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Streptococcus uberis (S. uberis) is an environmentally important pathogenic bacterium and is the main pathogenic microorganism responsible for mastitis, which causes significant economic losses worldwide. Currently, there is no particularly effective treatment other than antibiotic therapy. Angiotensin-converting enzyme 2 (ACE2) plays an anti-inflammatory as well as an anti-injury role in numerous inflammatory diseases. Therefore, this study aimed to assess the hypothesis that S. uberis-induced mammary epithelial cells injury associated with ACE2, angiotensin II (Ang II) as well as angiotensin 1-7 (Ang-(1-7)) imbalance and that overexpression of ACE2 can repair S. uberis-induced mammary epithelial cells injury. We observed that the expression level of ACE2 was significantly downregulated after treatment of EpH4-Ev cells with S. uberis. Next, this assay verified the role of ACE2 in S. uberis-induced inflammatory injury in EpH4-Ev cells by overexpressing the ACE2 gene as well as its silencing. The results showed that overexpression of the ACE2 gene significantly activated the interleukin-10/signal transducer and activator of transcription 3/suppressors-of-cytokine-signaling 3 (IL-10/STAT3/SOCS3) pathway, thereby inhibiting the nuclear factor-κB (NF-κB) as well as pyroptosis pathways. Furthermore, overexpression of the ACE2 gene reversed the downregulation of zonula occludens 1 (ZO-1), Occludin, Claudin-1, and Claudin-2 caused by S. uberis, suggesting that ACE2 could promote to repair the blood-milk barrier. However, siRNA silencing of the ACE2 gene produced the opposite effect. These results suggest that ACE2 ameliorates S. uberis-induced mammary epithelial cells injury. AVAILABILITY OF DATA: All data generated or analyzed during this study are included within the article and its additional information file., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The presence of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in raw milk is a challenge for veterinarians and public health professionals. In this study, we investigated the presence and clonality of S. aureus and MRSA in milk of individual dairy goats with subclinical mastitis reared under the low-input farming system in Greece and determined the isolates' enterotoxin gene carriage and their ability to form biofilms. S. aureus was isolated from 162 out of the 559 milk samples examined (29 %) and one isolate per S. aureus-positive sample was further characterized. S. aureus isolates were very closely related even among farms of distant geographical regions. Nine S. aureus isolates carried a functional mecA gene and were classified as MRSA. The S. aureus protein A (spa) typing in the MRSA isolates showed that four belonged to spa type t127 (44.4 %), three to t2049 (33.3 %) and two to t7947 (22.2 %). The spa type t7947 is reported for the first time in Greece. The MRSA isolates originated from two very distantly located farms, one located in the island of Skopelos and the other in Central Macedonia. Four of the MRSA isolates carried the staphylococcal enterotoxin genes sea or sec. Most of the isolates (92 % of S. aureus and 77.8 % of the MRSA) possessed moderate or weak biofilm-formation ability. Raw milk from low-input goat herds may serve as a potential vector of antimicrobial-resistant S. aureus to raw-milk consumers., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Although coagulase-negative staphylococci are the primary aetiological agents of subclinical mastitis in ewes, there is little information regarding vaccination against that infection. The objective of this study was to evaluate the efficacy of a vaccine against staphylococcal mastitis in ewes under experimental conditions. The antigen in the vaccine is based on a bacterin of Staphylococcus aureus strain, expressing the exopolysaccharide poly-N-acetylglucosamine (PNAG), which is involved in biofilm formation by these bacteria. Ewes in groups A (n = 17) or B (n = 6) were given an initial vaccination 5 weeks before expected lambing, followed by a repeat administration 21 days later. Ewes in groups C (n = 8) or D (n = 6) were unvaccinated controls. Ewes in group A (n = 17) or C (n = 8) were challenged with a biofilm-forming S. chromogenes; animals in subgroups A1 or C1 were challenged on the 10th and those in A2 or C2 on the 50th day after lambing. Ewes in groups B or D were uninoculated controls. Clinical examinations of ewes, ultrasonographic examinations of udder, milk yield measurements, blood sampling for detection of anti-PNAG specific antibodies and milk sample collection for bacteriological and cytological examinations were performed up to 52nd day post-challenge. Finally, biopsies were performed for mammary tissue collection for histopathological examination. Among group A ewes, 29% developed systemic signs and 59% signs in the inoculated gland; the respective figures for group C were 50% and 100% (P =  0.040 for mammary signs). The median total clinical score was 2.0 for A and 5.5 for C ewes (P =  0.025). For A, but not for C, clinical scores decreased progressively during the study (P =  0.018 and P =  0.47, respectively). The duration of mastitis was shorter in A (4 days) than in C (17.5 days) ewes (P =  0.022). Bacterial counts were lower in milk samples from A than from C ewes, for samples collected from the inoculated and the uninoculated (P <  0.01) mammary glands of these ewes. Somatic cell counts in samples from inoculated and uninoculated mammary glands of A ewes were higher than in samples of C ewes (P <  0.02). There were differences for gray-scale evaluations during ultrasonographic examination and for milk yield measurements between groups (P <  0.01). Median bacterial counts in tissue samples from A ewes (0 cfu g -1 ) were lower than in ones from C (6.5 cfu g -1 ) ewes (P =  0.041). The median score for histopathological findings in tissue samples from inoculated glands of A was lower than that for C ewes: 1 versus 2 (P =  0.014). It is concluded that mastitis was less severe in vaccinated animals, as indicated by a wide array of measures., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Aerococcus viridans, a firmicutes bacteria widespread in the environment, is increasingly isolated from humans and animals, especially cows with mastitis. However, its pathogenicity in the bovine mammary gland is unclear. The objective was to explore pathogenic potential of putative virulent and avirulent A. viridans in murine systemic and intramammary infection and mechanistically in cultured bovine mammary epithelial cells (bMECs). Virulence of 9 strains of A. viridans, isolated from subclinical cases of mastitis, was tested for their ability to kill mice when systemically inoculated. Two A. viridans strains, causing highest and lowest survival rate in mice, were selected further as putative avirulent and virulent strains, respectively. Staphylococcus aureus N305 was used as a positive control. After intramammary inoculation, the virulent strain survived and replicated in the murine mammary gland for 9 d, whereas the avirulent strain was eliminated within 3 d. The virulent strain induced a robust inflammatory reaction in the mammary gland, characterized by acute histopathological changes, increased myeloperoxidase activity and higher expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) compared to the avirulent strain. The virulent strain produced CAMP factor and exhibited strong cytotoxic effects (LDH release) and adhering and invasive abilities in contact with bMECs. Adhesion and invasion of virulent strain to bMECs was further confirmed by scanning and transmission electron microscopy; there was severe damage, including cytomembrane disruption, swollen mitochondria and loss of organelles. In conclusion, the putative virulent strain of A. viridans activated a strong neutrophil-based inflammatory response in the mammary gland, attributed to its ability to adhere to and invade mammary epithelium., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The objectives of this study were to 1) correlate pre-partum teat skin colonization with non-aureus staphylococcal (NAS) intramammary infection (IMI) in early lactation, and 2) evaluate infection dynamics of subclinical NAS IMI in goats during lactation in a small ruminant lentivirus-infected herd. Pre-partum teat skin swabs (41 goats, 82 halves) and post-partum half-level milk samples (106 goats, 203 halves) were collected at various intervals starting at ≤10 days in milk (DIM) until ≥120 DIM. Teat skin colonization and IMI were defined by culture and strain-typing. The association between the pre-kidding udder-half teat skin sample status and early lactation IMI status for a given species was investigated using McNemar's exact test or logistic regression. Time to IMI elimination and time to new IMI were evaluated by discrete-time survival analysis. Halves with S. caprae isolated from teat skin prior to kidding had increased odds of S. caprae IMI ≤ 10 DIM. Time to IMI elimination varied as a function of NAS species. Intramammary infections detected >10 DIM had a higher hazard of elimination (hazard ratio [HR] 5.6, 95% CI 2.8-11.2) than IMI detected ≤10 DIM. The presence of an IMI in the contralateral half was associated with a higher hazard of new IMI (HR 2.1, 95% CI 1.3-3.4) in an uninfected half. Further studies on interventional strategies targeting early IMI and IMI caused by persistent species are warranted., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective was to recognise areas potentially of high risk for increased frequency of subclinical mastitis in ewes. Milk samples were collected, for bacteriological and cytological examination, from 2198 clinically healthy ewes in 111 farms in all administrative regions of Greece. Data on farms were located in the field using hand-held Global Positioning System Garmin units. Collected data were analysed by an Ecological Niche Model under the framework of a geographic information system. Two separate analyses were performed: one for subclinical mastitis independently of causal agent (prevalence in population sampled: 0.260) and one for subclinical mastitis caused specifically by slime-producing staphylococci (prevalence in population sampled: 0.153). A model was constructed in which sheep farms were divided into two clusters, according to prevalence of subclinical mastitis: farms in the upper three quartiles of prevalence were used as occurrence points for the Ecological niche modelling procedure ('infected farms'); farms in the lower quartile of prevalence within each category were (pseudo)negative points. Significant differences in environmental parametres prevailing in locations of farms into the study, were identified for up to 13 parametres between locations of farms according to management system applied in farms. When farms in each management system were considered separately, differences became evident between farms in each management system, as well as between the two infections. The factor with the highest relative contribution in the analyses was the distance from other sheep farms; other factors also of importance in the predictive models were the altitude, the maximum temperature of warmest month and the total precipitation of driest month. Verification of the model revealed that ≥ 0.760 of infected farms' were located in areas predicted as high risk for prevalence of subclinical mastitis or slime staphylococcal subclinical mastitis. The paper describes for the first time potential association of mastitis with environmental factors and presents predictive models for mastitis in ewes taking into account environmental parametres., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mammary epithelial cells (MECs) play an important role in debating Streptococcus uberis (S. uberis) infection. Toll like receptor (TLR) engagement leads to the recruitment of phosphatidylinositol 3 kinases (PI3K). In order to investigate the relationship of TLRs/NF-κB and PI3K/Akt/mTOR signaling pathways in S. uberis infection in MECs, we challenged MECs (EpH4-Ev) with S. uberis 0140 J and quantified the adaptor molecules in these two signaling pathways, as-well-as proinflammatory cytokines and cell damage. The results indicate that the host's responses to virulent S. uberis infection are complex. In MECs, both TLR2 and TLR4 are detecting S. uberis infection and TLR2 is the principal receptor. The role of the PI3K/Akt/mTOR pathway in inflammatory regulation is independent of the activation of TLRs/NF-κB. Cross-talk between PI3K/Akt/mTOR and TLRs/NF-κB signaling pathways promote inflammation. This study increases our understanding of the molecular defense mechanisms of MECs in S. uberis mastitis, and provides theoretical support for the prevention of this disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hitherto, research work in slime production from staphylococcal strains of mastitis origin has focused in laboratory properties of these organisms. Objective of present work was to study subclinical mastitis in sheep, caused specifically by slime-producing staphylococci: to investigate its frequency and to identify potential factors playing a role therein. Slime production was evaluated in 708 staphylococcal isolates recovered from cases of subclinical mastitis in a field study in 2198 ewes performed in an extensive countrywide field investigation across Greece. Isolates were studied by means of microbiological and molecular methods. Of these strains, 262 were characterised as slime-producing, 227 as weak slime-producing and 219 as non slime-producing. Most frequently detected genes were eno and icaB; Staphylococcus aureus possessed more genes than coagulase-negative strains; greater number of genes was detected in slime-producing than in weak slime-producing or non-slime-producing strains. Subclinical mastitis caused specifically by slime-producing staphylococci was detected in 337 ewes: prevalence in population sampled was 0.153. A multivariable mixed-effects model revealed that milking mode (highest prevalence in hand-milked flocks) and flock management system (highest prevalence in semi-intensive flocks) were the two factors associated with increased prevalence of mastitis in flocks. The results confirmed the significance of slime producing staphylococcal strains of importance in the aetiology of subclinical mastitis of sheep. Hand-milking was identified as the most important factor predisposing to that infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Contagious agalactia is a mycoplasmosis that affects small ruminants, is associated with loss of milk production and high morbidity rates, and is highly deleterious to dairy industries. The etiological agents are four mycoplasma (sub)species, of which the relative importance depends on the countries and the animal host. Tetracyclines are non-expensive, broad-spectrum antimicrobials and are often used to control mastitis in dairy herds. However, the in vitro efficiency of tetracyclines against each of the etiological agents of contagious agalactia has been poorly assessed. The aims of this study were i) to compare the tetracycline susceptibilities of various field isolates, belonging to different mycoplasma (sub)species and subtypes, collected over the years from different clinical contexts in France or Spain, and ii) to investigate the molecular mechanisms behind the decreased susceptibility of some isolates to tetracyclines. The Minimum Inhibitory Concentrations (MICs) of tetracyclines were determined in vitro on a set of 120 isolates. Statistical analyses were run to define the significance of any observed differences in MICs distribution. As mutations in the genes encoding the tetracycline targets (rrs loci) are most often associated with increased tetracycline MICs in animal mycoplasmas, these genes were sequenced. The loss of susceptibility to tetracyclines after year 2010 is not significant and recent MICs are higher in M. agalactiae, especially isolates from mastitis cases, than in other etiological agents of contagious agalactia. The observed increases in MICs were not always associated with mutations in the rrs alleles which suggests the existence of other resistance mechanisms yet to be deciphered., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Trueperella pyogenes is an opportunistic pathogen of many animal species. It causes economic losses worldwide, through mastitis, metritis and mainly endometritis in dairy cows. The ability of this bacterium to form biofilms is implicated in chronic infections through hampering immune system recognition and antibiotic penetration. Since it is difficult to eradicate T. pyogenes infections with antibiotics, phage therapy presents itself as a non-toxic, effective and economically viable alternative. The present study evaluated the use of the bacteriophage vB&#95;EcoM-UFV13 (UFV13) in the prevention of T. pyogenes biofilm development. Based upon two different approaches (crystal violet and sessile cell counting) we observed that only a multiplicity of infection (MOI) of 10 showed a statistically significant reduction in biofilm formation. Although the exact mechanisms of biofilm disruption and cell-adhesion inhibition have not been determined, genome sequence analysis of the Escherichia phage UFV13 revealed a repertoire of virion-associated peptidoglycan hydrolases (VAPGHs). The present study presents new findings regarding the disruption of biofilm formation of a Gram-positive bacterium. Subsequent transcriptomic and proteomic research will help us to understand the exact interaction mechanisms between UFV13 and T. pyogenes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this research, 330 Staphylococcus aureus isolates, collected in Sardinia (Italy) in the period 1986-2015 from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines, were analyzed. Susceptibility to 12 antimicrobial agents was tested by disk diffusion, according to CLSI recommendations. Resistance genes were detected by PCR assays. The most of isolates (85.2%) were susceptible to all antimicrobials tested, suggesting that did not exist change of resistance over time. Two isolates were multidrug-resistant (MDR), one of them (isolate 1496) showed resistance to seven antibiotics including oxacillin and erythromycin. This MRSA harboured SCCmec type IV and the erm(C) gene. Isolates were characterized by spa typing and MLST. Isolates belonged to 29 spa types: t1773 (n=186), t2678 (n=53), t7754 (n=14), t1532 (n=5), t524 (n=5) and t6060 (n=4) were the most frequent spa types found in Sardinia. The majority of ovine isolates (t1773, t7754 and t1532) was grouped in MLST CC130 (n=205) followed by CC133 (n=57). MRSA 1496 was classified as t3896, ST1 and CC1, a clonal complex common in human and also reported in cattle and pig. This study suggests that the CC130/ST700/t1773 is the prevalent S. aureus lineage associated with ovine mastitis in Sardinia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is one of the most frequently isolated major pathogens from intramammary infections (IMI) worldwide. The mechanisms by which S. aureus IMI are established and maintained in dairy cows involve both bacterial escape strategies and modulation of the host immune response. Moreover, it was shown that different S. aureus strains have varying effects on the immune response. The aim of this study was to investigate the immune response in a mouse mastitis model of two S. aureus strains isolated from bovine IMI with different clinical manifestation (persistent-P or non-persistent-NP), phenotypic and genotypic profile. Both strains were capable of establishing an IMI after 264h post inoculation (pi). Strain A (NP) showed a more aggressive behaviour than strain B (P) at early stages of IMI, while strain B multiplied initially at a lower rate but increased its replication capacity from 120h pi to the end of the study (264h pi). Strain A triggered a stronger initial inflammatory response compared with strain B inducing higher gene and protein expression of TLR2, NF-κB activation and higher gene expression of IL-1α at initial stage of IMI (6-12h pi) but inducing extensive mammary tissue damage. Immune cells response was different for each S. aureus strain throughout the course of infection, showing mammary glands inoculated with strain A greater initial immune cells stimulation compared with strain B and then a second immune cells stimulation (from 120 to 264h pi) represented by monocytes-macrophages, T and B lymphocytes, mainly stimulated by strain B, consistent with inflammatory process becoming chronic. Strain-specific pathogenicity observed underscores the importance of pathogen factors in the progression of the infectious process. These results contribute to increase the available information on host-pathogen interaction and point out for the need of further research to expand the knowledge about these interactions for developing new strategies to intervene in the IMI progress., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aetiology and epidemiology of outbreaks of clinical mastitis in sheep under extensive pastoral conditions are incompletely understood. The objective of this study was to conduct a detailed investigation of a clinical mastitis outbreak that affected more than 10% of 230 at-risk ewes on a sheep and grain producing property in south east Australia during drought conditions in 2009. Milk samples were collected aseptically from all affected ewes and plated on sheep blood agar for bacterial identification. M. haemolytica was isolated from 80% of the samples that yielded cultivable microorganisms and thus was the main microorganism responsible for the outbreak. Analysis of the restriction endonuclease cleavage patterns of the isolates using pulsed field gel electrophoresis revealed some evidence of clonality, suggesting the possibility of horizontal transmission, but there was also considerable diversity between the clusters of closely related isolates. Multilocus sequence typing of the M. haemolytica isolates revealed most of the isolates belonged to ST1 with no association between the PFGE and MLST fingerprints of the isolates. Resistance to neomycin, streptomycin and sulphafurazole was detected in some of the isolates, but they were all susceptible to penicillin, ampicillin, ceftiofur, amoxycillin/clavulanic acid, ciprofloxacin, tetracycline, erythromycin and trimethoprim. This is the first published record of a comparison of the strains of M. haemolytica involved in a clinical mastitis outbreak in sheep and demonstrates the importance of this pathogen in sheep production systems, particularly during adverse climatic conditions and increased stocking rate., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Staphylococcus aureus (S. aureus) is a major pathogen involved in ruminant mastitis and present worldwide. Clinical signs of S. aureus mastitis vary considerably and are largely dependent on strain-specific factors. A comparison of two S. aureus strains that reproducibly induced either severe (O11) or mild (O46) mastitis in ewes revealed that the transcriptional regulator sigS was mutated in O46 (Le Maréchal et al., 2011. PLoS One. 6 (11) e27354. doi:10.1371/journal.pone.0027354). In the present paper, we analysed the sigS sequence in 18 other S. aureus strains isolated from goat or ewe mastitis and found a 4-bp deletion similar to that of the O46 sigS gene in three strains associated with subclinical ewe mastitis. This sigS gene was disrupted in strain O11 (O11ΔsigS), so our aim was to investigate its involvement in the severity of infections in the context of mastitis. The wild type (wt) and mutant strains were then characterized in vitro to determine the involvement of sigS in the response S. aureus under various stress conditions, and assess its influence on the cytotoxicity of the pathogen, its invasive capacity and biofilm formation. The strains were compared in vivo in an experimental mouse mastitis model in which clinical signs and cytokine production were evaluated at 24h post-infection. While no significant differences in the effect on bacterial growth between O11 and O11ΔsigS were observed either in vitro or in vivo, a significantly weaker in vivo production of interleukin (IL)-1α, IL-1β, and Tumor Necrosis Factor (TNF)-α was measured in the mammary glands infected with the mutant strain, suggesting that infection with O11ΔsigS induced an attenuated local innate immune response. These results suggest an impact of sigS disruption on S. aureus pathogenesis in a ruminant mastitis context. This disruption is probably involved in, and may partly explain, the milder symptoms previously observed in S. aureus O46-induced mastitis in ewes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lamb suckling has been suggested to be an important way of infecting a ewe's udder with different bacteria, including Mannheimia haemolytica. To test the potential role of lambs in transferring Mannheimia species to the ewe's udder, the restriction endonuclease cleavage patterns of isolates obtained from nasopharyngeal swabs were compared with those obtained from cases of mastitis. Sterile cotton swabs were used to collect nasopharyngeal samples from 50 ewes and 36 lambs from three flocks. M. haemolytica and Mannheimia glucosida as well as haemolytic Mannheimia ruminalis-like organisms were detected in the upper respiratory tract of lambs and ewes. Comparison of the restriction endonuclease cleavage patterns of the isolates suggested that the M. haemolytica isolates obtained from different milk samples from ewes with mastitis were more clonal than those obtained from the nasal swabs. However, some nasal isolates within both Mannheimia species had restriction endonuclease cleavage patterns identical to those obtained from milk samples from ewes with mastitis, indicating that lambs may have a role in transferring these organisms to the udder. More clonality was observed between the M. glucosida isolates than between M. haemolytica isolates., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bacterial mastitis is a significant welfare and financial problem in sheep flocks. This paper reviews the recently published literature, including publications that highlight the significance and virulence factors of the causal agents, especially Staphylococcus aureus and Mannheimia haemolytica, the primary causes of the disease. Research has also contributed to the understanding of risk factors, including genetic susceptibility of animals to infections, supporting future strategies for sustainable disease control. Pathogenetic mechanisms, including the role of the local defenses in the teat, have also been described and can assist formulation of strategies that induce local immune responses in the teat of ewes. Further to well-established diagnostic techniques, i.e., bacteriological tests and somatic cell counting, advanced methodologies, e.g., proteomics technologies, will likely contribute to more rapid and accurate diagnostics, in turn enhancing mastitis control efforts., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is one of the most relevant pathogens causing clinical and subclinical, chronic mastitis in dairy animals. Routinely, mastitis pathogens are isolated and classified to genus or species level, and regarded as single entities. However, S. aureus includes a broad range of genotypes with distinct pathogenic and epidemiologic characteristics. The objective of the present study was to assess the host-specificity of S. aureus causing mastitis in dairy animals, based on phylogenetic and genotypic characterization as well as the presence of virulence and antimicrobial resistance genes in the pathogen genome. S. aureus isolates from mastitis in cows, sheep and goats in Israel, and from cows in Germany, the USA and Italy, were compared by the following methods: a. Bayesian phylogenetic comparison of sequences of genes nuc, coa, lukF and clfA, b. genotyping by spa and agr typing, and assignment to MLST Clonal Complexes (MLST CC), and c. the presence of a broad array of virulence and antimicrobial resistance genes. Overall, phylogenetic, virulence and genotyping approaches agreed with each other. Cow isolates could be differentiated from sheep and goat isolates with all three methods, with different resolution. In two phylogenetic clusters, segregation was found also between cow isolates from Israel and abroad. Sheep and goats' isolates showed less variability than isolates from cows in all methods used. In conclusion, different S. aureus lineages are associated to cows in contrast to goats and sheep, suggesting co-evolution between pathogen and host species. Modern diagnostics approaches should aim to explore molecular data for a better understanding and cost-effective management of mastitis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bacterial infections causing mastitis in sheep can result in severe economic losses for farmers. A large survey of milk samples from ewes with mastitis in Sardinia, Italy, indicated an increasing prevalence of Pseudomonas aeruginosa infections. It has been shown previously that during chronic, biofilm-associated infections P. aeruginosa populations diversify. We report the phenotypic and genomic characterisation of two clonal P. aeruginosa isolates (PSE305 and PSE306) from a mastitis infection outbreak, representing distinct colony morphology variants. In addition to pigment production, PSE305 and PSE306 differed in phenotypic characteristics including biofilm formation, utilisation of various carbon and nitrogen sources, twitching motility. We found higher levels of expression of genes associated with biofilm formation (pelB) and twitching motility (flgD) in PSE305, compared to the biofilm and twitching-defective PSE306. Comparative genomics analysis revealed single nucleotide polymorphisms (SNPs) and minor insertion/deletion variations between PSE305 and PSE306, including a SNP mutation in the pilP gene of PSE306. By introducing a wild-type pilP gene we were able to partially complement the defective twitching motility of PSE306. There were also three larger regions of difference between the two genomes, indicating genomic instability. Hence, we have demonstrated that P. aeruginosa population divergence can occur during an outbreak of mastitis, leading to significant variations in phenotype and genotype, and resembling the behaviour of P. aeruginosa during chronic biofilm-associated infections., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Species within the genus Mannheimia are among the most important causes of ovine mastitis. Isolates of these species can express leukotoxin A (LktA), a primary virulence factor of these bacteria. To examine the significance of variation in the LktA, the sequences of the lktA genes in a panel of isolates from cases of ovine mastitis were compared. The cross-neutralising capacities of rat antisera raised against LktA of one Mannheimia glucosida, one haemolytic Mannheimia ruminalis, and two Mannheimia haemolytica isolates were also examined to assess the effect that variation in the lktA gene can have on protective immunity against leukotoxins with differing sequences. The lktA nucleotide distance between the M. haemolytica isolates was greater than between the M. glucosida isolates, with the M. haemolytica isolates divisible into two groups based on their lktA sequences. Comparison of the topology of phylogenetic trees of 16S rDNA and lktA sequences revealed differences in the relationships between some isolates, suggesting horizontal gene transfer. Cross neutralisation data obtained with monospecific anti-LktA rat sera were used to derive antigenic similarity coefficients for LktA from the four Mannheimia species isolates. Similarity coefficients indicated that LktA of the two M. haemolytica isolates were least similar, while LktA from M. glucosida was most similar to those for one of the M. haemolytica isolates and the haemolytic M. ruminalis isolate. The results suggested that vaccination with the M. glucosida leukotoxin would generate the greatest cross-protection against ovine mastitis caused by Mannheimia species with these alleles., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Despite being one of the main pathogens involved in ruminant mastitis, little is known about what proteins Staphylococcus aureus does express, in vivo, during the infection. Here, two S. aureus strains were isolated from curds formed within the udder of two ewes suffering from gangrenous mastitis. Protein samples were prepared from cell fractions and were analyzed using 1D-LC MS/MS. Results were compared to 1D-LC MS/MS analysis of the same S. aureus strains grown in ewe milk. A total of 365 proteins were identified. Most of them were related to cellular metabolism, cellular division and stress response. Half of the proteins were found in both conditions but a substantial number were specifically found in in vivo conditions and gave indications about the active metabolic status and the stresses encountered by S. aureus within the cistern during a gangrenous mastitis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Streptococcus uberis is a major environmental mastitis-causing pathogen. The infections are predominantly subclinical and are frequently undetected and untreated for extended periods of time. More information about the pathogenesis of S. uberis mastitis would be useful. To our knowledge, no experimental studies into the mastitis pathogenesis caused by S. uberis have been described in lactating goats. The aim of this study was to reproduce an experimentally induced S. uberis subclinical mastitis in lactating goats aimed to evaluate the inflammatory response, dynamics of infection and the pathological findings within the first hours of intramammary inoculation with S. uberis. Six Saanen goats in mid-lactation were inoculated with 1.7 × 10(8)cfu of S. uberis. Bacterial growth peaked in milk from challenged right mammary halves (RMH) at 4h PI. Shedding of viable bacteria showed a marked decrease at 20 h PI. Mean somatic cell counts in milk from the RMH peaked at 20 h PI. Inoculation with S. uberis was followed by a decrease in the mean total number of leukocytes. Signs and systemic symptoms were not evoked by intramammary inoculation. S. uberis could be isolated in tissue from all RMH. Histological examination of specimens of the RMH and lymph nodes of the goats showed an increased inflammatory response throughout the experiment. The histological findings correlated with the immunohistochemical detection of S. uberis in RMH. In conclusion, the experimental inoculation of S. uberis in lactating goats is capable of eliciting an inflammatory response and causing pathological changes, resulting in a subclinical mastitis. This investigation shows that goat might to represent a valuable model for the study of the mastitis pathogenesis caused by S. uberis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mannheimia haemolytica is known to be an important cause of intramammary infection in sheep. It usually causes severe clinical mastitis, followed by toxaemia and gangrenous necrosis of the udder. However there are limited data available on the epidemiology and pathogenesis of mastitis associated with Mannheimia species. These organisms can be more significant as a cause of mastitis than Staphylococcus aureus in some flocks. Some data suggest the possibility of horizontal transmission of Mannheimia species between ewes via lamb sucking. There is no vaccine available for prevention, and the sudden onset of mastitis and its peracute nature renders most treatments unsuccessful. This review examines the significance of the species within this genus in sheep mastitis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Coliform mastitis (CM) is not only a serious economical and animal welfare touching problem in dairy cattle, but also in sows after farrowing. Due to this disease, the essential adequate supply with colostrum for the growth and the health of the piglets is not ensured. Besides other influencing factors, Escherichia (E.) coli is of great importance as a causative agent of this multifactorial disease. In this study, E. coli isolates from milk samples of healthy and CM-affected sows were examined for the presence of virulence genes associated with extraintestinal E. coli strains, enterotoxigenic E. coli and other pathogenic E. coli. The isolated E. coli harbored mainly virulence genes of extraintestinal E. coli strains (especially fimC, ompA, traT, hra, kpsMTII, iroN). The virulence gene spectrum for both samples from CM-affected and healthy sows did not differ significantly. Particular virulence gene profiles of E. coli isolates from diseased sows were not detected. This study provides novel insights into the role of E. coli in association with mastitis in sows since it is the first time E. coli isolates from CM-affected sows' milk were analysed for virulence genes. Because there were no differences in the prevalence of E. coli and their virulence-associated genes between healthy and diseased sows, other causative factors seem to have greater influence on the pathogenesis of porcine CM., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective of the work was to study whether Mannheimia haemolytica may be transmitted from the mouth of the lambs into the teat of the dam during sucking. We compared bacterial populations within the teat duct and milk of ewes immediately before and immediately after sucking by the lambs. Tonsils of lambs of the ewes were swabbed. M. haemolytica strain DAG21T recovered from a teat duct of a ewe was compared to strain DAG21R recovered from the tonsils of her lamb by using 16s rRNA sequencing. We used those two isolates and another one of known pathogenicity, for challenging ewes: (i) 2 mm deep into healthy teats, (ii) 2 mm deep into teats with chapping lesions or (iii) into the cistern of healthy mammary glands. Of samples collected before suckling, 20/792 were bacteriologically positive, and of those after, 50/792 were bacteriologically positive (P<0.001); in 37 cases, a negative sample became positive. One M. haemolytica (DAG21T) was recovered after suckling from a teat duct of a ewe. The organism was isolated from 57/90 tonsillar swabs from lambs. Risk of infection of ewe' teats was 0.004 throughout lactation, being greatest (0.021) during the 3rd week of lactation. The 16s rRNA sequences of strains DAG21T and DAG21R were identical over 1450 nucleotides. Phylogenetic analysis showed that the two isolates clustered together with isolates of M. haemolytica. Organism deposition into healthy teats caused subclinical mastitis; deposition into teats with lesions or directly into mammary gland caused clinical mastitis. When results of inoculation of the three strains were compared between them, statistical significance was always P>0.9. Results provide clear evidence that suckling by lambs can lead to transmission of M. haemolytica into the teats of the ewes; the bacteria have the potential to cause mastitis if circumstances are favourable., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Intramammary infections are a serious problem for dairy sheep farms, and Staphylococcus epidermidis is one of the main etiological agents of ovine mastitis. In this work, 131 S. epidermidis isolates, collected from 2201 dairy Sarda sheep belonging to 14 flocks with high somatic cell count scores, were studied. The flocks were located in diverse geographical areas of Sardinia, Italy. The aim of study was to assess the susceptibility of isolates to 13 antimicrobial agents, many of which are frequently used in mastitis therapy. Oxacillin was used for detecting methicillin-resistant S. epidermidis (MRSE) by disk diffusion test. Thirty-eight percent of the isolates (n=50) were resistant to penicillin, 7.6% (n=10) were resistant to tetracycline, and 2.3% (n=3) were resistant to both penicillin and tetracycline (PTRSE). Two isolates were resistant to five antimicrobials including methicillin. Analysis of staphylococcal cassette chromosome mec (SCCmec) elements showed that both MRSE isolates harbored SCCmec type IVa. Based on pulsed-field gel electrophoresis (PFGE) typing by SmaI macrorestriction, S. epidermidis isolates were grouped into four clusters at 75% similarity level. The two multi-drug resistant MRSE isolates displayed distinct PFGE patters. This study indicates that S. epidermidis isolates from sheep milk samples may accumulate resistance markers for different antimicrobial agents. Furthermore, the occurrence of PTRSE and MRSE suggests to adopt adequate hygienic measures when handling animals with intramammary infections, in order to prevent spreading PTRSE and MRSE strains to humans through direct contact and/or consumption of contaminated food., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis in sheep and goats. In this study, 226 CNS isolates were collected from 2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All isolates were subjected to identification with the API Staph ID test, and then to the amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was subjected to restriction fragment length polymorphism analysis with the restriction endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting with the restriction endonucleases RsaI, PstI and AluI. When PCR-RFLP patterns of CNS isolates were different from those of their reference strains, gap gene amplicons were sequenced for definitive identification. The API Staph ID test, in alternative to the genotypic identification method, produced considerably different results in terms of species identified within each group. Using the PCR-RFLP assay, most of the isolates clustered together with the Staphylococcus epidermidis type strain (131, corresponding to 57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30, corresponding to 13.2%). In conclusion, the PCR-RFLP assay of 16S rRNA and gap genes is a more reliable and reproducible method than the API Staph ID test for the identification of CNS causing sheep mastitis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.

The objectives of the work were to study the features of experimentally induced canine mastitis and to present hypotheses regarding the pathogenesis of the disease. The right caudal abdominal mammary gland of six bitches was inoculated on day 8 after whelping with Staphylococcus intermedius to induce mastitis; adjacent mammary glands were used as controls. Clinical examination, bacteriological and cytological (whiteside test, Giemsa) examination of mammary secretion, as well as haematological tests were performed from 5 days before until 34 days after challenge. Mastectomy was sequentially performed 1, 2, 4, 18, 26 and 34 days after challenge in each of the bitches, in order to carry out a pathological examination of mammary glands. All animals developed clinical mastitis: challenged glands became painful, hot, enlarged and oedematous; secretion was brownish, purulent, with flakes or clots, subsequently becoming yellowish and thick. Staphylococci were isolated from all inoculated glands (up to 22 days). WST was positive in 41/46 samples from inoculated glands and 66/138 samples from control glands; neutrophils predominated during the acute stage. Blood leukocyte counts increased, whilst platelet counts decreased. Gross pathological findings initially included congestion, purulent discharge and subcutaneous oedema; then abscesses, brownish areas and size decrease were seen. Salient histopathological features were initially neutrophilic infiltration, haemorrhages, destruction of mammary epithelial cells and alveoli, and then infiltration by lymphocytes, shrunken alveoli, loss of glandular architecture and fibrous tissue proliferation. We conclude that in bitches, intrammamary inoculation of Staphylococcus intermedius can induce clinical mastitis, followed by subclinical disease. The disorder is characterized by bacterial isolation and leukocyte influx in challenged glands, by leukocyte presence in adjacent mammary glands, by increased blood leukocyte counts and by destruction of mammary parenchyma.

Three hundred and eighty-four Staphylococcus aureus isolates obtained from mammary secretions from 332 ewes kept for meat production were typed by pulsed-field gel electrophoresis (PFGE). The ewes were from 242 flocks located in 13 counties distributed in four regions of Norway. In total, 64 different pulsotypes were identified, 31 of these were represented by a single isolate. Fifty-nine percent of the isolates belonged to one of five closely related pulsotypes. This group of pulsotypes occurred in all the counties. Although widely disseminated, the proportions of the prevalent and closely related pulsotypes differed between the regions. Nine pulsotypes were unique to single regions but the number of isolates belonging to each of these pulsotypes was low. Resistance to penicillin was found in only 3 of the 384 S. aureus isolates. These represented three different single banding patterns, not related to any of the prevalent pulsotypes found.

Intramammary infection in seven commercial goat herds was studied using premilking and postmilking samples for purposes as bacteriological diagnosis. Using a positive result on both premilking and postmilking samples as the definitive diagnosis, we compared the efficacy of single samples collected either premilking or postmilking. With this aim, 2268 bacteriological culture results were compared. The kappa values (0.60) showed moderate agreement between the two samples. Specificity and positive predictive value were higher for postmilking samples than for premilking samples. Specificity of postmilking samples was 99.4% for coagulase-negative staphylococci, 99.9% for Gram-negative bacilli, 100% for streptococci, 99.9% for corynebacteria and 100% for mixed cultures. Premilking samples specificity was 96.6, 99.5, 99.7, 99.8 and 99.8%, respectively. False positive diagnoses were more frequent for premilking samples. The results suggest that postmilking samples should be used to diagnosis of goat intramammary infection.

Three techniques for the diagnosis of mammary aspergillosis in ewes were compared: indirect ELISA to detect the level of anti-Aspergillus IgG in serum, determination of galactomannan (Platelia procedure), and detection of DNA of Aspergillus in serum by a nested PCR. Twenty sera from proven cases of aspergillosis in ewes were positive using ELISA (100%), 80% were positive using PCR, but only 55% were positive using Platelia. All 20 control sera were negative using ELISA and PCR, whereas using Platelia methodology one was positive and the other doubtful. The detection of antibody by ELISA in sera is therefore a reliable criterion for the diagnosis of mammary aspergillosis in ewes. Platelia showed the same deficiencies reported in humans, with the appearance of false positives and negatives. The use of PCR was promising and might have valuable application in human medicine.

We investigated the genetic diversity of 179 Staphylococcus aureus isolates recovered from various sites in 10 farms producing cheeses manufactured with raw ewe's milk. Isolates were collected from handcrafted cheeses, bulk tank milk, milk from half-udders, skin abscesses on the udder if present, hands and anterior nares of farmers, and air of the milking area. The isolates were typed using pulsed-field gel electrophoresis (PFGE) of DNA SmaI digests and compared to other isolates of S. aureus isolated in different hosts or in different locations. The results showed that nine farms were contaminated by S. aureus isolates with identical banding patterns (named OV) or by genetically related isolates (named OV'). These dominant banding patterns were found in a variable proportion of the samples from each farm (range: 11-100%). Most of the strains isolated from nasal carriage or strains isolated from other regions or from other animal species had different PFGE patterns to OV or OV', except for three strains. These results show that a single clone of S. aureus is widely distributed both in infected mammary glands and in cheese produced from raw milk. This study confirms that infected mammary glands are the main source of the contamination of dairy products in sheep.

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at -20 and at -80 degrees C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at -20 and -80 degrees C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at -20 vs. -80 degrees C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At -20 degrees C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at -80 degrees C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified.

Staphylococcus aureus is the most persistent pathogen causing ovine mastitis. This study investigated S. aureus binding to cultured epithelial cells obtained from the mammary gland. A staphylococcal 145kDa cell wall adhesin, originally isolated from a bovine mastitis strain, was detected in lysostaphin-solubilized ovine mastitis strains and in the encapsulated strain A. This adhesin was able to bind to cultured ovine mammary gland epithelial cells (MGEC) and to a rat intestinal epithelial cell line (RIE-1), exhibiting different electrophoretic mobilities that could be attributable to protein polymorphism. Inhibition assays using antibodies against 145kDa adhesin and against whole bacteria showed the specificity of the binding to cells. The role of this protein in adherence was assessed by adherence inhibition tests carried out in vitro with radiolabeled bacteria and cultured epithelial cells. Preincubation of bacteria with antibodies against adhesin 145kDa or against strain c195 resulted in a statistically significant decrease of adherence. These experiments suggest that adherence of S. aureus to MGEC may be critical for colonization.

In each of two experiments, the effects of inoculation of Listeria monocytogenes into the ovine mammary gland were studied. In the first experiment, ewes were challenged with one or other of five different Listeria spp. isolates to study differences in their pathogenicity. In the second, ewes were challenged with L. monocytogenes serotype 1/2a to study the sequential features of the infection. The reaction of the mammary glands was assessed by bacteriological, cytological and histological methods. No distinct variation in the pathogenicity of L. monocytogenes isolates was evident: all produced subclinical mastitis, independently of their origin or serotype; a L. innocua isolate caused only a transient increase of milk somatic cell counts. After challenge, L. monocytogenes was isolated for 88 days from the milk of inoculated glands, whose milk somatic cell counts were greater than 1.0 x 10(6) cells ml-1. The organism was also isolated from the mammary lymph nodes, but not from any internal organ of any inoculated ewe. In early stages of the infection neutrophilic infiltration was the predominant histological feature, but hyperaemia, and degeneration of alveolar epithelial cells were also recorded. Later, chronic inflammatory features predominated, with lymphocytes as the principal cell types, destruction of alveoli and fibrous tissue proliferation. In the final stage of the experiment, fibrosis was the salient finding. It is concluded that L. monocytogenes can cause subclinical mastitis after intramammary inoculation into ewes.

Milk from mastitis-free goats from French herds was examined for the presence of coagulase-negative staphylococci (CNS), and 165 positive isolates were evaluated for their capacity to produce exoproteins. Most isolates were identified as Staphylococcus caprae (N = 91) or S. xylosus (N = 36), but members of at least nine other species were present. Overall, some 57% of isolates produced toxins with phenotypic properties of alpha-hemolysin, and 75% produced toxins resembling S. aureus beta and/or delta-hemolysins. Thermostable desoxyribonuclease (TNAse) was secreted by 29% of isolates and thermolabile DNAse by 66%. Slime was produced by 42% of our cultures and, although none of them showed activity to insoluble elastase, over 70% lysed the soluble substrate. No clinical consequences were observed to correlate with exoprotein production, which proved to be inconstant within individual CNS species.

A polymerase chain reaction (PCR)-based test was developed for the detection of Mycoplasma agalactiae in sheep milk samples. Two oligonucleotide primers were designed to amplify a 375 bp fragment of M. agalactiae chromosomal DNA. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization using a fluorescein labeled 528 bp probe. The primers allowed the amplification of fragment of M. agalactiae DNA and did not amplify any specific fragment of other mycoplasmal DNAs (M. capricolum, M. mycoides subsp. mycoides, M. mycoides subsp. capri, M. putrefaciens, M. arginini and M. bovis) or other bacterial DNAs (S. aureus, S. epidermidis, P. haemalytica, E. coli, S. agalactiae, S. dysgalactiae, S. uberis, B. cereus, P. aeruginosa, S. durans, L. lactis, L. lactis var. diacetilactis, L. mesenteroides, S. thermophilus, L. bulgaricus and L. casei). The limit of detection of PCR assay was between 2.5 and 25 fg of purified DNA and 10(2) CCU ml-1 on mycoplasma cultures. These results indicate that the PCR technique can be used as a rapid and specific diagnostic method for detection of M. agalactiae.

A total of 359 Micrococcaceae strains isolated from goat milk samples were differentiated with the commercially available ATB 32 Staph differentiation system. Of these strains, 303 (84.4%) were identified. Six strains were sensitive in the bacitracin resistance test, and accordingly classified as Micrococcus spp. Staphylococcal species isolated of goat milk were S. epidermidis, S. aureus, S. caprae, S. lentus, S. simulans, S. capitis, S. lugdunensis, S. xylosus, S. chromogenes, S. hominis, S. arlettae, S. warneri, S. sciuri, and S. saprophyticus. Highest somatic cell count (SCC) in milk and the highest prevalence of clinical udder alterations were associated with coagulase-positive S. aureus. Increases in milk SCC as well as pathological udder findings were observed in infections with coagulase-negative staphylococci such as novobiocin-sensitive S. epidermidis, S. simulans, S. lugdunensis, S. chromogenes, and S. warneri.

Twelve mutants were isolated from a Staphylococcus aureus strain derived from bovine mastitis after mutagenesis by ultraviolet light. These mutants were found to be deficient for several characteristics such as production of most exoproteins and had altered phage type and/or colonial morphology in serum-soft agar medium. They also differed in virulence when assayed in mice by intraperitoneal administration; the ratio of the LD50 of the mutants vs. that of the parental strain ranged from 1 to 123. The different virulence of the mutants could not be associated with lack of production of exoproteins or altered colonial morphology. On the other hand, a clear correlation was evidenced between lowered virulence and slower growth rate at 37 degrees C. Three mutants were assayed in the mouse mastitis model. One of them, which was about 40 times less virulent when assayed by intraperitoneal administration, induced a histopathological lesion similar to that produced by the parent strain; the other two mutants, which were about 70 to 120 times less virulent by intraperitoneal administration, induced only a very slight lesion. Mice were vaccinated by the intraperitoneal route with two of the less virulent mutants; the LD50 in the vaccinated mice that were challenged with the parental strain increased 11 to 14 times compared with that for the unvaccinated mice.

Staphylococcus aureus is one of the most prevalent causes of ruminant mastitis. The interaction of this microorganism with the host is strongly dependent on its cell surface properties, specially concerning the presence of the exopolysaccharide-containing outer layers (glycocalyx), which appear to play an important role in virulence. In this article, the definition and recognition of the types of exopolysaccharide layers are described, together with their likely role in the pathogenesis of mastitis.

Staphylococcus aureus isolated from mastitis (14 bovine and 11 ovine strains) exhibited an ability to adhere to epithelial primary cultures from ovine mammary gland and to a rat epithelial cell line, RIE-1. Strain differences in the degree of adherence were observed in both cases. These differences were maintained when comparing different epithelial sources (rat vs. ovine). RIE-1 cells can thus be used as a model for studying staphylococcal adherence to epithelial cells. Changes in bacterial adherence were observed according to the bacterial growth phase. The magnitude of these changes differed among strains. Bacterial cell surface hydrophobicity was not related to the degree of adherence to mammalian epithelial cells.

Bacterial adhesion to mammary gland epithelial cells (EC) may play a role in the pathogenesis of mastitis. In vitro adherence systems have been developed to study mastitis in cattle but little has been done in sheep. In this work, a method is described for obtaining mammary gland cell preparations containing greater than or equal to 65% EC from live or dead ewes, using a Ficoll-Hypaque flotation method (cell viability = 70-90%). An in vitro adhesion assay procedure was also developed to study the interaction between EC and ovine mastitis bacterial strains. It was observed that, under the test conditions, adherence increased as the incubation time was prolonged from 30 to 120 min (P less than 0.05). Adhesion was greater at incubation temperature of 37 degrees C than at 22 degrees C (P less than 0.001). An acidic pH (5.9) was associated with an increase in adhesion, when compared with a higher pH (7.2; P less than 0.05). Tween 20, Tween 80 and bovine serum albumin helped to eliminate a background of unbound bacteria from the test slides, but they also inhibited adhesion to some strains. Strain differences in adhesion and in ability to form a background were also observed. Some of these findings may have in vivo implications.

Twelve strains of M. bovigenitalium and two of three strains of M. canadense caused an infection resulting in a pathogenic effect when experimentally inoculated into the ovine mammary gland. Differences in the pathogenesis were quantified by the duration of continuous mycoplasma excretion and the duration of high milk cell levels, but variation in the susceptability of the experimental animals prevented the establishment of firm conclusions on the relative virulence of the strains. Seven M. bovigenitalium and two M. canadense strains were eventually eliminated naturally from the infected glands, but four M. bovigenitalium strain infections ultimately became sub-clinical with intermittent mycoplasma excretion and low milk cell levels.

The bacteriostatic activity of bovine lactoferrin (Lf) against mastitic bacteria was assessed with an in vitro microassay. The most susceptible species was Escherichia coli; all of the 35 isolates tested were susceptible to bacteriostasis by apo-Lf (0.1 mg ml-1), although a few strains showed a lower degree of inhibition. Heterogeneity among strains was more pronounced among 10 isolates of Staphylococcus aureus, four of which were apparently unaffected by apo-Lf (1 mg ml-1). Under the same conditions, Streptococcus agalactiae (six isolates) and Str. uberis (five isolates) resisted the bacteriostatic action of apo-Lf.

The virulence of isolates of Mycoplasma ovipneumoniae and M. arginini from pneumonic and unaffected ovine lungs was compared in a mouse mammary gland model. The isolates varied in their ability to induce a neutrophilic response in the mammary gland. A moderate to severe form of mastitis was induced by 3 M. ovipneumoniae isolates recovered from pneumonic lungs, while the remaining M. ovipneumoniae isolates from pneumonic lungs and those from unaffected lungs induced a very mild histopathological response. The severity of the mastitis could not be increased by the simultaneous inoculation of a mixture of 5 mycoplasma isolates. Mycoplasma arginini isolates induced only a very mild histopathological response despite having been isolated from pneumonic lungs. The finding that the 3 most virulent M. ovipneumoniae isolates were initially recovered from pneumonic ovine lungs suggested that these virulent isolates may contribute to ovine pneumonia. However, the isolation of M. ovipneumoniae from pneumonic ovine lungs does not necessarily imply that these organisms are the causal agents, since M. ovipneumoniae isolates may vary in virulence.

Infection of udder halves by coagulase-negative staphylococci in seven commercial goat herds was studied in conjunction with the California Mastitis Test (CMT). Nine different species were identified and only 10% of strains belonged to groups which could not be identified with any of the known Staphylococcus species. The most prevalent species were Staphylococcus epidermidis (47.7%) and Staphylococcus caprae (19.7%). About half of the coagulase-negative staphylococcal infections gave negative CMT scores. The score was independent of the species of staphylococci involved and the stage of lactation. About 60% of the coagulase-negative staphylococcal species isolated were reisolated in the identical half udder during the following lactation.

Seven out of eight ovine ureaplasma strains inoculated into the mammary gland of suckling ewes produced a mastitis. The pattern of infection was single phase in 5 ewes, persisting for 12-41 days, and biphasic in 3 ewes, persisting in 2 of them until weaning at 60 days and 3 months post-infection. Sucking lambs did not become infected in the eye or nasal areas, and did not transfer infection to the control contralateral glands.

Various methods of inducing mastitis in the ovine mammary gland with two bovine ureaplasma strains were investigated. The most successful method was by inoculation of fresh broth cultures on two successive days, 24 h apart. Eight more bovine strains were inoculated by this means and three successfully infected the glands.

Mycoplasma (M.) capricolum has been frequently isolated from diseased as well as from healthy sheep in Morocco. In order to determine its pathogenicity for sheep, experiments were performed in three trials with the Moroccan isolate 012. The following results were obtained: Ewes inoculated intramammarily developed acute mastitis; the organism was transmitted to lambs suckling these ewes. Only a mild mastitis appeared after a second inoculum, performed 5 weeks after the first. Young lambs, 1 month of age, fed four times with M. capricolum broth culture, died during the septicemic phase, showing a generalized septicemia, polyarthritis and a diffuse interstitial pneumonia. Lambs, 2 and 3 months of age inoculated intrabronchially developed a disease, which appeared to be age and weight dependent. The more susceptible animals died within 1-2 weeks after infection. The older lambs recovered gradually from the disease, which was characterized by pneumonia, conjunctivitis and arthritis. A significant increase of antibodies against M. capricolum developed in the older animals in the complement fixation test.