunochromatographic strip (ICS) test to detect coliforms in milk by comparing the results with the bacteriological culture method. We investigated the performance of the ICS test as compared with the bacteriological culture method using 308 milk samples from clinical bovine mastitis. First, to determine the optimal ICS test cutoff point for detecting coliform mastitis, we developed a receiver-operating characteristic curve. The result showed that the cutoff point was at 0.5 of our index. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of the ICS test were 81.3%, 84.8%, 69.2%, and 91.54%, respectively. As the clinical signs increased in severity, the F-measure, a weighted harmonic mean of the sensitivity and overall PPV performance, increased. Because it is especially important to treat clinical mastitis appropriately in the early stages of detection, the ICS test, which can be used by both dairy farmers and veterinarians on dairy farms, is considered to be a useful tool for detecting coliform mastitis, which often presents with severe signs.     

Genetic diversity and antimicrobial susceptibility profiles among mastitis-causing Staphylococcus aureus isolated from bovine milk samples

OBJECTIVE: To determine whether particular antimicrobial susceptibility profiles of bovine mastitis-causing Staphylococcus aureus isolates were associated with specific S aureus genotypes. SAMPLE POPULATION:357 S aureus isolates recovered from milk samples submitted for diagnostic bacteriologic testing from 24 dairy herds. PROCEDURES: Antimicrobial susceptibility of S aureus isolates was assessed by determining minimum inhibitory concentrations (MICs) to 14 antimicrobial agents. After digestion of S aureus genomic DNA by SmaI, electrophoretic patterns were obtained via pulsed-field gel electrophoresis (PFGE) and used to classify isolates into types. Gels were analyzed, and data were used to prepare dendrograms. RESULTS: 308 of 357 (86%) S aureus isolates were susceptible to all antimicrobials evaluated. Forty-nine S aureus isolates were resistant to 1 or more antimicrobials; of these isolates, 37 were resistant only to penicillin, 9 were resistant to penicillin and erythromycin, 2 were resistant to tetracycline, and 1 was resistant to erythromycin. Isolates were assigned to 7 PFGE types. An association was found between PFGE type and antimicrobial susceptibility profile. Organisms with resistance to at least one of the tested antimicrobial agents were identified in only 4 of the 7 types of S aureus. CONCLUSIONS AND CLINICAL RELEVANCE: Antimicrobial resistance was uncommon among the mastitis-causing S aureus isolates identified in the milk samples. A limited number of genotypes were associated with mastitis in these herds. Antimicrobial resistance phenotypes were associated with particular S aureus PFGE types; this association may have implications for future treatment and control of S aureus-associated mastitis in cattle.     

Recovery of Streptococcus agalactiae and Staphylococcus aureus from fresh and frozen bovine milk

Three hundred seventy-three milk samples were screened for Streptococcus agalactiae and Staphylococcus aureus. After sample storage at -20 C for 23 days, the frequency of Str agalactiae isolation increased 2.50 times. The frequency of S aureus isolation increased 1.48 times in the same interval. Increases in the proportion of these isolates were highly significant (P = 0.000006 and 0.0001, respectively). Results of the study indicate that optimal procedures for microbiological testing for these mastitis pathogens may include preculture freezing. The magnitude of the increase in the proportion of isolates indicates the existence of an important population of infected cattle shedding bacteria at concentrations not detected by use of standard microbiological techniques.     

High milk neutrophil chemiluminescence limits the severity of bovine coliform mastitis

Polymorphonuclear neutrophil (PMN) function changes during mastitis. To investigate the contribution of milk PMN to the severity of Escherichia coli (E. coli) mastitis, chemiluminescence (CL) of blood and milk PMN and their efficiency to destroy coliform bacteria in the mammary gland were examined following the induction of E. coli mastitis in early lactating cows. To better assess and define the degree of mastitis severity, cows were classified as moderate and severe responders according to milk production loss in the non-infected quarters at post-infection hour (PIH) 48. There was an inverse relationship between pre-infection milk PMN CL and colony-forming units at PIH 6. In moderate cows, the pre-infection blood and milk PMN CL was approximately 2-fold higher than that of severe cows. The probability of severe response increased with decreasing pre-infection PMN CL. At the beginning of the infection blood and milk PMN CL was consistently higher, and milk PMN CL increased faster after infection in moderate cows. At PIH > 48 milk PMN CL in severe cows exceeded that of moderate cows. The somatic cell count (SCC) in moderate cows increased faster than colony-forming units, whereas in severe cows the results were reversed. The kinetics of CL activity for blood and milk PMN before and during the early phase of infection confirmed an impairment in PMN CL activity for severe responding cows. High pre-infection blood and milk PMN CL and the immediate increase of milk PMN CL and SCC after infection limited bacterial growth thereby facilitating the recovery of E. coli mastitis in moderate cows. Our study strengthens the idea that pre-existing milk PMN (a static part of the udder's immune defense) functions as a "cellular antibiotic" before and during infection, and low milk PMN CL is a risk factor for bovine coliform mastitis.     

Arachidonic acid metabolites in milk of cows during acute coliform mastitis

Concentrations of prostaglandin F2 alpha (PGF2 alpha) and thromboxane B2 (TXB2) were evaluated in the milk of cows with naturally occurring (n = 3) and experimentally induced (n = 5) acute coliform mastitis. These arachidonic acid metabolites were measured by radioimmunoassay in unextracted milk. Experimental infections were induced by inoculating 600 to 1,200 colony-forming units of Escherichia coli into 1 mammary quarter per experimental cow. In the experimental cows, milk was collected before inoculation and at 12, 24, 36, 48, 60, and 72 hours after inoculation. Somatic cell concentrations, bovine serum albumin, and concentrations of PGF2 alpha and TXB2 were determined in milk collected at each sampling. Mild-to-moderate increases in milk PGF2 alpha and TXB2 concentrations were observed in cows with naturally occurring mastitis. the increases corresponded to the clinical severity and course of mastitis. In the experimental cows, increases in milk PGF2 alpha and TXB2 concentrations were observed, but the increases were not significant, using a statistical model that included factors of treatment, cows, hours after inoculation, cows-by-treatment and hours-by-treatment interactions, and random error (residual). Results of the present study indicated a large biological variability in milk arachidonic acid metabolite concentrations in cows with acute coliform mastitis, and that arachidonic acid metabolites may be important in the pathophysiologic process of acute coliform mastitis.     

Performance of a Johne's disease enzyme-linked immunosorbent assay adapted for milk samples from goats.

Antibody detection-based tests for paratuberculosis offer speed and economy, 2 diagnostic test attributes important to animal industries with narrow profit margins. Application of such tests to individual milk samples instead of serum samples can further improve testing efficiency and decrease testing cost. Accuracy of a commercial bovine paratuberculosis enzyme-linked immunosorbent assay (ELISA) adapted for use on goat serum and milk samples was determined. Fecal, blood, and milk samples were collected from 159 goats belonging to 2 Wisconsin goat herds with a prior history of paratuberculosis and 1 herd of 50 goats from a paratuberculosis-free Wisconsin herd. Fecal samples were cultured using the modified BACTEC 12B media. Sera were tested according to the manufacturer's instructions for bovine samples. Milk samples were centrifuged and mixed with the ELISA kit's Mycobacterium phlei-containing diluent at a ratio of 1:2. Using fecal culture as the "gold standard," the sensitivity of the ELISA on goat serum was 64% and the sensitivity of the ELISA on goat milk was 48%. The milk ELISA had higher agreement with fecal culture results (kappa = 0.525) than the serum ELISA (kappa = 0.425). ELISA specificity was 100% on both serum and milk. Regression analysis also showed good correlation between serum and milk S/P values (r2 = 0.67). Although less sensitive, the ELISA on goat milk samples appears to offer a useful, low-cost alternative for detection of goats with paratuberculosis that have progressed to the stage of shedding M. paratuberculosis in their feces.     

Limulus test--a rapid and simple method for the detection of endotoxins produced by gram-negative bacteria in mastitis milk

According to the present study the limulus amebocyte lysate test (LAL) seems to be a convenient test to detect endotoxin in milk from udder quarters with and without inflammation. The correlation between endotoxin concentration and the results from the bacteriological investigation of 79 milk samples was good (Table I). Determination of endotoxin in 20 milk samples from cases of acute clinical mastitis with high cell count and a negative bacteriological culture showed that all but one had an endotoxin concentration of greater than 1.0 ng/ml milk (Table II). By using a micromethod of the LAL it is possible to detect cases of mastitis caused by gram-negative bacteria about one hour after the sample has reached the laboratory. In a preliminary field study milk from 13 cases of acute clinical mastitis were tested by a modified limulus amebocyte lysate (LAL) test ("cowshed test"). A 100% correlation to bacteriological findings was observed (Table IV). By using the LAL test to detect mastitis cases caused by gram-negative bacteria great economic advantages and less risk for resistance problems can be achieved by using proper antibiotics. This is the fact in Sweden where the frequency of acute clinical mastitis caused by streptococci (100% of strains sensitive for penicillin) and Staphylococcus aureus (about 90% of strains sensitive for penicillin) is high (70-80%) and about 20% are caused by gram-negative bacteria, mostly E. coli.     

Detecting Mycoplasma bovis in milk by enzyme-linked immunosorbent assay, using monoclonal antibodies

Enzyme-linked immunosorbent assay, using monoclonal antibodies, was used to detect Mycoplasma bovis in milk samples from a dairy experiencing an epizootic of mastitis. This method was specific (100%) for M bovis. Broth enrichment increased the sensitivity from 65% to 86%, compared with standard culture methods.     

Effect of intramammary injection of RbIL-8 on milk levels of somatic cell count, chemiluminescence activity and shedding patterns of total bacteria and S. aureus in Holstein cows with naturally infected-subclinical mastitis

Summary The effect of intramammary injection of recombinant bovine interleukin-8 (rbIL-8, 1 mg/10 ml of saline) on quarter milk levels of somatic cell count (SCC), chemiluminescence (CL) activity and counts of total bacteria and Staphylococcus aureus (S. aureus) was investigated, using 10 Holstein cows with an early stage or a late stage of subclinical mastitis naturally infected with S. aureus. In the late-stage group, milk SCC and CL activity had significant rises with maximum levels at 6 h, following maintained high levels thereafter post-cytokine injection. The counts in milk total bacteria and S. aureus were insignificantly decreased, being increased back on day 7 post-cytokine injection. Thus, the cytokine was inefficient for the late-stage subclinical mastitis. However, in the early-stage group milk SCC and CL activity declined to under pre-injection levels on day 7 after marked and significant rises at 6 h and day 1 post-cytokine injection. The milk total bacterial count decreased significantly on days 0.25 and 2. Furthermore, the milk S. aureus count was decreased significantly on days 1, 2, 3 and 7 by the cytokine injection. These results suggest that the rbIL-8 has a potential as a therapeutic agent of the subclinical mastitis of dairy cows, if the cytokine is applied at an initial stage of infection.