Experimental infection of bovine mammary glands with Streptococcus agalactiae during the nonlactating period

Two experiments were done to determine the rate of intramammary infection (IMI) in dairy cows during the nonlactating period. In experiment 1, all glands were equally exposed to Klebsiella pneumoniae and Streptococcus agalactiae for the first 3 weeks after the start of the nonlactating period. Nearly all new IMI was caused by S agalactiae. In experiment 2, the susceptibility of nonlactating mammary glands to new IMI by S agalactiae after direct inoculation into the lactiferous sinus was determined. During the 1st month of the nonlactating period, 30% of inoculated glands became infected. During the last month of the nonlactating period, 90% of inoculated glands became infected.     

Ecologic study of the risk factors for environmental mastitis in cows

An index was developed to measure the proportion of intramammary infections caused by environmental microorganisms on dairy farms. This environmental index can be interpreted as the probability that an intramammary infection was caused by an environmental pathogen, rather than by a contagious pathogen. Using the environmental index as the outcome variable, risk factors for environmental mastitis were studied on 10 dairy farms in New York. Turning the cows outside was associated with lower environmental index, and having cows drink from a stream increased the environmental index. Selective (rather than uniform) nonlactating cow intramammary treatment was related to a lower environmental index (apparently because the farms practicing selective nonlactating cow treatment suffered from epizootics of contagious mastitis).     

Efficacy of intramammary treatment in unbred and primigravid dairy heifers

A total of 73 breeding-age and primigravid Jersey heifers in 4 herds was randomly allotted to treatment and control groups according to expected calving date. Thirty-five heifers were injected intramammarily with a nonlactating cow product containing penicillin/streptomycin. Thirty-eight heifers served as untreated controls. Of the 35 treated heifers, 34 (97.1%) were infected at time of treatment. In the untreated control group, all 38 heifers (100%) were infected at treatment time. At parturition, prevalence of intramammary infection in treated heifers decreased to 40%, whereas in the control group, prevalence remained about the same (97.4% of heifers). Prevalence of Staphylococcus aureus mastitis in treated heifers was reduced from 17.1% to 2.9% after treatment. In the control group, prevalence of S aureus mastitis decreased from 26.3% to 15.8%. Heifers treated during the second trimester of pregnancy had the greatest reduction in prevalence of mastitis and in somatic cell count at parturition, compared with controls. Findings indicated that intramammary treatment during pregnancy in primigravid heifers was effective in reducing prevalence of mastitis and somatic cell counts at parturition.     

Persistence of antibiotics in bovine mammary secretions following intramammary infusion at cessation of milking

A study was conducted to determine the persistence of antibiotic preparations for use in nonlactating cows in bovine mammary secretions following intramammary infusion at cessation of milking. Five commercially available antibiotic formulations were evaluated using 311 cows. All quarters of each cow were sampled once only during the nonlactating period and most cows were sampled at or near parturition. Antibiotic residues were detected qualitatively by the Bacillus stearothermophilus disc assay. Great variation between different antibiotics in persistence in mammary secretion was observed. In general, mammary secretions from most mammary glands infused with cloxacillin or penicillin-dihydrostreptomycin were positive at 28–35 days after infusion and some were positive at 42–49 days after infusion. On the other hand, <13% of mammary secretions at 7 days after infusion of novobiocin and 50% of mammary secretions at 14 days after infusion of penicillin-novobiocin were positive for antibiotics. Cephapirin benzathine persisted for about 21 days after infusion. Some samples that were positive for antibiotics after initial testing were negative following heating of samples, suggesting that component(s) of dry secretion can inhibit growth of B. stearothermophilus and influence the interpretation of results. Colostrum samples from all quarters except one were negative for antibiotics. These data suggest that nonlactating-cow antibiotic formulations persist primarily during the early to mid-nonlactating period. Based upon present methods of formulation, it would appear that antibiotic preparations for use in nonlactating cows most likely provide little protection during the periparturient period, at a time when mammary glands are highly susceptible to new intramammary infections.     

Gram-negative bacterial infections of the mammary gland in cows

Naturally acquired gram-negative bacterial intramammary infections (n = 160) were studied in 99 cows over a 2-year period. Escherichia coli, Klebsiella spp, Serratia spp, Enterobacter spp, and unidentified gram-negative bacteria were isolated from 28.8, 39.4, 9.4, 5.0, and 11.2%, respectively, of infected mammary glands. A majority (61%) of intramammary infections were first detected during the nonlactating period. Gram-negative bacteria isolated during the first half of the nonlactating period were predominantly Klebsiella spp, Serratia spp, and Enterobacter spp. Onset of E coli intramammary infections was more prevalent during the second half of the nonlactating period and during the first 7 days of lactation. The majority (59%) of infections were less than 28 days in duration, but Klebsiella spp and Serratia spp infections were of significantly (P less than 0.05) greater duration than infections with E coli. The greatest percentage (47%) of gram-negative bacterial intramammary infections were first detected during the summer.     

Host response in bovine mastitis experimentally induced with Staphylococcus chromogenes

An experimental infection model was developed to study host response to intramammary infection in cows caused by Staphylococcus chromogenes. CNS intramammary infections have become very common in modern dairy herds, and they can remain persistent in the mammary gland. More information would be needed about the pathophysiology of CNS mastitis, and an experimental mastitis model is a means for this research. Six primiparous Holstein-Friesian cows were challenged with S. chromogenes 4 weeks after calving. One udder quarter of each cow was inoculated with 2.1 x 10(6)cfu of S. chromogenes. All cows became infected and clinical signs were mild. Milk production of the challenged quarter decreased on average by 16.3% during 7 days post-challenge. Cows eliminated bacteria in a few days, except for one cow which developed persistent mastitis. Milk indicators of inflammation, SCC and N-acetyl-beta-D-glucosaminidase (NAGase) returned to normal within a week. Milk NAGase activity increased moderately, which reflects minor tissue damage in the udder. Concentrations of serum amyloid A (SAA) and milk amyloid A (MAA) were both elevated at 12h PC. MAA was affected by the milking times, and was at its highest before the morning milking. In our experimental model, systemic acute phase protein response with SAA occurred as an on-off type reaction. In conclusion, this experimental model could be used to study host response in CNS mastitis caused by the main CNS species and also for comparison of the host response in a mild intramammary infection and in more severe mastitis models.     

Experimental infection of lactating bovine mammary glands with Streptococcus uberis in quarters colonized by Corynebacterium bovis

Twenty-seven quarters of 18 lactating dairy cows were inoculated intramammarily with 3.6 X 10(4) colony-forming units (CFU) of a strain of Streptococcus uberis isolated from a cow with clinical mastitis. Before quarters were inoculated, 22 were considered as naturally colonized with Corynebacterium bovis, and 5 were considered bacteriologically negative. Streptococcus uberis was isolated from all quarters within 2 days after inoculation, and all quarters developed clinical mastitis by 3 days after inoculation. Mastitis was acute, and most cows had increased rectal temperatures. The number of somatic cells increased significantly (P less than 0.05), and milk production decreased significantly. In many cows, rectal temperatures remained increased, and Str uberis was isolated from infected glands after intramammary and systemic antimicrobial treatments were given. A decreased number (110 CFU) of the same strain of Str uberis caused equally severe mastitis in 3 quarters colonized with C bovis and in 1 bacteriologically negative quarter in 2 cows. Streptococcus uberis was isolated from all inoculated quarters, and all quarters developed clinical mastitis by 2 days after inoculation. Two quarters colonized with C bovis and 2 bacteriologically negative quarters were inoculated once with 25 CFU and once with 240 CFU of a different strain of Str uberis (ATCC 27958). Streptococcus uberis was never isolated from inoculated quarters, and changes in milk yield or number of somatic cells were not observed.     

Frequency of isolation of environmental mastitis-causing pathogens and incidence of new intramammary infection during the nonlactating period

Quarter samples (n = 6,328) of mammary secretions were collected from 160 cows during physiologic transitions of the udder to determine the frequency of isolation of mastitis-causing pathogens and the incidence of new intramammary infections (IMI) during the nonlactating period. None of the cows in the herd was infected with Streptococcus agalactiae, and the prevalence of Staphylococcus aureus was low. Cows were not treated with antibiotics at cessation of milking. A threefold increase in the percentage of quarters infected with major mastitis-causing pathogens developed from late lactation to early involution. Coliforms and streptococci other than Str agalactiae accounted for 94% of major pathogen infections. The number of quarters infected with coagulase-negative staphylococci increased slightly from late lactation to early involution, whereas the number of quarters infected with Corynebacterium bovis decreased markedly. Major pathogens caused 101 of 153 IMI at parturition and greater than 90% were caused by streptococci and coliforms. At parturition, 51 of 52 minor pathogen IMI were caused by coagulase-negative staphylococci. During early lactation, there was a marked decrease in quarters infected with major pathogens; however, the number of quarters with major pathogen IMI during early lactation was 2.3 times higher than the number of quarters infected before cessation of milking. The number of quarters with minor pathogen IMI during early lactation was the same as at parturition, but a marked decrease in quarters infected with coagulase-negative staphylococci and a marked increase in C bovis IMI developed from parturition to early lactation.     

Natural and experimental bovine intramammary infection with Prototheca zopfii

A naturally occurring case of chronic mastitis caused by Prototheca zopfii was detected in a dairy cow. The cow was observed for 12 weeks, and milk samples were cultured each week. Milk production was decreased, and high numbers (greater than 10(3)/ml) of the organism were detected in the milk. Six glands of 2 cows were inoculated with small numbers of P zopfii. All 3 cows were euthanatized, and at postmortem, a progressive pyogranulomatous inflammation was observed in all infected mammary glands. The organism was recovered from tissue and secretions from all inoculated glands and from lymph nodes draining these glands but not from other tissues.     

Mastitis in Post-Partum Dairy Cows

Transition from the dry period to lactation is a high risk period for the modern dairy cow. The biggest challenge at that time is mastitis. Environmental bacteria are the most problematic pathogens around parturition. Coliforms are able to cause severe infections in multiparous cows, and heifers are likely to be infected with coagulase-negative staphylococci. During the periparturient period, hormonal and other factors make the dairy cows more or less immunocompromised. A successful mastitis control programme is focused on the management of dry and calving cows and heifers. Clean and comfortable environment, proper feeding and adequate supplementation of the diet with vitamins and trace elements are essential for maintaining good udder health. Strategies which would enhance closure of the teat canal in the beginning of the dry period and would protect teat end from bacteria until the keratin plug has formed decrease the risk for mastitis after calving. Dry cow therapy has been used with considerable success. Yet, a selective approach could be recommended rather than blanket therapy. Non-antibiotic approaches can be useful tools to prevent new infections during the dry period, in herds where the risk for environmental mastitis is high. Vaccination has been suggested as a means to support the immune defence of the dairy cow around parturition. In some countries, implementation of Escherichia coli core antigen vaccine has reduced the incidence of severe coliform mastitis after calving.     

Efficacy of extended pirlimycin therapy for treatment of experimentally induced Streptococcus uberis intramammary infections in lactating dairy cattle

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, around the time of calving, and during early lactation. Strategies for controlling S. uberis mastitis have not received adequate research attention and are therefore poorly defined and inadequate. Objectives of the present study were to evaluate the efficacy of extended therapy regimens with pirlimycin for treatment of experimentally induced S. uberis intramammary infections in lactating dairy cows during early lactation and to evaluate the usefulness of the S. uberis experimental infection model for evaluating antimicrobial efficacy in dairy cows. The efficacy of extended pirlimycin intramammary therapy regimens was investigated in 103 mammary glands of 68 dairy cows that became infected following experimental challenge with S. uberis during early lactation. Cows infected with S. uberis in one or both experimentally challenged mammary glands were randomly allocated to three groups, representing three different treatment regimens with pirlimycin, including 2-day (n = 21 cows, 31 mammary quarters), 5-day (n = 21 cows, 32 quarters), and 8-day (n = 26 cows, 40 quarters). For all groups, pirlimycin was administered at a rate of 50 mg of pirlimycin hydrochloride via intramammary infusion. A cure was defined as an experimentally infected mammary gland that was treated with pirlimycin and was bacteriologically negative for the presence of S. uberis at 7, 14, 21, and 28 days after treatment. Experimental S. uberis intramammary infections were eliminated in 58.1% of the infected quarters treated with the pirlimycin 2-day regimen, 68.8% for the 5-day regimen, and 80.0% for the 8-day regimen. Significant differences (P <.05) in efficacy were observed between the 2-day and 8-day treatment regimens. The number of somatic cells in milk decreased significantly following therapy in quarters for which treatment was successful in eliminating S. uberis. However, there was no evidence to suggest that extended therapy with pirlimycin resulted in a greater reduction in somatic cell counts in milk than the 2-day treatment. The S. uberis experimental infection model was a rapid and effective means of evaluating antimicrobial efficacy during early lactation at a time when mammary glands are highly susceptible to S. uberis intramammary infection.     

Nocardia mastitis in cattle. 1. Clinical observations and diagnosis in 7 particular cases

Lately reports of nocardial mastitis have been increasing. Therefore the clinical aspects of 7 individual cases were investigated. Pathologic-anatomical changes of the udder were registered and bacteriological examination of tissue and milk probes were performed. All of the cows had a mastitis during previous lactation periods which had to be treated with antibiotics. The infected quarters recovered in no cases. Shortly after parturition 6 cows had a severe acute mastitis. The cows were treated intramammarily and parenterally with various antibiotics during several days. The infected quarter became indurated and strongly enlarged. These changes were not influenced by therapy, and the cows lost weight. 14 days after the outbreak of the illness, all 6 cows were culled. One cow had a chronic Nocardia mastitis with involution of the affected quarter. Though treated several times, this cow did not recover either. The histopathological examination of the affected quarters revealed an acute to chronic, necrotic to granulomatous mastitis. Nocardia asteroides was histologically and bacteriologically proven to be the causative agent. The reason for the frequent infections with Nocardia and the way of infection is not clear yet. Therapy with antibiotics brought no recovery in any cases. The infected cows should be removed from the herd or culled.     

Clinical and bacteriological response to treatment of clinical mastitis with one of three intramammary antibiotics

AIM: To compare the proportions of clinical and bacteriological cure of glands of dairy cows diagnosed with clinical mastitis, following treatment with one of three different intramammary antibiotic preparations. METHODS: Cows from dairy cow herds (n=28) across New Zealand which were diagnosed with clinical mastitis in one or more glands at any stage of lactation were randomly assigned at the cow level within sequentially presented groups of three animals to be treated with one of three intramammary antibiotics. The treatments were 1 g procaine penicillin, 0.25 g cefuroxime, and a combination of 1 g procaine penicillin and 0.5 g dihydrostreptomycin (DHS). All treatments were infused on three occasions at 12-hourly intervals. Duplicate milk samples were collected for bacteriology before initial treatment, and 21-42 days later. Logistic regression or generalised linear mixed models were used to analyse the proportion of cows or quarters retreated for mastitis within 30 days of initial treatment ('clinical treatment failure'), and the proportion of glands from which bacteria were isolated initially but from which the same bacterial species was not re-isolated ('bacteriological cure'). RESULTS: The annual herd average cumulative incidence rate of clinical mastitis was 12.7 cases/100 cows. The incidence rate was higher in young (2-year-old) and old (> or = 9-year-old) cows relative to 3- and 4-year-old cows, and was higher in Friesian than Jersey or crossbred cows. Streptococcus uberis was the pathogen most commonly isolated, and its relative prevalence declined with time postpartum. Cows treated with cefuroxime were more likely (p<0.01) to be re-treated for clinical mastitis in the 30 days after initial treatment than cows treated with procaine penicillin or procaine penicillin and DHS. Bacteriological cure occurred in 74% of treated glands and there was no difference in the proportion of cures among the treatments (p=0.4). The proportion of cures was lower when treatment occurred 28-72 days after calving (p<0.01) and if a major pathogen was isolated (p<0.001). CONCLUSIONS AND CLINICAL RELEVANCE: There was no benefit in terms of clinical or bacteriological cure rate in treating clinical mastitis cases with the combination of procaine penicillin and DHS compared to treatment with procaine penicillin alone. The proportion of clinical mastitis cases re-treated differed among herds, and more cows treated with cefuroxime were retreated within 30 days of initial treatment. However, the bacteriological cure proportion was the same among the treatments. Cure proportions were lower in cows from which major mastitis pathogens were isolated and when treatment commenced 28-72 days after calving.     

Antimicrobial resistance and genotypic characterization of coagulase-negative staphylococci over the dry period

Coagulase-negative staphylococci (CNS) have become the most frequently isolated organisms from bovine intramammary infections in recent years. While antimicrobial resistance (AR) is not considered a major problem among mastitis pathogens, concerns over emerging AR in general are increasing worldwide. Little information exists about the association between AR and one of the most common mastitis control measures, antibiotic dry cow therapy. The primary objective of the current study was to determine the prevalence of AR in CNS isolated before and after antimicrobial dry cow therapy. An additional objective was to genotypically characterize selected CNS isolates using pulsed-field gel electrophoresis (PFGE), to assess diversity and persistence of organisms over the dry period. Resistance against 10 antimicrobials was determined using a broth microdilution method and compared between CNS isolates collected at dry-off and at calving and from cows treated or not treated with intramammary antimicrobial products at dry-off (752 cows in total). Results suggested that increasing age of a cow and dry cow treatment when combined with high milk somatic cell count at dry-off and positive clinical mastitis history, were associated with increased AR to most beta-lactam antimicrobials and sulfadimethoxine. PFGE results suggested considerable diversity among the tested isolates as well as some clusters within cows and herds. PFGE would be useful in distinguishing between potentially persisting infections and cures and reinfections with different CNS strains.     

Morphometric analysis of proinflammatory cytokines in mammary glands of sows suggests an association between clinical mastitis and local production of IL-1beta, IL-6 and TNF-alpha

Twelve healthy primiparous sows received intramammary inoculation with Escherichia coli (serotype O127) during the 24-h period preceding parturition. Mammary gland biopsy samples were taken immediately before inoculation (0 h) and from the inoculated and the contralateral non-inoculated glands 24 h after inoculation. The analyses of interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) by immunohistochemistry revealed that the production of these proinflammatory cytokines significantly increased in the inoculated mammary glands of sows that developed clinical signs of mastitis (affected group, n=4) 24 h after inoculation. This was also true for IL-8 in the inoculated mammary glands of sows that did not develop clinical signs of mastitis (nonaffected group, n=8). Sows that developed clinical signs of mastitis displayed significantly lower constitutive production of IL-1beta than did sows that remained clinically healthy. The data indicate that the development of clinical symptoms of coliform mastitis in the sow is associated with a locally increased proinflammatory cytokine production in response to intramammary E. coli infection.     

Induced staphylococcal infections in the bovine mammary gland

In a study to develop and define a practical model of bovine mastitis caused by Staphylococcus aureus, induced infections were attempted in 203 bovine mammary glands of 41 cows, using 12 strains of S aureus. Approximately 100 colony-forming units of S aureus in saline solution were injected after milking, and milk samples were collected daily from test glands for 14 days to monitor the progress of infections and inflammatory responses. Relationships were examined for cow-related factors and for various characteristics of the strains of S aureus used to the development of a persistent intramammary infection. A dairy cow that was useful in this model was defined as follows: (1) the 2nd to 7th month in the 1st to 5th lactation; (2) producing milk from all mammary glands that contained less than 6 x 10(5) somatic cells/ml; and (3) having mammary glands that were free of any primary mastitis pathogen, as well as micrococci and Corynebacterium bovis. From the present study, it was not possible to define clearly a strain of S aureus which would be useful in the model, but 5 strains of S aureus were identified as being capable of producing persistent subacute infections with a high degree of repeatability.     

The prophylactic effect of a teat sealer on bovine mastitis during the dry period and the following lactation

AIM: To determine the prophylactic efficacy of a teat sealer, administered at drying off, in reducing new intramammary infections in the dry period and the following lactation. METHODS: A total of 528 cows with late lactation somatic cell counts <2 00,000 cells/ml was identified in three commercial herds. Of these, bacteriological examination showed 482 cows were uninfected in all four quarters and 46 were infected in only one quarter. At drying off, uninfected quarters were randomly allocated to the following treatments: no infusion (negative controls), infusion with a bismuth subnitrate based teat sealer, infusion with teat sealer + antibiotic, or infusion with a cephalonium-based dry cow antibiotic (positive control). New infections were identified during the dry period by periodic udder palpations and at calving by bacteriological culture. RESULTS: All three infused treatments reduced the incidence of new intramammary infections due to Streptococcus uberis, both during the dry period and at calving, by about 90% (p <0.01). The majority of the infections were due to Streptococcus uberis. For all treatments, a 50% lower incidence of clinical mastitis over the first 5 months of the ensuing lactation was reported by farmers. X-ray imaging of 19 teats showed that the teat sealer material was retained, at least in part, in the lower teat sinus over about 100 days of the dry period. CONCLUSIONS: Closure of the teat canal from day one of the dry period as achieved by the teat sealer was as effective in reducing new dry period infections as the infusion of a long-acting dry cow antibiotic formulation. The lower incidence of new infections in the ensuing lactation among the infused quarters implies that fewer subclinical infections persisted from the dry period. Use of teat sealers at drying off appears to offer the same prophylactic efficacy as the dry cow antibiotic approach.     

Coagulase-positive staphylococcal mastitis in a herd with low somatic cell counts

An increase in clinical mastitis infections was observed in a high-producing 77-cow Holstein herd. Low bulk tank somatic cell counts and individual cow Dairy Herd Improvement Association somatic cell counts observed before, during, and after the epizootic were suggestive of herd environmental mastitis. However, bacteriologic culture survey of the total herd indicated that, in addition to infections possibly attributable to environmental pathogens, 22% (17/77) of the cows were infected with coagulase-positive Staphylococcus spp. Conceivably, investigative efforts and management changes, without bacteriologic culturing, might have resulted in reduction of the clinical infection rate in this herd. However, the continued high prevalence of a contagious pathogen with potential future implications would have gone unnoticed. Somatic cell count in milk from individual cows generally is a useful tool for monitoring the probability of intramammary infection, but must be complemented with bacteriologic culture of milk to determine whether contagious or environmental pathogens are responsible.     

Microbiologic investigation of an epizootic of mastitis caused by Serratia marcescens in a dairy herd.

An epizootic of subclinical and clinical mastitis caused by Serratia marcescens was investigated in a 1,000-cow dairy farm in California. Serratia marcescens was isolated from 13 to 18% of composite milk samples obtained from lactating dairy cows. During monthly milk sampling performed during a 4-month period, S marcescens was isolated from 38.8 to 62.3% of composite milk samples obtained from cows from which S marcescens was previously isolated. Few cows infected with S marcescens had evidence of clinical mastitis. Somatic cell count value was associated with isolation of S marcescens. Cows with somatic cell counts greater than 500,000 were 5.48 times as likely to have intramammary infections with S marcescens, compared with cows with somatic cell count less than or equal to 500,000. Lactation number also was associated with S marcescens intramammary infection. After adjusting for the effect of lactation number, cows with high somatic cell count values were 2.98 times as likely to have intramammary infection with S marcescens, compared with cows with low somatic cell counts. Infection with S marcescens was independent of days in lactation, production string, and daily milk production. Eleven months after the beginning of the epizootic, S marcescens was isolated from organic bedding samples obtained from the dairy. Despite numerous attempts, other sources of S marcescens could not be identified on this dairy.     

Management strategies to decrease the prevalence of mastitis caused by one strain of Staphylococcus aureus in a dairy herd

The dairy herd at Washington State University had an outbreak of mastitis caused by a single strain of Staphylococcus aureus. The outbreak strain, termed novel, could not be controlled with routine contagious mastitis pathogen control procedures (incidence, 3.4 infections/100 cow months; peak prevalence > 22%). Our objective was to implement mastitis control measures that would decrease the incidence and prevalence of intramammary infection (IMI) caused by S aureus in the herd. The following intervention strategies were successfully implemented: strict segregation of cattle with IMI caused by S aureus, intensified culling of cattle with multiple-quarter IMI caused by S aureus, and inducing cessation of lactation of infected quarters in single-mammary-quarter infected cattle. One year after implementation of these control measures, incidence of IMI caused by S aureus was 0.35 infections/100 cow months, and prevalence had decreased from 20 to 8%.