Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome

Based on our clinical experience on bovine mastitis, we hypothesized that subtypes of Staphylococcus aureus (S. aureus) exist which differ in their contagious and pathogenic properties. In order to investigate this hypothesis, we analyzed strains of S. aureus isolated from spontaneous intramammary infection (IMI) with their virulence gene patterns and genotypes obtained by PCR amplification of the 16S-23S rRNA intergenic spacer (RS-PCR). The genotypes were then associated with epidemiological and clinical data including 26 herds. The results demonstrated a high association between genotypes and virulence gene patterns as well as between epidemiological and pathogenic properties of S. aureus. In particular, genotype B was related to high contagiosity and increased pathogenicity whereas the other types (C, OG) were found with infection of single cows. Because of the high clinical relevance, our results indicate the need to subtype the IMI-associated strains of S. aureus in the future.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland

Antimicrobial susceptibility testing was performed on a total of 581 clinical Escherichia coli isolates from diarrhea and edema disease in pigs, from acute mastitis in dairy cattle, from urinary tract infections in dogs and cats, and from septicemia in laying hens collected in Switzerland between 1999 and 2001. Among the 16 antimicrobial agents tested, resistance was most frequent for sulfonamides, tetracycline, and streptomycin. Isolates from swine presented significantly more resistance than those from the other animal species. The distribution of the resistance determinants for sulfonamides, tetracycline, and streptomycin was assessed by hybridization and PCR in resistant isolates. Significant differences in the distribution of resistance determinants for tetracycline (tetA, tetB) and sulfonamides (sulII) were observed between the isolates from swine and those from the other species. Resistance to sulfonamides could not be explained by known resistance mechanisms in more than a quarter of the sulfonamide-resistant and sulfonamide-intermediate isolates from swine, dogs and cats. This finding suggests that one or several new resistance mechanisms for sulfonamides may be widespread among E. coli isolates from these animal species. The integrase gene (intI) from class I integrons was detected in a large proportion of resistant isolates in association with the sulI and aadA genes, thus demonstrating the importance of integrons in the epidemiology of resistance in clinical E. coli isolates from animals.

Evaluation of a Novel Barrier—Type Teat Dip Product Applied to Ewes Post-Weaning

Teats of 36 ewes (72 udder halves or teats) were dipped with an experimental barrier - type teat dip product to evaluate product persistency post weaning. Persistency was evaluated one to two times/day and scored positive if the teat end orifice was covered and protected. Persistency or the percentage of teats covered/protected at 36, 54, 72, 96, 132, and 156 hours was 100%, 93%, 89%, 63%, 35%, and 24% respectively. Ewes will be dipped again pre-lambing and both persistency and bacteriology (mastitis prevention) will be evaluated compared to 36 control ewes.

Camel Streptococcus agalactiae populations are associated with specific disease complexes and acquired the tetracycline resistance gene tetM via a Tn916-like element

Camels are the most valuable livestock species in the Horn of Africa and play a pivotal role in the nutritional sustainability for millions of people. Their health status is therefore of utmost importance for the people living in this region. Streptococcus agalactiae, a Group B Streptococcus (GBS), is an important camel pathogen. Here we present the first epidemiological study based on genetic and phenotypic data from African camel derived GBS. Ninety-two GBS were characterized using multilocus sequence typing (MLST), capsular polysaccharide typing and in vitro antimicrobial susceptibility testing. We analysed the GBS using Bayesian linkage, phylogenetic and minimum spanning tree analyses and compared them with human GBS from East Africa in order to investigate the level of genetic exchange between GBS populations in the region. Camel GBS sequence types (STs) were distinct from other STs reported so far. We mapped specific STs and capsular types to major disease complexes caused by GBS. Widespread resistance (34%) to tetracycline was associated with acquisition of the tetM gene that is carried on a Tn916-like element, and observed primarily among GBS isolated from mastitis. The presence of tetM within different MLST clades suggests acquisition on multiple occasions. Wound infections and mastitis in camels associated with GBS are widespread and should ideally be treated with antimicrobials other than tetracycline in East Africa.

Concomitant lipopolysaccharide-induced transfer of blood-derived components including immunoglobulins into milk

During a mammary immune response, the integrity of the blood-milk barrier is negatively affected and becomes leaky. The aim of the present study was to demonstrate the blood origin, and to investigate changes in the concentration, of various constituents including immunoglobulins in blood and milk during the early phase of lipopolysaccharide (LPS)-induced mastitis. Five lactating dairy cows received continuous β-hydroxybutyrate (BHBA) clamp infusions to maintain elevated BHBA blood concentrations (1.5 to 2.0 mmol/L) from 48 h before and 8h after LPS administration. One udder quarter was infused with 200 μg of Escherichia coli LPS. A second quarter served as control. Milk and blood samples were taken hourly for 8h postchallenge (PC). The somatic cell count in LPS-challenged quarters was increased from 4h PC to the end of the experiment compared with control quarters. In LPS-challenged quarters, l-lactate, BHBA, lactate dehydrogenase (LDH), IgG(1), and IgG(2) were increased at 3h PC and remained elevated until the end of experiment (8h PC) compared with control quarters. In addition, the optical density values in milk in a nonquantitative ELISA for antibodies directed against bluetongue virus (used as a measure of nonspecific antibody transfer; all animals were vaccinated) increased and, thus, indicates an increase in these antibodies in response to LPS treatment. l-Lactate concentration also increased in blood 2h PC and in the milk of control quarters during the experiment from 3h PC. A second experiment was conducted in vitro to investigate a possible contribution from destructed milk cells to l-lactate concentration and activity of LDH in milk. Aliquots of milk samples (n=8) were frozen (-20°C) or disrupted with ultrasound, respectively. Freeze thawing and ultrasound treatment increased LDH in milk samples, but had no effect on l-lactate concentrations. Results suggest that intramammary infusion of LPS induces a systemic response, as evidenced by an elevation of blood l-lactate concentration. The concomitant changes of all investigated components suggest that they were blood derived. However, the increase in blood components in the milk is not necessarily supportive of the mammary immune system, and likely a side effect of reduced blood-milk barrier integrity.

Effects of induced energy deficiency on lactoferrin concentration in milk and the lactoferrin reaction of primary bovine mammary epithelial cells in vitro.

A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid-lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control-fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat-inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme-linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post-partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up-regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk-extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non-invasive milk-extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.

An inter-laboratory comparative study of serological tools employed in the diagnosis of Besnoitia besnoiti infection in bovines

Bovine besnoitiosis is considered an emerging chronic and debilitating disease in Europe. Many infections remain subclinical, and the only sign of disease is the presence of parasitic cysts in the sclera and conjunctiva. Serological tests are useful for detecting asymptomatic cattle/sub-clinical infections for control purposes, as there are no effective drugs or vaccines. For this purpose, diagnostic tools need to be further standardized. Thus, the aim of this study was to compare the serological tests available in Europe in a multi-centred study. A coded panel of 241 well-characterized sera from infected and non-infected bovines was provided by all participants (SALUVET-Madrid, FLI-Wusterhausen, ENV-Toulouse, IPB-Berne). The tests evaluated were as follows: an in-house ELISA, three commercial ELISAs (INGEZIM BES 12.BES.K1 INGENASA, PrioCHECK Besnoitia Ab V2.0, ID Screen Besnoitia indirect IDVET), two IFATs and seven Western blot tests (tachyzoite and bradyzoite extracts under reducing and non-reducing conditions). Two different definitions of a gold standard were used: (i) the result of the majority of tests ('Majority of tests') and (ii) the majority of test results plus pre-test information based on clinical signs ('Majority of tests plus pre-test info'). Relative to the gold standard 'Majority of tests', almost 100% sensitivity (Se) and specificity (Sp) were obtained with SALUVET-Madrid and FLI-Wusterhausen tachyzoite- and bradyzoite-based Western blot tests under non-reducing conditions. On the ELISAs, PrioCHECK Besnoitia Ab V2.0 showed 100% Se and 98.8% Sp, whereas ID Screen Besnoitia indirect IDVET showed 97.2% Se and 100% Sp. The in-house ELISA and INGEZIM BES 12.BES.K1 INGENASA showed 97.3% and 97.2% Se; and 94.6% and 93.0% Sp, respectively. IFAT FLI-Wusterhausen performed better than IFAT SALUVET-Madrid, with 100% Se and 95.4% Sp. Relative to the gold standard 'Majority of test plus pre-test info', Sp significantly decreased; this result was expected because of the existence of seronegative animals with clinical signs. All ELISAs performed very well and could be used in epidemiological studies; however, Western blot tests performed better and could be employed as a posteriori tests for control purposes in the case of uncertain results from valuable samples.

Epidemiologic survey in Swiss group-housed breeding rabbits: extent of lesions and potential risk factors

In Switzerland, group-housing for breeding rabbit does is not explicitly required by law, but label programmes, as well as the general public and animal welfare groups, are advocating it. Although group-housing is of great benefit to the gregariously living rabbits, the establishment of a social hierarchy within the group might lead to stress and lesions. In the present epidemiological study, lesions were scored twice on 30% of the breeding does on all 28 commercial Swiss farms with group-housed breeding does. Additionally, a detailed questionnaire was filled out with all producers to determine risk factors potentially associated with lesions. Data were analysed using hierarchical proportional odds models. About 33% of the does examined had lesions, including wounds that were almost healed and small scratches. Severe lesions were counted on 9% of the animals. Differences between seasons in lesions score were identified, with the extent of lesions being higher in summer than in spring. Fewer lesions occurred on farms on which mastitis was more common. More lesions were found on farms where the does were isolated between littering and artificial insemination than on farms without isolation. According to the producers, most of the aggression occurred directly after the isolation phase when the does were regrouped again. We conclude that lesions in group-housed breeding does might be reduced by appropriate reproductive management.

Bovine Staphylococcus aureus: diagnostic properties of specific media

As accurate discrimination between Staphylococcus (S.) aureus and NSA (non-S. aureus staphylococci) involved in bovine mastitis is essential in terms of clinical prognosis and outcome, the aim of this study was to reevaluate the classical bacteriological procedures to identify these agents. Various media and the coagulase tube test were investigated using 116 strains of S. aureus and 115 of NSA, all isolated from cows with spontaneous intramammary infections (IMI). Furthermore, 25 NSA reference strains were analyzed. The study demonstrated that a few media were appropriate for differentiating S. aureus from NSA, provided that the staphylococci were isolated from bovine IMI. Evaluation of hemolysis further revealed that double or incomplete hemolysis are specific for S. aureus and are, therefore, a decisive diagnostic criterion. For strains showing complete hemolysis, maximal discrimination between S. aureus and NSA was observed by subculturing them on CHROMagar Staph. aureus.

Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections.

BACKGROUND Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC. RESULTS The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster. CONCLUSIONS The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Lameness and foot lesions in Swiss dairy cows: I. Prevalence.

Prevalences of foot lesions and lameness were recorded in 1'449 Swiss dairy cows during routine claw-trimming on 78 farms from June 2010 until February 2011. Lameness was present in 14.8 % of cows and on 80.8 % of investigated farms. Highest prevalences were seen for widened white line (80.7 %/100 %), signalling foot lesion (65.6 %/98.7 %), heel-horn erosion (34.2 %/88.5 %), digital dermatitis complex (29.1 %/73.1 %), severe hemorrhages (27.9 %/87.2 %), and Rusterholz' sole ulcers (11.5 %/74.4 %) at cow and herd level, respectively. Lower prevalences were found for subclinical laminitis (5.4 %/47.4 %), chronic laminitis (3.3 %/25.6 %), white line disease (4.7 %/42.3 %), double soles (2.6 %/33.3 %), interdigital hyperplasia (3.1 %/33.3 %), sole ulcers (0.4 %/6.4 %), toe infections caused by faulty claw-trimming (3.9 %/39.7 %) and by injury (0.1 %/2.6 %), deep lacerations (0.4 %/6.4 %), and interdigital phlegmona (0.1 %/1.3 %). Lameness and foot lesions were shown to represent important health problems of dairy cows under the conditions of the typical grass-based production system in Switzerland. Digital dermatitis has developed to the most relevant foot disease with a high impact on welfare of Swiss dairy cows within the past 10 years.

Self-Reported Psychotic-Like Experiences Are a Poor Estimate of Clinician-Rated Attenuated and Frank Delusions and Hallucinations

Background: One reason for the decision to delay the introduction of an Attenuated Psychosis Syndrome in the main text of the fifth edition of the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders was the concern that attenuated psychotic symptoms (APS) might in fact be common features in adolescents and young adults from the general population of no psychopathological significance in themselves. This concern was based on reports of high prevalence rates of psychotic-like experiences (PLEs) in the general population and the assumption that PLEs are a good estimate of APS. Although the criterion validity of self-reported PLEs had already been studied with respect to clinician-rated psychotic symptoms and found insufficient, it had been argued that PLEs might in fact be more comparable with mild, subclinical expressions of psychotic symptoms and, therefore, with APS. The present paper is the first to specifically study this assumption. Sampling and Methods: The sample consisted of 123 persons seeking help at a service for the early detection of psychosis, of whom 54 had an at-risk mental state or psychosis, 55 had a nonpsychotic mental disorder and 14 had no full-blown mental disorder. PLEs were assessed with the Peters Delusion Inventory and the revised Launay-Slade Hallucination Scale, and psychotic symptoms and APS were assessed with the Structured Interview for Prodromal Syndromes. Results: At a level of agreement between the presence of any PLE (in 98.4% of patients) and any APS (in 40.7%) just exceeding chance (κ = 0.022), the criterion validity of PLEs for APS was insufficient. Even if additional qualifiers (high agreement or distress, preoccupation and conviction) were considered, PLEs (in 52.8%) still tended to significantly overestimate APS, and agreement was only fair (κ = 0.340). Furthermore, the group effect on PLE prevalence was, at most, moderate (Cramer's V ≤ 0.382). Conclusions: The prevalence of APS cannot be deduced from studies of PLEs. Thus, the high population prevalence rate of PLEs does not allow the conclusion that APS are common features of no pathological significance and would lack clinical validity as an Attenuated Psychosis Syndrome in the Diagnostic and Statistical Manual of Mental Disorders, 5th edition. Rather, the population prevalence rate of APS has to be assumed to be largely unknown at present but is likely lower than indicated by epidemiological studies of PLEs. Therefore, dedicated studies are warranted, in which APS are assessed in a way that equates to their clinical evaluation.

A longitudinal study investigating the prevalence of Staphylococcus aureus genotype B in seasonally communal dairy herds

Abstract Staphylococcus aureus is a major mastitis-causing pathogen. Various genotypes have been recently identified in Switzerland but Staph. aureus genotype B (GTB) was the only genotype associated with high within-herd prevalence. The risk of introducing this Staph. aureus genotype into a herd may be increased by frequent animal movements. This may also be the case when cows from different herds of origin are commingled and share their milking equipment for a limited period of time. The aim of the present study was to determine the prevalence of Staph. aureus GTB in seasonally communal dairy herds before and after a summer period when dairy farming is characterized by mixing cows from different herds of origin in 1 communal operation. In addition, the environment was investigated to identify potential Staph. aureus GTB reservoirs relevant for transmission of the disease. A total of 829 cows from 110 herds of origin in 9 communal operations were included in the study. Composite milk samples were collected from all cows during the first or second milking after arrival at the communal operation and again shortly before the end of the season. Swab samples from the environment, involved personnel, and herding dogs present were collected before the cows arrived. At the end of the season, sampling of personnel was repeated. All samples were analyzed for the presence of Staph. aureus GTB using an established quantitative PCR. At the beginning of the season, Staph. aureus GTB-positive cows were identified in 7 out of 9 communal operations and the within-communal operation prevalence ranged from 2.2 to 38.9%. At the second sampling, all communal operations were Staph. aureus GTB positive, showing within-communal operation prevalence from 1 to 72.1%. The between-herd of origin prevalence increased from 27.3 to 56.6% and the cow-level prevalence increased from 11.2% at the beginning of the season to 29.6% at the end of the season. On 3 different communal operations, Staph. aureus GTB-positive swabs from seasonally employed personnel were identified at the end of the season. The results indicate that Staph. aureus GTB can easily spread in communal operations when cows from different herds of origin are mixed during the summer season. Effective management measures need to be designed to prevent the spread of Staph. aureus GTB in seasonally communal herds. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved. KEYWORDS: Staphylococcus aureus; biosecurity; communal herd; epidemiology