Pathogen group-specific risk factors for intramammary infection in water buffalo.

A cross-sectional study was conducted to estimate the prevalence of intramammary infection (IMI) associated bacteria and to identify risk factors for pathogen group-specific IMI in water buffalo in Bangladesh. A California Mastitis Test (CMT) and bacteriological cultures were performed on 1,374 quarter milk samples collected from 763 water buffalo from 244 buffalo farms in nine districts in Bangladesh. Quarter, buffalo, and farm-related data were obtained through questionnaires and visual observations. A total of 618 quarter samples were found to be culture positive. Non-aureus staphylococci were the predominant IMI-associated bacterial species, and Staphylococcus (S.) chromogenes, S. hyicus, and S. epidermidis were the most common bacteria found. The proportion of non-aureus staphylococci or Mammaliicoccus sciuri (NASM), S. aureus, and other bacterial species identified in the buffalo quarter samples varied between buffalo farms. Therefore, different management practices, buffalo breeding factors, and nutrition were considered and further analyzed when estimating the IMI odds ratio (OR). The odds of IMI by any pathogen (OR: 1.8) or by NASM (OR: 2.2) was high in buffalo herds with poor milking hygiene. Poor cleanliness of the hind quarters had a high odds of IMI caused by any pathogen (OR: 2.0) or NASM (OR: 1.9). Twice daily milking (OR: 3.1) and farms with buffalo purchased from another herd (OR: 2.0) were associated with IMI by any pathogen. Asymmetrical udders were associated with IMI-caused by any bacteria (OR: 1.7). A poor body condition score showed higher odds of IMI by any pathogen (OR: 1.4) or by NASM (OR: 1.7). This study shows that the prevalence of IMI in water buffalo was high and varied between farms. In accordance with the literature, our data highlight that IMI can be partly controlled through better farm management, primarily by improving hygiene, milking management, breeding, and nutrition.

Evaluation of Bayesian Hui-Walter and logistic regression latent class models to estimate diagnostic test characteristics with simulated data.

Estimation of the accuracy of diagnostic tests in the absence of a gold standard is an important research subject in epidemiology (Dohoo et al., 2009). One of the most used methods the last few decades is the Bayesian Hui-Walter (HW) latent class model (Hui and Walter, 1980). However, the classic HW models aggregate the observed individual test results to the population level, and as a result, potentially valuable information from the lower level(s) is not fully incorporated. An alternative approach is the Bayesian logistic regression (LR) latent class model that allows inclusion of individual level covariates (McInturff et al., 2004). In this study, we explored both classic HW and individual level LR latent class models using Bayesian methodology within a simulation context where true disease status and true test properties were predefined. Population prevalences and test characteristics that were realistic for paratuberculosis in cattle (Toft et al., 2005) were used for the simulation. Individual animals were generated to be clustered within herds in two regions. Two tests with binary outcomes were simulated with constant test characteristics across the two regions. On top of the prevalence properties and test characteristics, one animal level binary risk factor was added to the data. The main objective was to compare the performance of Bayesian HW and LR approaches in estimating test sensitivity and specificity in simulated datasets with different population characteristics. Results from various settings showed that LR models provided posterior estimates that were closer to the true values. The LR models that incorporated herd level clustering effects provided the most accurate estimates, in terms of being closest to the true values and having smaller estimation intervals. This work illustrates that individual level LR models are in many situations preferable over classic HW models for estimation of test characteristics in the absence of a gold standard.

Colonization of Extramammary Sites with Mastitis-Associated S. aureus Strains in Dairy Goats.

Staphylococcus aureus (S. aureus), a major mastitis pathogen in dairy goats, is classified as a contagious pathogen. Although previous research has shown that extramammary body sites can be colonized with S. aureus, it is unknown whether these sites are reservoirs for intramammary infections. The aim of this research was to determine whether extramammary sites can be colonized with mastitis-associated S. aureus strains in dairy goats. Milk samples were collected from 207 primiparous goats and from 120 of these goats, extramammary site samples (hock, groin, nares, vulva and udder) were collected from a large commercial dairy goat herd in the Netherlands during four sampling visits. Extramammary site swabs and milk samples were (selectively) cultured and S. aureus isolates were spa genotyped. The prevalence of colonization of the extramammary sites at goat level was 51.7% and the prevalence of S. aureus intramammary infections was 7.2%. The nares were colonized most frequently (45%), while the groin area was colonized the least (2.5%). Six spa genotypes were identified in this herd and there was no significant difference in the distribution of spa genotypes between the milk or the extramammary sites (p = 0.141). Both in the extramammary sites and in the milk, spa genotypes t544 (82.3% and 53.3%) and t1236 (22.6% and 33.3%) were the dominant genotypes. These results show that in goats, extramammary sites, particularly the nares, are frequently colonized with mastitis-associated S. aureus strains. Extramammary sites may, thus, be a source of S. aureus intramammary infections that are not targeted by the intervention measures aimed at preventing transmission from infected udder glands.

The prevalence and risk factors of subclinical mastitis in water buffalo (Bubalis bubalis) in Bangladesh.

Subclinical mastitis (SCM) in water buffalo is responsible for reduced milk yield and quality. This cross-sectional study was carried out to a) estimate the prevalence of SCM, b) identify risk factors associated with SCM, and c) identify farm-level risk factors associated with bulk milk somatic cell count (BMSCC). The buffalo farms included in this study represented five rearing systems: free-range, semi-free-range, household, semi-intensive, and intensive, providing a total of 3491 functional quarters of 880 lactating buffalo on 248 farms. The California mastitis test score was used to identify SCM. Bulk milk samples (n = 242) were used for farm-level BMSCC. Quarter and buffalo-level risk factors for SCM were measured using questionnaires and observations. The overall SCM prevalence was high at 27.9% at the quarter-level (25th and 75th percentiles: 8.3% and 41.7%) and 51.5% at buffalo-level (25th and 75th percentiles: 33.3% and 66.7%). The geometric mean BMSCC was 217,000 cells/mL of milk (ranging from 36,000-1,213,000 cells/mL), which is low on average, but some farms could improve substantially. The buffalo rearing system, udder location (left versus right), teat shape, udder asymmetry, number of milkers, and having a quarantine facility were associated with buffalo udder health. Our findings suggest that mainly using free-range rearing systems may help decrease the prevalence of SCM primarily by employing buffalo breeding and better farm biosecurity, and udder health control strategies can be designed based on our findings.

Association of Staphylococcus aureus genotypes with milk or colonization of extramammary sites in Dutch dairy cattle indicates strain variation in reservoirs for intramammary infections.

Staphylococcus aureus (S. aureus) is a major mastitis pathogen with a detrimental impact on udder health in dairy cattle. Although S. aureus is generally classified as a contagious mastitis pathogen, control measures aimed at preventing contagious transmission are not always effective. Previous studies showed that various extramammary sites can be colonized with S. aureus and could be a reservoir for S. aureus intramammary infections (IMI). The aim of this study was to determine the prevalence of S. aureus extramammary site colonization in Dutch dairy cattle and to compare the spa genotypes of S. aureus isolates from IMI to spa genotypes of isolates from extramammary sites. Six herds were visited and from cows with a composite milk somatic cell count ≥200,000 cells/mL quarter milk samples and swabs from various extramammary sites (hock, groin, udder cleft, nares, and feces) were taken. Extramammary site samples were processed by a two-step high salt selective culture and presence of S. aureus was confirmed by femA PCR. S. aureus isolates from milk and extramammary sites were compared by spa typing. The cow level colonization varied from 0% to 73%, and the prevalence of IMI in the sampled cows varied from 0% to 61% between herds. The extramammary site with the highest prevalence of colonization was the hock (23%) and the lowest prevalence of colonization was found for the nares (5%) and feces (5%). Spa typing of S. aureus isolates from either extramammary sites or milk showed that in most herds there were one or two predominant S. aureus spa genotypes present. Different S. aureus spa genotypes could be categorized into three groups based on the distribution between milk or extramammary sites: i) predominantly milk associated, ii) associated with both milk and extramammary sites, and iii) associated with extramammary sites. In conclusion, we showed that the prevalence of extramammary site colonization differed significantly between herds and extramammary sites and that specific S. aureus spa genotypes were associated with milk (IMI) or extramammary site colonization. Extramammary S. aureus reservoirs could be a source for IMI that cannot be eradicated by intervention measures aimed at contagious mastitis pathogens.

Subclinical mastitis in dairy cows in south-Asian countries: a review of risk factors and etiology to prioritize control measures.

Mastitis is a major production disease, causing significant economic losses for dairy farmers in South-Asian countries, as well as other parts of the world. Udder health control programs (UHCP) have been established in developed countries as an effective strategy for mastitis control but have not yet been introduced in South-Asian low-income countries like Bangladesh, India, Pakistan, and Sri Lanka. To launch UHCP successfully in dairy herds in South-Asia, it is important to know the current prevalence and risk factors for subclinical mastitis (SCM). Therefore, a narrative literature review was conducted with the aim to describe the dairy sector, the prevalence of SCM and its causal agents, risk factors for mastitis occurrence and the control measures suggested by different studies conducted in the selected countries. The literature revealed that India had the highest cattle population. Milking was mainly done by hand in all of the studied countries. Stall feeding was done in Bangladesh and Sri Lanka and limited access to grazing was also reported in some farms in India and Pakistan. There was substantial variation in the prevalence of SCM between studies in all 4 countries, ranging from about 20% to about 80%, but the average prevalence across all studies was high (50%). The most common causal agents for SCM were non-aureus staphylococci (NAS), Staphylococcus (S.) aureus, Streptococcus spp. and Escherichia (E.) coli. The management related risk factors reported for SCM were stall feeding of cows, a higher stock density, cracked floors, open drains, the presence of flies, poor drainage, peri-parturient diseases, infrequent dung removal and earth floors. The control measures suggested in these studies were to improve the hygiene and sanitation of cows, to improve the cleanliness of farms and milker's hands, to apply dry cow therapy, supplementing micronutrients and routine screening for SCM combined with taking intervention measures like isolation of cows or milking infected cows last, and proper treatment. Also, full hand milking, complete milking, machine milking, and providing feed and water immediately after milking have been recommended. Finally, we show that current literature often studies the same set of (non-manageable) risk factors, so more research is needed to obtain a comprehensive picture of the determinants of SCM. Randomized controlled trials are needed to truly quantify the effect of intervention under field conditions. Altogether, our work gives an overview of the udder health situation in South-Asia and provides the basis for the design of UHCP in this region.

A stochastic modelling approach to determine the effect of diverse Staphylococcus aureus strains on the economic and epidemiological outcomes of mastitis intervention strategies in dairy cattle.

Staphylococcus aureus (S. aureus) strains with considerable genetic and phenotypic differences have previously been identified. The economic and epidemiologic impact of S. aureus mastitis has been investigated, but none of these studies took differences between strains into account. Here we aimed to investigate how differences between S. aureus strains affect the economic and epidemiologic outcome of various intervention strategies against clinical and subclinical intramammary infections. Five S. aureus strains were modelled using a stochastic bio-economic model simulating a dairy herd of 200 cows using single-day time steps. The strain characteristics of the five simulated S. aureus strains (general, contagious, spill-over, clinical and persistent) were based on divergent phenotypes as described in literature. Outcomes of the model included incidence (both clinical and subclinical), number of antibiotic treatment days, number of culled cows, and net income. Intervention strategies against clinical and subclinical intramammary infections were based on (variations of) intramammary antibiotic treatment, testing, and culling. Both single and multiple pathogen (intramammary infection caused by S. aureus, Escherichia coli, and non-aureus staphylococci) scenarios were simulated to determine the effect of the five S. aureus strains on the impact of 19 different intervention strategies. The results showed that the incidence (both clinical and subclinical), number of treatment days, number of culled cows, and net income varied considerably for the different S. aureus strains. Comparison of the model outcomes within and between strains showed that for most intervention strategies the relative impact differed per strain. However, the intervention strategy with the best outcome for most variables and strains was the culling of cows with a recovery probability lower than 50%. This shows that the relative economic and epidemiologic impact of most of the modelled intervention strategies were strain-dependent, while some intervention strategies were not strain-dependent. From this, we conclude that, depending on the intervention strategy applied on a farm, it could be advantageous to type S. aureus to determine whether it would be economically and epidemiologically beneficial for the existing intervention strategy to be changed.

Incidence, Etiology, and Risk Factors of Clinical Mastitis in Dairy Cows under Semi-Tropical Circumstances in Chattogram, Bangladesh.

Clinical mastitis (CM) is an important production disease in dairy cows, but much of the knowledge required to effectively control CM is lacking, specifically in low-income countries where most farms are small and have specific dairy management, such as regular udder cleaning and practicing hand milking. Therefore, we conducted a 6-month-long cohort study to (a) estimate the incidence rate of clinical mastitis (IRCM) at the cow and quarter level, (b) identify risk factors for the occurrence of CM, (c) describe the etiology of CM, and (d) quantify antimicrobial susceptibility (AMS) against commonly used antimicrobial agents in S. aureus and non-aureus Staphylococcus spp. (NAS) in dairy farms in the Chattogram region of Bangladesh. On 24 farms, all cows were monitored for CM during a 6-month period. Cases of CM were identified by trained farmers and milk samples were collected aseptically before administering any antimicrobial therapy. In total, 1383 lactating cows were enrolled, which totaled 446 cow-years at risk. During the study period, 196 new cases of CM occurred, resulting in an estimated crude IRCM of 43.9 cases per 100 cow-years, though this varied substantially between farms. Among the tested CM quarter samples, Streptococci (22.9%) followed by non-aureus staphylococci (20.3%) were the most frequently isolated pathogens and resistance of S. aureus and NAS against penicillin (2 out of 3 and 27 out of 39 isolates, respectively) and oxacillin (2 out of 3 and 38 out of 39 isolates, respectively) was common. The IRCM was associated with a high milk yield, 28 to 90 days in milk, and a higher body condition score. Our results show that there is substantial room for udder health improvement on most farms.

Transmission dynamics of Staphylococcus aureus and Streptococcus agalactiae in a Dutch dairy herd using an automatic milking system.

Staphylococcus aureus and Streptococcus agalactiae are important contagious mastitis pathogens and are considered to mainly transmit between cows through the milking machine. Controlling contagious mastitis on dairy farms requires a reduction of the transmission rate or the duration of intramammary infections (IMI), or both. These parameters may differ in dairy herds milked with an automatic milking system (AMS) as compared to those milked with a conventional milking system (CMS). The aims of this prospective longitudinal study were to estimate the transmission rate, the median duration of IMI and the basic reproduction number (R0) of Staph. aureus and Strep. agalactiae in a Dutch AMS herd. Bacteriological cultures of quarter milk samples were collected every 2 wks. Using 3 different definitions of IMI, we estimated the transmission rate for Staph. aureus to be within the range of 0.002 (95 % CI: 0-0.005) quarter-day-1 to 0.019 (95 % CI: 0.010-0.032) quarter-day-1, and for Strep. agalactiae of 0.007 (95 % CI: 0.005-0.010) quarter-day-1 to 0.019 (95 % CI: 0.011-0.032) quarter-day-1, the median duration of chronic IMI at 95 (95 % CI: 72-125) days for Staph. aureus and at 86 (95 % CI: 67-111) days for Strep. agalactiae, and the R0 between 0.16 (95 % CI: 0.05-0.27) and 0.34 (95 % CI: 0.20-0.48) for Staph. aureus, and between 0.64 (95 % CI: 0.41-0.87) and 0.68 (95 % CI: 0.48-0.88) for Strep. agalactiae. Transmission of these two contagious pathogens in this herd was limited and theoretically the IMI would not sustain, given that R0 of both pathogens was lower than 1. The estimated transmission rate of Staph. aureus in this AMS herd was found to be comparable to those described for CMS herds, while for Strep. agalactiae, it was slightly higher than in CMS herds. The duration of Staph. aureus IMI was in line with results from CMS farms, while the duration of Strep. agalactiae was lower than what has been described in CMS herds. The R0 of these contagious pathogens was found to be lower than the estimates in CMS herds. Our study suggests that the transmission rate of these two contagious pathogens in this AMS herd were comparable to what has been reported about well-performing CMS herds that have a low rate of transmission.

Effects of a mastitis treatment strategy with or without on-farm testing.

The etiology of mastitis is crucial information to use antimicrobials prudently for control and treatment. This study aimed to evaluate the effects of mastitis diagnosis and treatment strategies with on-farm testing, on cure, new intramammary infections (IMI), somatic cell count (SCC), and antimicrobial use, compared with farmers' current diagnosis and treatment strategies. The on-farm tests used, CHROMagar Mastitis (CHROMagar, Paris, France) and Minnesota Easy Culture System II Tri-plate (University of Minnesota, St. Paul, MN), both had etiological groups of IMI as result, being gram-positive growth, gram-negative growth, or culture negative. Two randomized controlled trials were conducted on 15 herds: trial 1 prospectively enrolled 155 cows with clinical mastitis, and trial 2 cross-sectionally included 78 cows with subclinical mastitis. In both trials, cows were randomly distributed over 3 equal-sized groups: a test group using CHROMagar, a test group using Minnesota, and a control group not using on-farm tests. Farmers decided whether or not to treat, and which antimicrobial treatment would be applied, using information available on the day of enrollment (control group), complemented with the on-farm test result 1 d after enrollment (both test groups). For clinical mastitis, an antimicrobial treatment was given in 58% of cases that used CHROMagar, in 80% that used Minnesota, and in 86% of the controls. For subclinical mastitis, an antimicrobial treatment was given in 50% of cases that used CHROMagar, in 54% that used Minnesota, and in 4% of the controls. Bacteriological cure rate of clinical mastitis was lowest in the CHROMagar group [odds ratio 0.18 (95%CI 0.03-0.99)] compared with the controls. Using the Minnesota on-farm test for subclinical mastitis diagnosis and treatments resulted in fewer new IMI on d 21 [odds ratio 0.06 (95%CI 0.00-0.74)] compared with the controls. Clinical cure rate, percentage of new IMI, and SCC on d 21 of clinical mastitis were comparable among the groups. Using on-farm tests in farmers' decision-making process resulted in more treatments in accordance with the etiology of mastitis than without on-farm testing. A diagnosis and treatment strategy with on-farm testing is advised in cows with clinical mastitis to enhance prudent antimicrobial use. For subclinical mastitis, however, on-farm testing may lead to an unacceptable increase in use of antimicrobials and thus should not be advised as the common approach.

Dynamics of intramammary infections in suckler ewes during early lactation.

Mastitis is an important problem in meat-producing sheep, but few studies have investigated the transmission dynamics of mastitis pathogens in these animals. The objective of this study was to describe the pathogens causing intramammary infections (IMI) in suckler ewes, their effect on somatic cell count, and the dynamics of these IMI in early lactation. We enrolled 15 sheep flocks early after lambing and selected ewes in each flock that were sampled twice with a 3-wk interval. Milk samples from both glands of each ewe were bacteriologically cultured, and somatic cell count was measured. Non-aureus Staphylococcus spp. were the most prevalent culture results. Somatic cell counts were most strongly increased in ewes infected with Mannheimia haemolytica, whereas staphylococci, including Staphylococcus aureus, were associated with a moderate increase in somatic cell count. The proportion of udder halves that remained culture-positive with Staphylococcus spp. during the 3-wk sampling interval was moderate, but M. haemolytica infections were stable during this time period. A substantial number of new infections were seen in the early lactation study period for non-aureus Staphylococcus spp., Staph. aureus, and Corynebacterium spp., but not for M. haemolytica or Streptococcus spp. The number of new IMI of Staph. aureus was associated with the number of Staph. aureus-infected udder halves in the flock at the first sampling moment, indicative of contagious transmission. Altogether, we show that substantial transmission happens in early lactation in suckler ewes, but that the dynamics differ between pathogen species. More research is needed to further describe transmission in different stages of lactation and to identify transmission routes, to develop effective interventions to control mastitis.

Urinary excretion of advanced glycation end products in dogs and cats.

The present study was conducted with privately owned dogs and cats to investigate whether a relationship exists between the dietary AGEs and the urinary excretion of AGEs, as indication of possible effective absorption of those compounds in the intestinal tract of pet carnivores. For this purpose, data were collected from both raw fed and dry processed food (DPF) fed to dogs and cats, through spot urine sampling and questionnaires. Raw pet food (RF, low in AGE diets) was fed as a primary food source to 29 dogs and DPF to 28 dogs. Cats were categorized into 3 groups, which were RF (n = 15), DPF (n = 14) and dry and wet processed pet food (DWF, n = 25). Urinary-free carboxymethyllysine (CML), carboxyethyllysine (CEL) and lysinoalanine (LAL) were analysed using ultrahigh-performance liquid chromatography (UHPLC)-mass spectrometry, and were standardized for variable urine concentration by expressing the AGE concentrations as a ratio to urine creatinine (Ucr) concentration (µg/µmol Ucr). Urinary excretion of CML, CEL and LAL in dogs fed with DPF was 2.03, 2.14 and 3 times higher compared to dogs fed with RF (p < .005). Similar to the dogs, a significant difference in CML:Ucr, CEL:Ucr and LAL:Ucr between the three diet groups was observed in cats (p-overall < 0.005, ANOVA), in which the RF fed group excreted less AGEs than the other groups. Linear regression coefficients and SE of CML:Ucr, CEL:Ucr and LAL:Ucr showed that body weight and neuter status were significantly correlated with CML and CEL excretion, but not to LAL excretion. Our results revealed a significant correlation between dietary AGEs and urinary excretion of free CML, CEL and LAL, and also showed that endogenous formation of these AGEs occurs in both dogs and cats under physiological conditions.

Genomic analysis of European bovine Staphylococcus aureus from clinical versus subclinical mastitis.

Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38-9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis.

Performance of Online Somatic Cell Count Estimation in Automatic Milking Systems.

Somatic cell count (SCC) is one of the most important and widely used mastitis diagnostics. For detecting (sub)clinical mastitis, online SCC related measurements are more and more used in automatic milking systems (AMS). Sensors such as an automated online California Mastitis Test (O-CMT) allow for high frequency screening of high SCC cows within a herd, which makes it potentially powerful to identify episodes of mastitis. However, the performance of O-CMT measurements, as compared to SCC determined in the laboratory (L-SCC), has only scarcely been described. The aims of this study were (1) to assess the agreement between the O-CMT measurement averaged over different time windows and the corresponding L-SCC measurements; (2) to determine the optimal time window for averaging O-CMT as compared to L-SCC; (3) to explore the added value of time-series of frequent O-CMT measurements in individual cow udder health monitoring compared to L-SCC measurements. Data were collected from 50 farms in 6 different countries that were equipped with AMS using O-CMT measurements and also performed regular L-SCC testing. We found that the overall concordance correlation coefficient (CCC) between O-CMT and L-SCC was 0.53 but differed substantially between farms. The CCC between O-CMT and L-SCC improved when averaging O-CMT over multiple milkings, with an optimal time-window of 24 h. Exploration of time series of daily O-CMT recordings show that this is an effective screening tool to find episodes of high SCC. Altogether, we conclude that although O-CMT agrees moderately with L-SCC, because of its high measurement frequency, it is a promising on-farm tool for udder health monitoring.

Activation of a Bovine Mammary Epithelial Cell Line by Ruminant-Associated Staphylococcus aureus is Lineage Dependent.

Bovine mastitis is a costly disease to the dairy industry and intramammary infections (IMI) with Staphylococcus aureus are a major cause of mastitis. Staphylococcus aureus strains responsible for mastitis in cattle predominantly belong to ruminant-associated clonal complexes (CCs). Recognition of pathogens by bovine mammary epithelial cells (bMEC) plays a key role in activation of immune responsiveness during IMI. However, it is still largely unknown to what extent the bMEC response differs according to S. aureus CC. The aim of this study was to determine whether ruminant-associated S. aureus CCs differentially activate bMEC. For this purpose, the immortalized bMEC line PS was stimulated with S. aureus mastitis isolates belonging to four different clonal complexes (CCs; CC133, CC479, CC151 and CC425) and interleukin 8 (IL-8) release was measured as indicator of activation. To validate our bMEC model, we first stimulated PS cells with genetically modified S. aureus strains lacking (protein A, wall teichoic acid (WTA) synthesis) or expressing (capsular polysaccharide (CP) type 5 or type 8) factors expected to affect S. aureus recognition by bMEC. The absence of functional WTA synthesis increased IL-8 release by bMEC in response to bacterial stimulation compared to wildtype. In addition, bMEC released more IL-8 after stimulation with S. aureus expressing CP type 5 compared to CP type 8 or a strain lacking CP expression. Among the S. aureus lineages, isolates belonging to CC133 induced a significantly stronger IL-8 release from bMEC than isolates from the other CCs, and the IL-8 response to CC479 was higher compared to CC151 and CC425. Transcription levels of IL-8, tumor necrosis factor alpha (TNFα), serum amyloid A3 (SAA3), Toll-like receptor (TLR)-2 and nuclear factor κB (NF-κB) in bMEC after bacterial stimulation tended to follow a similar pattern as IL-8 release, but there were no significant differences between the CCs. This study demonstrates a differential activation of bMEC by ruminant-associated CCs of S. aureus, which may have implications for the severity of mastitis during IMI by S. aureus belonging to these lineages.

Comparing four diagnostic tests for Giardia duodenalis in dogs using latent class analysis.

BACKGROUND: To accurately diagnose giardiosis in dogs, knowledge of diagnostic test characteristics and expected prevalence are required. The aim of this work was to estimate test characteristics (sensitivity and specificity) of four commonly used diagnostic tests for detection of Giardia duodenalis in dogs. METHODS: Fecal samples from 573 dogs originating from four populations (household dogs, shelter dogs, hunting dogs and clinical dogs) were examined with centrifugation sedimentation flotation (CSF) coproscopical analysis, direct immunofluorescence assay (DFA, Merifluor Cryptosporidium/Giardia®), a rapid enzyme immunochromatographic assay (IDEXX SNAP Giardia®) and qPCR (SSU rDNA) for presence of G. duodenalis. Bayesian latent class analysis was used to determine test performance characteristics and to estimate G. duodenalis prevalence of each of the four dog populations. RESULTS: All tests were highly specific. IDEXX SNAP Giardia® showed the highest specificity (99.6%) and qPCR the lowest (85.6%). The sensitivities were much more variable, with qPCR showing the highest (97.0%) and CSF the lowest (48.2%) sensitivity. DFA was more sensitive than IDEXX SNAP Giardia®, but slightly less specific. Prevalences of G. duodenalis differed substantially between populations, with the hunting dogs showing the highest G. duodenalis prevalence (64.9%) and the household dogs the lowest (7.9%). CONCLUSIONS: This study identifies qPCR as a valuable screening tool because of its high sensitivity, whereas methods using microscopy for cyst identification or cyst wall detection should be used in situations where high specificity is required. G. duodenalis is a prevalent gastro-intestinal parasite in Dutch dogs, especially in dogs living in groups (hunting and shelter dogs) and clinical dogs.

High Production of LukMF' in Staphylococcus aureus Field Strains Is Associated with Clinical Bovine Mastitis.

Staphylococcus aureus, a major cause of bovine mastitis, produces a wide range of immune-evasion molecules. The bi-component leukocidin LukMF' is a potent killer of bovine neutrophils in vitro. Since the role of LukMF' in development of bovine mastitis has not been studied in natural infections, we aimed to clarify whether presence of the lukM-lukF' genes and production levels of LukMF' are associated with clinical severity of the disease. Staphylococcus aureus was isolated from mastitis milk samples (38 clinical and 17 subclinical cases) from 33 different farms. The lukM-lukF' genes were present in 96% of the isolates. Remarkably, 22% of the lukM-lukF'-positive S. aureus isolates displayed a 10-fold higher in vitro LukMF' production than the average of the lower-producing ones. These high producing isolates were cultured significantly more frequently from clinical than subclinical mastitis cases. Also, the detection of LukM protein in milk samples was significantly associated with clinical mastitis and high production in vitro. The high producing LukMF' strains all belonged to the same genetic lineage, spa-type t543. Analysis of their global toxin gene regulators revealed a point mutation in the Repressor of toxins (rot) gene which results in a non-functional start codon, preventing translation of rot. This mutation was only identified in high LukMF' producing isolates and not in low LukMF' producing isolates. Since rot suppresses the expression of various toxins including leukocidins, this mutation is a possible explanation for increased LukMF' production. Identification of high LukMF' producing strains is of clinical relevance and can potentially be used as a prognostic marker for severity of mastitis.

Identification of a staphylococcal complement inhibitor with broad host specificity in equid Staphylococcus aureus strains.

Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome (e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal complement inhibitor (SCIN), a secreted protein that interferes with activation of the human complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded complement inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts.

The intention of North-Western Ethiopian dairy farmers to control mastitis.

Understanding the intentions of dairy farmers towards mastitis control is important to design effective udder health control programs. We used the Theory of Planned Behavior (TPB) to explore the intentions of North-Western Ethiopian dairy farmers towards implementing non-specified mastitis control measures (nsMCMs) and towards implementing 4 specific MCMs. Face to face interviews were held with 134 dairy farmers to study associations between their intentions and any of three factors (attitude, subjective norm and perceived behavioral control) that, according to the TPB, determine intentions. The majority of the farmers (93%) had a positive intention to implement nsMCMs, whereas a smaller majority of farmers had the intention to implement the specific MCMs to improve udder cleaning (87%), to improve stall hygiene (78%), to improve feeding of cows (76%), and to perform foremilk stripping (74%). Farmers had a more positive attitude, but lower subjective norm and lower perceived behavioural control towards implementing nsMCMs compared with implementing most specific MCMs, although the subjective norms for stall hygiene and perceived behavioural control for improving feeding of cows were also low. Attitude was positively associated with intentions to implement nsMCMs, to improve cleaning of the udders, to improve stall hygiene and to implement foremilk stripping. Both the intention to improve udder cleaning and to implement foremilk stripping, were positively associated to subjective norms towards these MCMs. Our data can help tailor intervention programs aiming to increase the intention of Ethiopian dairy farmers to implement MCMs and thus to improve udder health in this country. We show that such programs should primarily focus on changing attitude and secondarily on improving the farmers' subjective norms.