Teat skin normal flora and colonization with mastitis pathogen inhibitors

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial floral became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow tests with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.     

In vitro growth inhibition of major mastitis pathogens by Staphylococcus chromogenes originating from teat apices of dairy heifers

Earlier field observations suggest that teat apex colonization by Staphylococcus chromogenes pre-partum in dairy heifers protects udder quarters against elevated somatic cell counts early post-partum. To explain these findings, the in vitro inhibitory capability of S. chromogenes from teat apices of heifers towards some major mastitis pathogens was tested using a modified cross-streaking method. Two out of 10 S. chromogenes isolates, both originating from two different teats from the same heifer, consistently inhibited growth of all Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis strains, but none of the Escherichia coli strains. The present study, therefore, supports the protective effect of teat apex colonization by S. chromogenes by in vitro production of inhibitory substances.     

The bacterial flora in the teat duct of ewes can protect against and can cause mastitis

We studied the possible effects of bacterial populations within the teat duct, in the pathogenesis of ovine mastitis. In experiment I, 32 ewes were allocated into group A (ewes from which we isolated (+++ growth) coagulase-negative staphylococci), B (ewes from whose duct we isolated (+ growth) coagulase-negative staphylococci) or C (ewes from which we isolated Bacillus spp.) and subdivided into A1, B1, C1 (n=4; challenged by deposition of 1.250 cfu of Mannheimia haemolytica into the teat duct) or A2, B2, C2 (n=4; used as uninoculated controls); group D (n=8) contained ewes with no bacteria in their teat ducts and were challenged as above. There were less bacteriological isolations of flora (P = 0.018) and challenge (P<0.05) organisms from A1 than from A2 and D ewes; the severity of pathological findings in A1 (summed up score: 27) ewes was smaller than in D (summed up score: 36) ewes (P = 0.038). No such findings were evident with B1 or C1 ewes (P>0.4). In experiment II, ewes (groups E and F, n=6) from whose duct we isolated coagulase-negative staphylococci (+ growth) were used; in group G (n=6) ewes with no bacteria in their teat ducts were included. Teat chapping was applied in E and G ewes. All E ewes developed acute clinical mastitis within 24 h after teat chapping, although we had carried out no challenge; there were more bacteriological isolations of flora organisms from E than from F and G ewes (P < 0.001); the severity of pathological findings in E (score: 28) was greater than in F (score: 3) or G (score: 14) ewes. In experiment III, eight ewes with no bacteria in their teat ducts were allocated into group H or I (n=4) and challenged into the teat (group H) or into the gland (group I) with 10(6) cfu of a Staphylococcus simulans recovered from the teat duct of a group E ewe. Group H ewes developed transiently clinical followed by subclinical mastitis (based on bacteriological and cytological evidence), whilst group I ewes developed severe clinical disease. We conclude that staphylococcal flora present in high numbers within the teat duct of ewes can afford some protection against invading microorganisms. However with impeded defence mechanisms of the teat, the same flora may invade the mammary parenchyma and cause clinical mastitis.     

Coagulase-negative staphylococci-emerging mastitis pathogens

Coagulase-negative staphylococci (CNS) have become the most common bovine mastitis isolate in many countries and could therefore be described as emerging mastitis pathogens. The prevalence of CNS mastitis is higher in primiparous cows than in older cows. CNS are not as pathogenic as the other principal mastitis pathogens and infection mostly remains subclinical. However, CNS can cause persistent infections, which result in increased milk somatic cell count (SCC) and decreased milk quality. CNS infection can damage udder tissue and lead to decreased milk production. Staphylococcus simulans and Staphylococcus chromogenes are currently the predominant CNS species in bovine mastitis. S. chromogenes is the major CNS species affecting nulliparous and primiparous cows whereas S. simulans has been isolated more frequently from older cows. Multiparous cows generally become infected with CNS during later lactation whereas primiparous cows develop infection before or shortly after calving. CNS mastitis is not a therapeutic problem as cure rates after antimicrobial treatment are usually high. Based on current knowledge, it is difficult to determine whether CNS species behave as contagious or environmental pathogens. Control measures against contagious mastitis pathogens, such as post-milking teat disinfection, reduce CNS infections in the herd. Phenotypic methods for identification of CNS are not sufficiently reliable, and molecular methods may soon replace them. Knowledge of the CNS species involved in bovine mastitis is limited. The dairy industry would benefit from more research on the epidemiology of CNS mastitis and more reliable methods for species identification.     

The role of Hydrotaea irritans in the transmission of summer mastitis

A series of experiments to investigate the possible role of the sheep head fly, Hydrotaea irritans, in the transmission of summer mastitis is reported. These showed that the pathogens Actinomyces pyogenes and Peptococcus indolicus may be harboured in the gut of the fly for up to 96 h following a contaminated meal; A. pyogenes survived on the surface of the fly for a similar period. H. irritans fed on the pathogens can contaminate a subsequent meal by regurgitation. However, no infections resulted from repeated exposure of the teats of heifers or cows to H. irritans fed on blood containing A. pyogenes and P. indolicus or on pus from a case of summer mastitis. In these experiments the frequency of feeding of the flies on the teat was high but subsequent bacterial contamination of the teat skin was low. Artificial contamination of the teat skin with greater than 10(6) cfu A. pyogenes led to a low incidence of mastitis even in the absence of cutaneous damage. Repeated applications of lower doses (less than 10(4) cfu) did not produce infection.     

Culture-independent exploration of the teat apex microbiota of dairy cows reveals a wide bacterial species diversity

Due to their close proximity to the mammary gland tissue, the bacterial communities lining the teat apex of the udders from lactating cows influence udder health. Denaturing gradient gel electrophoresis of the amplified V3 variable region of the 16S rRNA gene was used as a culture-independent method to reveal the bacterial composition of 48 samples originating from the teat apices of twelve Friesian-Holstein dairy cows suffering from clinical mastitis in one quarter. The microbiota belonged to four bacterial phyla: the Actinobacteria (32% of all genera), the Bacteroidetes (1%), the Firmicutes (42%), and the Proteobacteria (25%), encompassing 17 bacterial genera. Some differences in occurrence of these genera were seen when comparing quarters that were non-infected (n=22), subclinically infected (n=14), or clinically infected (n=12). Besides commensal skin-associated bacteria, opportunistic pathogenic bacteria, and mastitis-causing pathogens were found as well. The species diversity varied considerably among the most prevalent bacterial genera. While Corynebacterium and Staphylococcus displayed a large diversity among the recovered sequences, indicating the possible presence of a variety of different species, only a single bacterial species (represented by one sequence) was obtained for the genera Aerococcus, Acinetobacter, and Psychrobacter. In conclusion, introducing culture-independent analysis of teat apical skin swabs in mastitis research revealed an unexpected wide bacterial diversity, with variations between quarters with a different clinical status. In addition to potential mastitis-causing pathogens, it exposed the yet poorly mapped presence of skin-associated and other bacteria residing in close proximity to the mammary gland tissue. PCR-DGGE may thus be considered as a useful tool for the entanglement of animal skin microbiota, in casu the teat apices of dairy cows.     

Intramammary infections and risk factors for clinical mastitis in herds with low somatic cell counts in bulk milk

Ten herds with low somatic cell counts in bulk milk had an incidence of clinical mastitis of only 2.2 per 100 cows whereas 10 other herds with similarly low cell counts had an incidence of 53.6 per 100 cows. The major pathogens in the herds with a high incidence were Escherichia coli, Streptococcus uberis, Staphylococcus aureus and the coagulase-negative staphylococci. The percentage of uninfected quarters in the herds with a high incidence of clinical mastitis was 21.4 per cent compared with 12.2 per cent in the herds with a low incidence of clinical mastitis. The prevalence of coagulase-negative staphylococci, Corynebacterium bovis and Micrococcus species was higher in the herds with a low incidence of clinical mastitis. There was a significant linear relationship between the percentage of uninfected quarters and the incidence of clinical mastitis in the herds with a high incidence of clinical mastitis. In herds with a low incidence of clinical mastitis significantly less teat disinfection after milking was practised. The results suggest that infections with minor pathogens tend to protect cows against mastitis, and that teat disinfection after milking may increase the percentage of uninfected quarters and lead to an increased risk of clinical mastitis in herds with low somatic cell counts in bulk milk.     

Growth of Staphylococcus aureus and Streptococcus species in bovine mammary secretions during the nonlactating and peripartum periods following intramammary infusion of lipopolysaccharide at cessation of milking.

The objective was to determine if induced mammary inflammation at cessation of milking influenced growth of gram-positive mastitis pathogens in mammary secretions, particularly during early involution. Growth of all mastitis pathogens evaluated was similar in cell-free fat-free mammary secretions from LPS-infused and control glands. These data indicate that intramammary infusion of LPS at cessation of milking did not alter growth of gram-positive mastitis pathogens in mammary secretion during the nonlactating period. Stage of lactation and the nonlactating period influenced bacterial growth and marked differences between bacteria and among strains of a bacterial species were observed. Staphylococcus aureus grew well in secretions collected during late lactation, but growth decreased during early- and mid-involution and increased again in secretions obtained near parturition. Streptococcus agalactiae and Strep. uberis grew better in mammary secretion obtained during involution than in secretions collected during late or early lactation. Streptococcus dysgalactiae grew well in mammary secretions at all time periods. These data demonstrate the variability of mastitis pathogen growth during physiologic transitions of the bovine udder.     

Summer mastitis in heifers: studies on the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus and Bacteroidaceae in clinically healthy cattle in Denmark.

With the aim of investigating the seasonal occurrence of Actinomyces pyogenes, Peptostreptococcus indolicus, Bacteroides melaninogenicus ss. levii and Fusobacterium necrophorum, and thus the potential for development of summer mastitis, clinically healthy Danish Holstein-Friesian heifers due to calve in the autumn were sampled from the teat tip, the conjunctiva and the oral cavity at 2-6 week intervals from 1979 to 1981. The overall isolation rates of F. necrophorum, P. indolicus and B. melaninogenicus ss. levii, in order of significance, were significantly higher during the pasture period whereas no differences in isolation rates of A. pyogenes between housed and pastured animals were detected. F. necrophorum was recovered almost exclusively from the oral cavity, P. indolicus and A. pyogenes occurred most frequently in samples from the teat skin, whereas isolates of B. melaninogenicus ss. levii were evenly distributed between conjunctiva and teat tip samples. A distinct seasonal pattern of the isolation rates of summer mastitis pathogens was recorded, which corresponded closely to the seasonal activity of symbovine insects, in particular the headfly Hydrotaea irritans (Fallén). However, the high proportion of clinically healthy bacterial carriers as compared with the incidence of clinical disease strongly suggests that as yet unknown contributing or triggering factors, apart from the mere presence of the relevant bacterial species, are required for the establishment and development of clinical summer mastitis.     

Milk flow and udder health in cows after treatment of covered teat injuries via theloresectoscopy: 52 cases (2000-2002)

OBJECTIVE: To determine milk flow, somatic cell counts (SCCs), and the incidence of clinical mastitis in cows that had undergone theloresectoscopy for treatment of teat stenosis caused by mucosal detachment in the region of the streak canal or Fürstenberg's rosette. DESIGN: Retrospective study. ANIMALS: 52 cows with teat stenosis that were treated via theloresectoscopy. PROCEDURE: Medical records of eligible cows were reviewed. Additional data regarding milking ease, SCC, development of clinical mastitis of the affected gland, and whether the cow remained in the herd were collected via owner-completed questionnaires. RESULTS: 49 of 52 questionnaires were completed. At referral, teat sinusitis was diagnosed in 29 of 52 cows. Milk flow was normal in 38 of 41 treated teats at discharge and in 24 of 40 during the next lactation. Thirteen of 49 cows were culled during the next lactation because of abnormal udder health. High SCC and teat sinusitis at referral and development of clinical mastitis during the 10-day period after surgery resulted in high SCCs in the remainder of the current lactation. The incidence of clinical mastitis during the remainder of the current and during the next lactation was higher in cows that had teat sinusitis at the time of surgery, compared with those that did not. CONCLUSIONS AND CLINICAL RELEVANCE: Teat stenoses resulting from mucosal lesions in the region of the streak canal or Fürstenberg's rosette may be successfully treated via theloresectoscopy. Inflammation of the teat sinus and gland at the time of surgery may adversely affect udder health.     

Relationship between antimicrobial susceptibility of clinical mastitis pathogens and treatment outcome in cows

OBJECTIVE: To determine whether there was any association between results of in vitro antimicrobial susceptibility testing of pathogens isolated from cows with mild or moderate clinical mastitis and outcome of treatment. DESIGN: Observational study. ANIMALS: 133 cows with mild or moderate mastitis in a single quarter. PROCEDURE: Cows were treated by means of intramammary infusion of pirlimycin (50 mg) in the affected quarter once daily for 2 days; additional intramammary treatments with the same product were administered if the milk continued to appear abnormal. Duration of treatment and days until clinical cure were recorded. Bacterial isolates were tested for antimicrobial susceptibility by means of a broth micro-dilution technique. RESULTS: Environmental streptococci, coliforms, and coagulase-negative Staphylococcus spp were the most commonly isolated pathogens. Duration of treatment and days until clinical cure were not significantly different for cows from which pathogens that were susceptible or resistant to pirlimycin were isolated. Bacteriologic cure rates 14 and 21 days after treatment were not significantly different for cows with mastitis caused by susceptible or resistant bacteria. Similar results were found when data only from cows with mastitis caused by gram-positive isolates were analyzed. CONCLUSIONS AND CLINICAL RELEVANCE: In the present study, differences in clinical outcome for cows with mild or moderate mastitis that could be attributed to differences in results of in vitro susceptibility testing were not identified. The use of in vitro susceptibility testing to guide intramammary mastitis treatment cannot be recommended on the basis of results of this study.     

Growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution of the bovine mammary gland: relation to secretion composition

Mammary secretions from 12 Holstein dairy cows were collected to evaluate growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution and during physiologic transitions of the mammary gland. Mammary secretions obtained during late lactation poorly inhibited growth of E coli and K pneumoniae. However, as involution progressed, mammary secretions increasingly inhibited growth of both coliform mastitis pathogens. Greatest inhibition of E coli and K pneumoniae growth was observed when mammary glands were fully involuted. Growth inhibition remained high until 7 days before parturition, and then it decreased significantly (P less than 0.05) to that observed during late lactation. Inhibition of coliform mastitis pathogen growth was associated with high concentrations of lactoferrin and immunoglobulin G, decreased citrate concentration, and a low citrate to lactoferrin molar ratio. These data suggested that differences in susceptibility or resistance to new intramammary infection with coliform mastitis pathogens during the nonlactating period may be attributable, in part, to marked changes in mammary secretion composition that develop during physiologic transitions of the mammary gland. Resistance of the fully involuted mammary gland to coliform infection may be associated with high concentrations of natural protective factors.     

A review of prevention and control of heifer mastitis via non-antibiotic strategies

Clinical and subclinical mastitis is a significant problem in primiparous dairy cattle (heifers) with a higher prevalence and incidence in heifers than cows, especially early in lactation. Differences in management (e.g. nutrition, pasturing, no use of dry cow therapy) as well as differences in physiological status (e.g. continuing growth in heifers) are likely contribute to the observed differences between heifers and cows. These differences may result in the requirement for different approaches for mastitis management in heifers than for cows. Mastitis is a multifactorial disease, hence control requires an understanding of the risk factors before effective interventions can be defined. Control strategies are aimed at reducing the incidence of new intramammary infections and eliminating existing infections. Potential strategies can include improved environmental and animal hygiene, application of internal and external teat sealants, prepartum application of teat antiseptics, prepartum milking and control of horn fly in environments where it acts as vector. Other less well-proven strategies to control heifer mastitis include management of heifers as a physically separate group from older cows and not feeding mastitic milk to calves. It is concluded that several well-proven strategies are available to manage heifer mastitis. However, further research is likely to improve understanding of heifer mastitis and lead to novel managerial approaches to mastitis control in this age group.     

Immune mechanisms of the bovine udder: an overview

Understanding basic defenses of the udder is instrumental in developing measures to prevent mastitis. The teat canal is the first defense against pathogens, providing a physical barrier and antimicrobial substances. When bacteria breach the teat canal, milk leukocytes provide a second defense by ingesting pathogens. Intramammary devices have been used experimentally to increase leukocyte numbers and to enhance destruction of bacteria. Milk antibodies opsonize and lyse bacteria, neutralize toxins, and prevent adhesion to tissue. Vaccinating cows against mastitis generally has been unsuccessful; however, immunization is useful in controlling specific bacterial strains. Antibody-producing plasma cells preferentially accumulate in internal teat end tissues. Because bacteria contact these tissues to reach milk-producing areas of the udder, an immunostimulant to enhance locally the protective nature of plasma cells may decrease the occurrence of infection.     

Efficacy of blanket treatment of cows and heifers with an internal teat sealant in reducing the risk of mastitis in dairy cattle calving on pasture

AIM: To evaluate the benefit of blanket treatment of all milking cows at drying-off and all replacement heifers one month prior to the planned start of mating with an internal teat sealant on the incidence of mastitis, and somatic cell counts (SCC), postpartum in a 650-cow herd with a mastitis incidence in early lactation of 15% in cows and 26% in heifers. METHODS: Prior to drying-off, lactating cattle were divided on the basis of SCC and mastitis history. Cattle with records of individual SCC >150,000 cells/ml or mastitis in the previous lactation were treated with a cloxacillin-based dry-cow therapy (DCT), while the remaining cattle received no antibiotics. Cattle in each of the two groups were then randomly assigned to receive either an internal teat sealant or no further treatment. Additionally, one month prior to the planned start of calving the rising 2-year-old heifers were also randomly assigned to receive either an internal teat sealant or no treatment. All clinical mastitis cases were recorded for the first 10 weeks after the start of calving, while SCC were measured on three occasions during the subsequent lactation. RESULTS: Data were available from 466 cows and 206 heifers; treatment with the internal teat sealant significantly reduced the incidence of mastitis in both groups. For cows, the reduced risk of mastitis due to treatment with the teat sealant was not significantly different between cows, which, based on their mastitis history, required dry-cow antibiotics and those which did not. There was no effect of teat sealant on the mean SCC of any group at any time-point. CONCLUSIONS: On this farm, treatment of all cows and heifers prior to parturition with an internal teat sealant significantly reduced the risk of clinical mastitis. The benefits of an internal teat sealant were the same when used in combination with antibiotics in cows with a history of mastitis as when used alone in cows with no such history. CLINICAL RELEVANCE: These data support the proposition that blanket treatment with an internal teat sealant of all cows due to enter the milking herd can be a useful method of mastitis control. They also suggest that combined therapy with dry-cow antibiotics and internal teat sealants can be beneficial under New Zealand conditions. More detailed research on more farms in more areas of New Zealand is required to confirm these suggestions.     

Factors for the genesis of bovine subclinical mastitis

The infection risk of the bovine mammary gland is determined by susceptibility of the cow and contamination and invasion by mastitis pathogens. Genetic (e.g. yield) and lactational physiologic (e.g. stage of lactation) factors support the genesis of subclinical mastitis. This paper considers new data on cow individual and milking related influences on defence mechanisms in the teat end tissue acting in prevention of penetration through the teat canal by mastitis pathogens.     

Prevention of intramammary infections in dairy cows by the use of a premilking teat dip method with a foaming iodophor dip agent

In this study we investigated the efficacy of premilking teat dipping with a foaming iodophor teat dip in a negative controlled field study. Incidence of new intramammary infections (IMI), incidence of clinical mastitis, influence on somatic cell count (SCC) and the characteristics of udder tissue and teats were used as parameters to evaluate clinical efficacy. Predipping was compared with a negative control using a split-udder experimental design. Right teats were predipped with a foaming disinfectant containing 0.27% iodine while left teats served as controls. The latter were conventionally cleaned with damp cloth towels and dried manually with disposable paper towels ("best cleaning practice"). All teats were dipped after milking with the same dip. There were no differences between treated and control quarters with respect to incidence of new IMI during the study period (treated quarters: 6.6% vs. untreated: 6.95%), incidence of clinical mastitis (30 cases in the treatment group vs. 39 cases in the control group) and geometric mean of SCC of quarter milk samples. Spectrum of detected pathogens was also comparable. Condition of udder tissue and teat ducts did not differ between treated and control quarters.     

The association between teat end hyperkeratosis and teat canal microbial load in lactating dairy cattle

Most pathogens that cause bovine mastitis invade the udder lumen through the teat canal. Amino acids and intercellular lipids may support microbial colonisation of the teat canal epithelium by pathogenic microorganisms. The aim of this study was to investigate the association between teat end hyperkeratosis, which is induced by machine milking, and teat canal microbial load. Contralateral teats, which differed in teat end hyperkeratosis scores, were identified in a split-udder experiment. The teat canal's microbial load was evaluated using the wet and dry swab technique. Staphylococcus (S.) aureus, Streptococcus (Sc.) uberis, Escherichia (E.) coli and other coliforms were detected by agar plate cultures. The positive detection of E. coli and the log(10)-transformed E. coli load of a teat canal were significantly associated with the teat end hyperkeratosis score (P<0.05). There were significant differences with respect to positive findings for E. coli, as well as the microbial load of E. coli and Sc. uberis, between the less-calloused and the more-calloused teat of a pair. For S. aureus, no significant associations between hyperkeratosis score and teat canal microbial load were detected. In general, a teat with a highly calloused teat end had an increased teat canal microbial load compared with the contralateral teat, characterised by a lower callosity. The results of the present study indicate that the environmental pathogen load is associated with teat end hyperkeratosis. Further research is needed to identify factors that may affect teat canal microbial load in lactating dairy cattle.     

Microbiological colonisation of bovine teat canal--significance and influencing factors

The teat canal of lactating dairy cattle is of particular importance for the defence of facultative pathogenic and pathogenic microorganisms (e.g., staphylococci) invading the bovine udder. Furthermore the canal is usually colonized with microorganisms, too. In addition to microorganisms inhibiting mastitis pathogens the teat canal is colonized by staphylococci. The microbial colonization can be influenced by the environment of the animals, the care and disinfection of the teat skin and indirectly by the effects of forces being associated with machine milking. Because of vacuum fluctuations occurring under the teat tip microorganisms, which colonize the teat canal, can invade the bovine mammary gland and cause infections there. This paper gives a review of the microbial colonization of the bovine teat duct and of influencing factors on the microbial populations as well as of the significance of the teat canal colonization for the development of mastitis.