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PubMed2006

Detection of multiple virulence-associated genes in Listeria monocytogenes isolated from bovine mastitis cases.

Clinical samples (n=725) were collected from bovines (n=243) which were positive for mastitis using the California mastitis test (CMT) and somatic cell count (SCC). The clinical samples comprising blood (n=239), milk (n=243), and faecal swabs (n=243) were examined for the presence of pathogenic Listeria spp. Isolation of the pathogen was done using selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Confirmation of the isolates was based on biochemical tests and Christie, Atkins, Munch-Petersen (CAMP) test followed by pathogenicity testing. Pathogenicity of the isolates was tested by phosphatidylinositol-specific phospholipase C (PI-PLC) assay as well as in vivo tests namely, chick embryo and mice inoculation tests. The isolates were subjected to PCR assay for five virulence-associated genes, plcA, prfA, hlyA, actA and iap. Listeria spp. were isolated from 12 (1.66%) samples. Of these 4 (0.55%) and 1 (0.14%) were confirmed as Listeria monocytogenes and Listeria ivanovii, respectively. L. monocytogenes and L. ivanovii were recovered from milk samples (2) and faecal (3) of mastitic cattle (3) and buffaloes (2). L. monocytogenes recovered from the milk of mastitic cattle and L. ivanovii from the faecal swab of buffalo turned out to be pathogenic. However, the remaining three hemolytic isolates exhibiting positive CAMP test turned out to be negative in PI-PLC assay, chick embryo and mice inoculation. L. monocytogenes and L. ivanovii isolates characterized as pathogenic by PI-PLC assay and in vivo pathogenicity tests were found to possess all the five virulence-associated genes and three genes, plcA, prfA and actA respectively. The remaining three hemolytic but non-pathogenic L. monocytogenes isolates were negative for plcA by PCR. It seems that the plcA gene and its expression (in the PI-PLC assay) have an important role as virulence determinants in pathogenic Listeria spp. In conclusion, the PI-PLC assay and virulence genes targeted PCR (plcA, prfA and hlyA genes for L. monocytogenes and plcA, prfA and actA genes for L. ivanovii) hold a good promise as rapid and reliable in vitro alternatives to in vivo pathogenicity tests.

Animals↗
PubMed2006

Rembrandt's Bathsheba, possible lactation mastitis following unsuccessful pregnancy.

Deformity of the left breast and axilla observed in Rembrandt's famous painting "Bathsheba at her toilet" (1654, Louvre Paris) has been discussed by several researchers. Proposed diagnoses were breast cancer and abscess due to tuberculosis. The present article reviews previous articles written concerning the left breast abnormalities of Bathsheba and carefully examines other works of Rembrandt modeled by Hendrickje and painted around 1654. Previous diagnosis of breast cancer and tuberculous mastitis is less probable. Because Hendrickje survived for more then 9 years after the painting and in other works modeled by Hendrickje shows no signs of cachexia or permanent changes in the left breast. The most likely diagnosis of the left breast deformity of Bathsheba is a sequela of lactation mastitis abscess following miscarriage or premature childbirth without breast feeding.

Diagnosis, Differential↗
PubMed2005

Associations between somatic cell count patterns and the incidence of clinical mastitis.

Associations between clinical mastitis (CM) and the proportional distribution of patterns in somatic cell count (SCC) on a herd level were determined in this study. Data on CM and SCC over a 12-month period from 274 Dutch herds were used. The dataset contained parts of 29,719 lactations from 22,955 cows of different parities. In total, 207,079 SCC test-days were recorded with 5719 cases of CM; 1561 cases were associated with environmental pathogens (ENV_CM), and 2681 with contagious pathogens (CONT_CM). Definitions of patterns in SCC were based on 3, 4, or 5 consecutive test-day recordings of SCC that differentiated between short or longer periods of increased SCC, and also between lactations with and without recovery. The distribution of those patterns (relative to their maximum) varied among herds. The distribution of SCC patterns was correlated with the incidence rate of CM. Herds with a relatively frequent quick recovery pattern had a 2.5 times more chance of being classified in the upper quartile for CM. These herds also had 2.1 times more chance of being classified in the upper quartile for ENV_CM but only 0.4 times for CONT_CM. Herds with a relatively frequent no recovery pattern had less chance (odds ratio=0.5) of being classified in the lower quartile for CONT_CM. Since the distributions of SCC patterns were indicative for overall, environmental and contagious CM, the necessity to introduce pathogen-specific mastitis control programs in a herd could be determined based on the mean incidences of SCC patterns in that herd.

Animals↗
PubMed2006

The transmission and control of mastitis in dairy cows: a theoretical approach.

A multi-species model that incorporates the transmission of both major and minor mastitis pathogens as well as the interaction between them via coinfection of a quarter is fitted to data from seven dairy herds. The results suggest that major and minor pathogens can interact, on occasion, in a counter-intuitive way with implications for the control of clinical mastitis. The key finding is that delaying culling of cows with major pathogen infections for more than 100 days post infection could result in a higher prevalence of major pathogen infections, whereas early culling would reduce the levels. A theoretical exploration of current and proposed control strategies is carried out, informed by parameters estimated from the model and data. The results at each stage suggest of areas of further research such as: field-testing of the hypotheses presented; the exploration of a stochastic formulation of the model; analysis of the raw repeated measures data; application of control theory to determine the most effective combination of control strategies; inclusion of economic factors into the modelling framework.

Animals↗
PubMed2006

Incidence and economics of clinical mastitis in five Holstein herds in the Czech Republic.

Data on clinical mastitis (CM) collected between 1996 and 2003 on five Holstein dairy farms in the Czech Republic were analyzed. Lactational incidences of CM, averaged across farms and calculated only from cows with complete lactations, were 0.35, 0.45 and 0.57 for the first, second and third plus subsequent lactations, respectively. The mean numbers of CM cases per cow and lactation were 0.63, 0.94 and 1.22, and the incidence of CM cases per cow-year at risk were 0.68, 1.00 and 1.27 for the first, second and third plus subsequent lactations, respectively. Longitudinal analysis of CM prevalence based on daily records showed the highest proportion of infected cows in the first 10 days of lactation. The within-farm incidence of CM cases per cow per year, averaged over lactations, ranged from 0.53 to 1.56 with a mean value of 0.94 in the whole data set. Direct financial losses from CM per cow per year within farm ranged from 43.63 to 84.84 euros. They included losses from discarded milk, cost for drugs, veterinary service, herdsman's time, cost for an extra milking machine and cost for antibiotic drying of cows. The economic value of CM incidence (change in direct losses per cow per year when increasing CM incidence by one case above the average value) ranged from 58.3 to 80.1 euros per CM case per cow per year with the mean value of 62.6 euros per CM case per cow per year in the total data set. Daily prevalence rate of CM was shown to be the best among various indicators of CM susceptibility, because it accounted for the censored character of the data and for repeated cases of CM within lactations. In order to reduce the incidence of clinical mastitis for dairy cattle in the Czech Republic, we recommend that it should be included as a goal in the breeding program.

Animals↗
PubMed2006

Invasive potential of bacterial isolates associated with subclinical bovine mastitis.

This work describes differences in the invasive ability of bacterial isolates associated with mastitis. Invasion ability was determined by the uptake and survival in a primary culture of bovine mammary epithelial cells (BMEC). BMEC were isolated from a healthy lactating cow and characterized by their morphology, immunostaining for cytokeratin and the detection of beta- and kappa-casein mRNAs. Ten bacterial isolates comprising the staphylococcal species Staphylococcus aureus (3), Staphylococcus epidermidis (1), Staphylococcus haemolyticus (1), Staphylococcus equorum (2), Staphylococcus xylosus (1) and Brevibacterium stationis (2) obtained from raw milk of cows with mastitis from backyard farms were assayed for their ability to invade BMEC. Only two S. aureus and one S. epidermidis isolates were able to invade BMEC, at similar levels to the S. aureus control strain ATCC 27543. In conclusion, using the in vitro model of infection used in this study, differences in bacterial invasion capability may be detected.

Animals↗
PubMed2006

Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey.

The genetic relatedness of coagulase (coa) positive Staphylococcus aureus isolated from cows with subclinical mastitis in Turkey was investigated by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Among 700 milk samples positive in the California Mastitis Test (CMT), species specific PCR identified 200 (28.6%) isolates as S. aureus and 161 (80.5%) of these isolates were positive for the 3' end of the coa gene by PCR. Most isolates (n=135, 83.9%) produced a single band on coa PCR, with molecular sizes ranging from 500 to 1400bp, whereas a small number of isolates (n=26, 16.1%) yielded two amplification products. Coa RFLP analysis using AluI and Hin6I revealed 23 and 22 band patterns, respectively. The detection of double bands by coa PCR, previously reported in human isolates, suggests that milking personnel can play a role in the transmission of S. aureus.

Animals↗
PubMed2006

Mucosal genetic immunization against four adhesins protects against Staphylococcus aureus-induced mastitis in mice.

Staphylococcus aureus is the most common etiologic agent of mastitis in bovines and a major cause of economic losses in the dairy industry. Since adhesins, that anchor bacteria to the extracellular matrix (ECM), are among the most important S. aureus virulence factors, we used four adhesion factors (fibrinogen binding protein, fibronectin binding protein A, clumping factor A and collagen adhesin) as target in a DNA vaccine. Intranasal immunization with a pDNA mixture coding the four adhesins, triggered significant levels of specific serum and mucosal Ig that inhibited S. aureus adhesion to cow mammary gland epithelial cells in vitro. Splenocytes of immunized mice challenged in vitro with S. aureus extracts showed a strong proliferative response. Finally, immunized mice were significantly protected against intramammary challenge with S. aureus. Our data confirm the feasibility of a genetic mucosal vaccine targeting S. aureus adhesins and prompt further study to test the efficacy in preventing cow mastitis.

Adhesins, Bacterial↗
PubMed2005

Inflammatory cell infiltration as an indicator of Staphylococcus aureus infection and therapeutic efficacy in experimental mouse mastitis.

Staphylococcus aureus intramammary colonization of the mouse mammary gland induces migration of polymorphonuclear neutrophils (PMNs) similar to that observed during bovine mastitis. In the present study, a method combining acridine orange staining, fluorescence microscopy and computer-assisted image analysis has been developed to quantitate PMN infiltration in a mouse model of mastitis. This was carried out using paraffin embedded sections, and using this method, we showed that the presence of PMNs increased with the number of bacteria present in tissues. Nearly 400 and 1100 times more PMNs were counted in the mammary gland tissue after 12 and 24 h of infection, respectively, compared to mice infected for 6 h. Treatment with the antibiotic cephapirin at 10 or 25 mg/kg reduced PMN infiltration by 71 and 85%, respectively. In conclusion, this method can be used to quantitate PMN infiltration as a marker of inflammation and bacterial burden in infected tissue sections.

Acridine Orange↗
PubMed2005

The bovine innate immune response during experimentally-induced Pseudomonas aeruginosa mastitis.

Almost half of all clinical cases of mastitis are caused by Gram-negative bacteria. Among these bacteria, intramammary infection by Pseudomonas aeruginosa remains one of the most refractory to antibiotic therapy. The ability to recognize potentially harmful pathogens whether previously encountered or not, as well as the induction of an initial pro-inflammatory response to these pathogens, are critical components of host innate immunity. Although the innate immune response to another Gram-negative mastitis-causing pathogen, Escherichia coli, has been well-characterized, little is known about the response to other Gram-negative bacteria, including P. aeruginosa. The objective of the current study was to characterize the systemic and localized bovine innate immune response to intramammary infection with P. aeruginosa. The contralateral quarters of ten mid-lactating Holstein cows were challenged with either saline or P. aeruginosa. Following the establishment of infection, milk samples were collected and assayed for changes in cytokine and growth factor concentrations, complement activation, and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), two accessory molecules involved in host recognition of Gram-negative bacteria. Initial increases in milk somatic cell counts were evident within 12h of experimental challenge and remained elevated for >or=3 weeks. Increased permeability of the mammary gland vasculature, as evidenced by elevated milk levels of BSA, was initially observed 20 h post-infection and persisted for 2 weeks. Within 32 h of challenge, increased levels of IL-8, TNF-alpha, IL-10, and IL-12 were detected, however, the elevated levels of these cytokines were not sustained for longer than a 24h period. In contrast, elevations in IL-1beta, IFN-gamma, TGF-alpha, TGF-beta1, TGF-beta2, sCD14, LBP, and activated complement factor 5 (C5a) were sustained for periods of >48 h. Systemic changes were characterized by elevated body temperature, induction of the acute phase protein synthesis of serum amyloid A and LBP, and a transient decrease in circulating neutrophils and lymphocytes. Together, these data demonstrate the capability of the mammary gland to mount a robust innate immune response to P. aeruginosa that is characterized by the induction of pro-inflammatory cytokines, complement activation, and increased levels of accessory molecules involved in Gram-negative bacterial recognition.

Acute-Phase Proteins↗
PubMed2005

Immune responses to a Staphylococcus aureus GapC/B chimera and its potential use as a component of a vaccine for S. aureus mastitis.

Bovine mastitis caused by strains of S. aureus is the most economically important disease affecting the dairy industry worldwide. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggests that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Since it seems impractical to produce a vaccine containing antigens from all major S. aureus mastitis isolates, we took the approach of using two surface antigens GapB and GapC that appear to be conserved and constructed a GapC/B chimera as the basis for a vaccine. The humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins, alone or in combination. The GapC/B protein elicited strong humoral and cellular responses in mice as judged by the levels of total IgG, IgG1, IgG2a, and number of IL-4- and IFN-gamma-secreting cells. These results suggest that this chimeric protein could be an attractive target for further vaccine efficacy studies.

Animals↗
PubMed2004

Mouse mastitis model of infection for antimicrobial compound efficacy studies against intracellular and extracellular forms of Staphylococcus aureus.

Despite the general in vitro susceptibility of Staphylococcus aureus isolates that cause infectious bovine mastitis, the pathogen remains difficult to eradicate with the available antibiotics. The capacity to survive intracellularly has been proposed as a factor contributing to the persistence of S. aureus in the bovine mammary gland. The costs associated with the use of cows or goats to assess the in vivo efficacy of new antibacterial compounds constitute a major drawback. Therefore, in the present study, a mouse model of intramammary infection has been characterized for in vivo testing of new experimental drugs. An inoculum of 100 CFU of S. aureus per gland caused an important level of infection with minimal tissue damage as observed at 24 h post-inoculation. By microscopy, polymorphonuclear neutrophil cell infiltration of the infected mammary glands was observed to increase over time. At 12-24 h of infection, the pathogen was primarily found alive and dividing in neutrophils and occasionally within mammary epithelial cells. Intramuscular or intravenous injections of cephapirin at t = 0 and 10 h reduced the number of CFU/g of gland in a dose-dependent manner. In conclusion, the mouse model of infectious mastitis proposed here is suitable for primary evaluation of experimental drugs after parenteral treatment of intramammary infection with a pathogen such as S. aureus that presents both intracellular and extracellular phases of growth.

Animals↗
PubMed2006

Adherent and invasive Escherichia coli are associated with persistent bovine mastitis.

Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.

Analysis of Variance↗
PubMed2006

Characterization of cell wall associated proteins of a Staphylococcus aureus isolated from bovine mastitis case by a proteomic approach.

Staphylococcus aureus causes different pathologies in humans and animals. In particular, it is involved in intramammary infections in cows, causing economic losses and milk-safety problems. Although it is well-known that surface components (proteins and capsular polysaccharides) and exotoxins are virulence factors involved in the pathogenesis of bovine mastitis, less is known about the precise biochemical identity of such molecules. Therefore, mapping of surface proteins using specific disease- and environment-isolates provides a benchmark for strain comparison of pathogens with different pathogenic characteristics and antibiotic resistance mechanism and can aid in defining specific vaccine and therapeutic targets. In this study, we used a proteomic approach on protein extracts of lysostaphin-treated S. aureus in isotonic conditions, to produce a reproducible and well resolved 2-D electrophoresis (2-DE) reference map of surface associated proteins of isolated S. aureus from a case of bovine mastitis. The most abundant protein components were identified by Matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry.

Animals↗
PubMed2005

The effect of an experimentally induced acute mastitis on the test results of an Ostertagia ostertagi milk ELISA.

Antibody levels against Ostertagia ostertagi in the milk are a promising parameter to identify dairy cows or herds with production losses due to gastrointestinal nematodes. However, milk antibody levels can be influenced by udder-related factors. In this study, the effect of an experimentally induced mastitis on the test results (ODR) of a milk O. ostertagi ELISA was investigated. Twenty-five cows that were naturally infected with gastrointestinal nematodes, were inoculated in their left udder quarters with Escherichia coli P4:O32 and quarter milk samples were collected at several intervals from 24 h before until 144 h after experimental infection. The effect of the contribution of milk from one or more infected quarters on the bulk tank milk ODR was estimated, based on a titration experiment. The mean O. ostertagi antibody levels of the infected udder quarters were significantly (P < 0.001) higher than those of the uninfected udder quarters at each sampling time post-infection. The largest difference was observed at 24 h post-infection with a mean difference of 0.251 ODR (95%CI: 0.172; 0.330). There was also a significant increase (P < 0.001) in total IgG levels with the largest difference being observed at 24 h post-infection. Highly significant (P < 0.005) correlation coefficients were observed between O. ostertagi ODR, total IgG ODR, Na+ and Cl- ion concentration and log transformed somatic cell counts at 24 h post-infection. The results demonstrate that an acute mastitis causes a flow of specific and non-specific antibodies from serum to milk with a subsequent increase in the O. ostertagi ODR values. The effect of the contribution of milk from infected quarters on the bulk tank milk O. ostertagi ODR was estimated to be minor if the relative number of infected quarters is small (< 3%).

Animals↗
PubMed1976

Chronic mastitis and carcinoma of the breast.

733 women in whom chronic mastitis had been diagnosed were followed for an average of 30-3 years. 49 died from breast cancer compared with an expected number of 18-8. The excess risk of dying from breast cancer persisted for at least 30 years after chronic mastitis was diagnosed.

Adult↗
PubMed2003

Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model.

Bovine mastitis caused by Staphylococcus aureus (S. aureus) is a most important infection disease that affects both the quality and the quantity of milk production. Antibiotic therapies formulated for intramammary use are generally unsuccessful in eliminating existing S. aureus infections. Vaccination is a logical approach to the control of S. aureus udder infections. However, to date commercially available S. aureus vaccine have shown limited efficacy under field conditions, mainly due to the paucity of information regarding relevant antigens which will induce a broad spectrum immunization. In the present paper the attempt to develop a new vaccine designated MASTIVAC I is described. MASTIVAC I is composed of three strains of S. aureus namely: VLVL8407; ZO3984 and BS449 which were isolated from clinical and sub-clinical cases of bovine mastitis. A mouse model was used to evaluate the S. aureus specific antibody production and protection of mice against virulent S. aureus strains. The results obtained showed that this vaccine exhibits a broad spectrum of antigenic and immunogenic properties that protects mice from homologues and hetrologous S. aureus challenge.

Animals↗
PubMed1998

Flow cytometric measurement of neutrophil alkaline phosphatase before and during initiation of an induced Escherichia coli mastitis in cattle.

In 12 healthy cows, neutrophil alkaline phosphatase (NAP) activity was measured by flow cytometer before and during an experimentally induced Escherichia coli mastitis, to study the role and increase of NAP in Gram-negative bacterial infections. Percentage of neutrophils containing alkaline phosphatase and intensity of NAP activity were measured. Preinfection percentage of neutrophils with enzyme activity varied between 64.0% and 84.4% and the intensity of enzyme activity was low in all cows. After induction of infection, percentage of neutrophils with enzyme activity showed a significant decrease on day 1 followed by an significant increase on day 3. NAP intensity increased significantly on the second and third day after infection. This increase of intensity was significantly, positively correlated with the severity of infection. From this study we may conclude that variation in susceptibility to E. coli mastitis could not be explained by preinfection NAP levels. The post-infection increase of NAP activity, that was found following an induced infection was more a result of increased enzyme intensity per neutrophil, then from an increase of percentage neutrophils with enzyme activity. Furthermore, a strong correlation was found between NAP intensity and severity of inflammation. There was evidence that the more severely diseased animals showed stronger NAP intensity increase.

Alkaline Phosphatase↗