Bert Verbist

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.

Intra-species diversity and epidemiology varies among coagulase-negative Staphylococcus species causing bovine intramammary infections

Although many studies report coagulase-negative staphylococci (CNS) as the predominant cause of subclinical bovine mastitis, their epidemiology is poorly understood. In the current study, the genetic diversity within four CNS species frequently associated with bovine intramammary infections, Staphylococcus haemolyticus, S. simulans, S. chromogenes, and S. epidermidis, was determined. For epidemiological purposes, CNS genotypes recovered from bovine milk collected on six Flemish dairy farms were compared with those from the farm environment, and their distribution within the farms was investigated. Genetic diversity was assessed by two molecular typing techniques, amplification fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD) analysis. Subtyping revealed the highest genetic heterogeneity among S. haemolyticus isolates. A large variety of genotypes was found among environmental isolates, of which several could be linked with intramammary infection, indicating that the environment could act as a potential source for infection. For S. simulans, various genotypes were found in the environment, but a link with IMI was less obvious. For S. epidermidis and S. chromogenes, genetic heterogeneity was limited and the sporadic isolates from environment displayed largely the same genotypes as those from milk. The higher clonality of the S. epidermidis and S. chromogenes isolates from milk suggests that specific genotypes probably disseminate within herds and are more udder-adapted. Environmental sources and cow-to-cow transmission both seem to be involved in the epidemiology of CNS, although their relative importance might substantially vary between species.

Sources other than unused sawdust can introduce Klebsiella pneumoniae into dairy herds.

A longitudinal study was carried out to detect intramammary infections caused by Klebsiella pneumoniae and to identify potential sources of this bacterial species in the environment of the cows. The study was performed in 6 well-managed Belgian dairy herds from May 2008 to May 2009. Monthly (n=13), unused and used sawdust bedding samples as well as individual quarter milk and feces samples were collected from 10 randomly selected cohort cows in each herd. Cases of clinical mastitis of all lactating cows in the 6 herds were also sampled (n=64). From the 3,518 collected samples, 153 K. pneumoniae isolates were obtained, of which 2 originated from milk (clinical mastitis cases). In feces (n=728), used bedding (n=73), and unused bedding (n=73), respectively, 125 (17.2%), 20 (27.4%), and 6 (8.2%) isolates were found. The isolates were fingerprinted by means of pulsed field gel electrophoresis. In total, 109 different pulsotypes were differentiated, indicating a high degree of genetic diversity within the isolates. All isolates from unused bedding belonged to pulsotypes other than those from the other sources, suggesting that sources other than unused sawdust may introduce K. pneumoniae into the herd. Only 2 pulsotypes contained isolates originating from different sources. Pulsotype 10 was found in milk and used bedding and pulsotype 21 was found in feces and used bedding. The 2 milk isolates originated from 2 cows in the same herd but they belonged to a different pulsotype. The results indicate that K. pneumoniae can be prevalent in the environment without causing significant mastitis problems. Most cows were shedding K. pneumoniae in feces, substantiating findings under very different conditions (i.e., American dairy herds). Contamination of used bedding in the cubicles with K. pneumoniae from feces was confirmed, whereas unused bedding was not an important source of K. pneumoniae for the environment of the cows.

Short communication: Associations between teat dimensions and milking-induced changes in teat dimensions and quarter milk somatic cell counts in dairy cows

Although many studies have examined the relation between a wide range of factors and quarter milk somatic cell count (qSCC), including physical characteristics of the teat and changes in teat tissue due to milking, the effect of short-term, milking-induced changes in teat dimensions on somatic cell count has not yet been investigated. To identify teat dimensions and milking-induced changes in teat dimensions associated with qSCC, we conducted a longitudinal study (n(herds)=6, n(cows)=72, n(measurements)=12). Parity, stage of lactation, teat barrel diameter, and changes in teat barrel diameter during milking were identified as factors associated with qSCC. Teats with wider barrels had higher qSCC. Negative changes in the diameter of the teat barrel during milking (i.e., thinner teats postmilking compared with premilking) were associated with lower qSCC, whereas positive changes (i.e., thicker teats postmilking compared with premilking) were associated with higher qSCC. Selection toward more optimal teat characteristics may therefore result in improved milk quality and udder health. However, a threshold might exist for the maximum reduction in teat barrel diameter below which udder health is negatively influenced. If so, changes in teat barrel diameter might serve as an indicator for suboptimal milking and incorrect choice of teatcup liner or milking machine settings and thus help improve management of the herd.

Short communication: Subtyping of Staphylococcus haemolyticus isolates from milk and corresponding teat apices to verify the potential teat-skin origin of intramammary infections in dairy cows

Coagulase-negative staphylococci (CNS) are a major cause of intramammary infections (IMI) in dairy cows and they colonize the teat skin. Staphylococcus haemolyticus, one of the more common CNS, has been identified as a highly versatile opportunistic species. The aim of the present study was to gain better insight into the adaptation of S. haemolyticus subtypes to the udder ecosystem with respect to IMI development. During a longitudinal observational study conducted over 13 mo on 6 Flemish dairy herds, S. haemolyticus isolates were recovered from milk and teat apices. A total of 44 S. haemolyticus isolates originating from milk (24 isolates) and teat apices (20 isolates) of 6 selected udder quarters were singled out and analyzed using a combined methodology of (GTG)5-PCR and amplified fragment length polymorphism (AFLP) fingerprinting to determine intraspecies differences. Combining both fingerprinting methods, 4 S. haemolyticus subtypes were obtained (I to IV). Subtypes I, II, and IV were recovered from both milk and teat apex samples and were found to be associated with persisting IMI. Subtype III, not apparently related to IMI, was isolated solely from teat apices and not from milk. In general, S. haemolyticus subtypes found in milk from infected quarters could be recovered from the corresponding teat apices, although the latter could be colonized with up to 3 different subtypes. Comparing subtypes from milk and teat apices indicates that the IMI-causing agent likely originates from the teat skin.