Sarne De Vliegher

Prevalence and distribution of mastitis pathogens in subclinically infected dairy cows in Flanders, Belgium

The main objective was to determine the prevalence of intramammary infections (IMI) in dairy cows in Flanders, Belgium. Data were obtained from quarter milk samples of dairy herds subjected to a mandatory yearly screening of all lactating cows. A total of 178,668 quarter milk samples were collected at 1087 cross-sectional dairy herd screenings performed in three consecutive years. Of the dairy cows, 40% had at least one culture-positive quarter. More than 50% of all IMI were caused by non-aureus staphylococci. Streptococcus agalactiae is almost eradicated in Flanders, whereas Staphylococcus aureus was isolated from 18% of the culture-positive quarters. In addition, the distribution of mastitis pathogens in quarter milk samples from selected dairy cows with an elevated somatic cell count (SCC) is described. From 6390 cows with a geometric mean composite SCC 250,000 cells/ml, nearly 65% had at least one culture-positive quarter. The majority of the IMI were caused by non-aureus staphylococci (41.1%), whereas Staph. aureus and aesculin-positive cocci were found in respectively 25% and 18% of the culture-positive milk samples. We conclude that more efforts are needed in the prevention and control of subclinical mastitis in Flanders. Non-aureus staphylococci are the predominant cause of IMI, warranting more research regarding the epidemiology and pathogenicity of those species.

Staphylococcus agnetis sp nov., a coagulase-variable species from bovine subclinical and mild clinical mastitis

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n=12) and a teat apex (n=1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA-DNA hybridization with S. hyicus DSM 20459(T) and S. chromogenes DSM 20674(T) confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C(15:0), ai-C(15:0), i-C(17:0) and C(20:0) and the peptidoglycan type was A3α L-Lys-Gly(5). Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4(T) (=DSM 23656(T)=CCUG 59809(T)) is the type strain.

A Staphylococcus xylosus Isolate with a new mecC allotype

ABSTRACT Recently, a novel variant of mecA known as mecC (mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.

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.

Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants.

Since phenotypic methods to identify coagulase negative staphylococci (CNS) from the milk of ruminants often yield unreliable results, methods for molecular identification based on gene sequencing or fingerprinting techniques have been developed. In addition to culture-based detection of isolates, culture-independent methods may be of interest. On the basis of molecular studies, the five CNS species commonly causing intramammary infections (IMI) are Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus simulans and Staphylococcus xylosus. Current knowledge suggests that S. chromogenes is a bovine-adapted species, with most cases of IMI due to this bacterium being opportunistic. S. haemolyticus also appears to be an opportunistic pathogen, but this bacterium occupies a variety of habitats, the importance of which as a source of IMI remains to be elucidated. S. xylosus appears to be a versatile species, but little is known of its epidemiology. S. epidermidis is considered to be a human-adapted species and most cases of IMI appear to arise from human sources, but the organism is capable of residing in other habitats. S. simulans typically causes contagious IMI, but opportunistic cases also occur and the ecology of this bacterium requires further study. Further studies of the ecology and epidemiology of CNS as a cause of IMI in cattle are required, along with careful attention to classification of these bacteria and the diseases they cause.

Influence of different centrifugation protocols on equine semen preservation.

Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 x g and 2400 x g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P<0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P<0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P<0.005), whereas CP3 and CP5 yielded a lower BCF (P<0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800 x g or 2400 x g for a shorter period of time (5 min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate.

The association between indicators of inflammation and liver variables during the transition period in high-yielding dairy cows : an observational study

During the transition period, cows are confronted with infectious and inflammatory challenges leading to an acute phase response (APR) marked by increased hepatic synthesis of positive acute phase reactants (+AP) and a decrease in negative acute phase reactants (-AP). The aim of this study was to quantify the APR in 21 high-yielding dairy cows studied from 9 days before until 42 days after calving, and to assess the association between the APR, disease incidence and indicators of liver function. Repeated blood samples were analyzed for -AP (retinol, albumin, cholesterol), +AP (haptoglobin, caeruloplasmin), paraoxonase, and liver-associated variables (aspartate aminotransferase, γ-glutamyl transferase, bilirubin). All cows displayed postpartum decreases in -AP and paraoxonase, and increases in +AP and liver variables. When retrospectively categorized, cows presenting a stronger -AP decline displayed higher +AP and liver variables, and a higher disease incidence compared to cows with a milder decline. Altogether, typical changes in -AP and +AP identify the transition period as a time of increased inflammatory load. Group differences in liver variables suggest that a more severe APR may be associated with altered liver function. However, no causal relationship can be proven based on this observational dataset, and results should be interpreted cautiously.

Optimization of the Isolation, Culture, and Characterization of Equine Umbilical Cord Blood Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation methods. The mononuclear cell recovery after Percoll separation, however, resulted in significantly more putative MSC colonies; and, therefore, this isolation method was used for all further experiments. Culture conditions such as cell density and medium or serum coating of the wells did not significantly affect putative MSC recovery. Isolated MSC using Percoll were subsequently differentiated toward the osteogenic, chondrogenic, and adipogenic lineage. In addition, MSC were phenotyped by multicolor flow cytometry based on their expression of different cell protein markers. Cultured MSC were CD29, CD44, and CD90-positive and CD79α, Macrophage/Monocyte and MHC II-negative. In conclusion, this study reports optimized protocols to isolate, culture, and characterize solid equine MSC from UCB.

Cost estimation of heifer mastitis in early lactation by stochastic modelling

Heifer mastitis, reflected by an elevated somatic cell count (SCC) in early lactation (SCCel), results in a decreased milk production, a higher risk for subclinical and clinical mastitis during lactation, and an elevated culling hazard. The aims of this study were to calculate the costs of heifer mastitis defined as an elevated SCC in early lactation, and to show the variation of these costs in the Dutch/Belgian dairy sector. A stochastic model, in which the variation and uncertainty of heifer mastitis are taken into the account, was developed with input data from literature and expertise. Costs were estimated, using default values. The mean costs for an elevated SCCel that cured were on average euro 13/heifer present on a farm (range: euro 0-118), for an elevated SCC at calving proceeding in subclinical mastitis on average euro 5/heifer present on a farm (range: euro 0-euro 82), and for a clinical heifer mastitis case associated with an elevated SCC after calving on average euro 270. On average this results in euro 13/heifer present on a farm (range: euro 0-137). Combined, these three cost aspects result in a total cost of on average euro 31/heifer present on a farm (range: euro 0-220). The large variation in the costs is very important regarding farm management and farm support. The difference in costs reflects also the difference in room for investment. When the costs that can be prevented are estimated at farm level, these data can be of help in setting goals in herd health advice and farm management.

Pathogen-specific incidence rate of clinical mastitis in Flemish dairy herds, severity, and association with herd hygiene

A one-year survey on clinical mastitis was conducted on 50 randomly selected commercial Flemish dairy herds to estimate the pathogen-specific incidence rate of clinical mastitis (IRCM). The severity of the cases and the potential associations with herd hygiene were studied. Participating producers sampled 845 cases and 692 dairy cows. The mean and median IRCM was estimated at 7.4 and 5.3 quarter cases per 10,000 cow-days at risk, respectively. A large between-herd variation was observed (range of 0-21.3). In general, the IRCM was lower in heifers compared with multiparous cows (2.9 vs. 11.0 quarter cases per 10,000 cow-days at risk). However, the overall IRCM in the first week after calving was higher in heifers compared with cows (43.4 vs. 31.6 quarter cases per 10,000 cow-days at risk). Streptococcus uberis (18.2% of the cases) and Escherichia coli (15.5%) were the most frequently isolated pathogens and no growth was observed in 19.9% of the cases. The majority of the cases (63.1%) were mild (only clots in milk). Moderate (hard quarter without general signs) and severe symptoms (systemic illness) were observed in 29.9 and 7.0% of the cases, respectively. Isolation of E. coli (vs. any other culture result) was more likely in moderate and severe cases compared with mild cases. Overall IRCM and E. coli IRCM were higher in dirty compared with clean herds based on udder hygiene scores (9.0 and 1.7 vs. 6.0 and 0.6 quarter cases per 10,000 cow-days at risk, respectively). This study broadens the knowledge on clinical mastitis in Flemish dairy herds and underlines the high risk of CM in early-lactation heifers, the role of the so-called environmental pathogens, and herd hygiene.