Staphylococcus aureus adlb gene is associated with high prevalence of intramammary infection in dairy herds of northern Italy: A cross-sectional study.

Staphylococcus aureus is a major mastitis pathogen in dairy cattle worldwide, responsible for substantial economic losses. Environmental factors, milking routine, and good maintenance of milking equipment have been described as important factors to prevent intramammary infections (IMI). Staphylococcus aureus IMI can be widespread within the farm or the infection can be limited to few animals. Several studies have reported that Staph. aureus genotypes differ in their ability to spread within a herd. In particular, Staph. aureus belonging to ribosomal spacer PCR genotype B (GTB)/clonal complex 8 (CC8) is associated with high within-herd prevalence of IMI, whereas other genotypes are generally associated with individual cow disease. The adlb gene seems to be strictly related to Staph. aureus GTB/CC8, and is a potential marker of contagiousness. We investigated Staph. aureus IMI prevalence in 60 herds in northern Italy. In the same farms, we assessed specific indicators linked to milking management (e.g., teat condition score and udder hygiene score) and additional milking risk factors for IMI spread. Ribosomal spacer-PCR and adlb-targeted PCR were performed on 262 Staph. aureus isolates, of which 77 underwent multilocus sequence typing. In most of the herds (90%), a predominant genotype was identified, especially Staph. aureus CC8 (30%). In 19 of 60 herds, the predominant circulating Staph. aureus was adlb-positive and the observed IMI prevalence was relevant. Moreover, the adlb gene was detected only in genotypes of CC8 and CC97. Statistical analysis showed a strong association between the prevalence of Staph. aureus IMI, the specific CCs, and carriage of adlb, with the predominant circulating CC and presence of the gene alone explaining the total variation. Interestingly, the difference in the odds ratio obtained in the models for CC8 and CC97 suggests that it is carriage of the adlb gene, rather than the circulation of these CCs per se, that leads to higher within-herd prevalence of Staph. aureus. In addition, the model showed that environmental and milking management factors had no or minimal effect on Staph. aureus IMI prevalence. In conclusion, the circulation of adlb-positive Staph. aureus strains within a herd has a strong effect on the prevalence of IMI. Thus, adlb can be proposed as a genetic marker of contagiousness for Staph. aureus IMI in cattle. However, further analyses using whole-genome sequencing are required to understand the role of genes other than adlb that may be involved in the mechanisms of contagiousness of Staph. aureus strains associated with high prevalence of IMI.

Penicillin resistance in bovine Staphylococcus aureus: Genomic evaluation of the discrepancy between phenotypic and molecular test methods.

Staphylococcus aureus is a major pathogen in humans and animals. In cattle, it is one of the most important agents of mastitis, causing serious costs in the dairy industry. Early diagnosis and adequate therapy are therefore 2 key factors to deal with the problems caused by this bacterium, and benzylpenicillin (penicillin) is usually the first choice to treat these infections. Unfortunately, penicillin resistance testing in bovine S. aureus strains shows discrepant results depending on the test used; consequently, the best method for assessing penicillin resistance is still unknown. The aim of this study was therefore to find a method that assesses penicillin resistance in S. aureus and to elucidate the mechanisms leading to the observed discrepancies. A total of 146 methicillin-sensitive S. aureus strains isolated from bovine mastitis were tested for penicillin resistance using a broth microdilution [minimum inhibitory concentration (MIC)] and 2 different disk diffusion protocols. Furthermore, the strains were analyzed for the presence of the bla operon genes (blaI, blaR1, blaZ) by PCR, and a subset of 45 strains was also subjected to whole genome sequencing (WGS). Discrepant results were obtained when penicillin resistance of bovine S. aureus was evaluated by disk diffusion, MIC, and PCR methods. The discrepancies, however, could be fully explained by WGS analysis. In fact, it turned out that penicillin resistance is highly dependent on the completeness of the bla operon promotor: when the bla operon was complete based on WGS analysis, all strains showed MIC ≥1 µg/mL, whereas when the bla operon was mutated (31-nucleotide deletion), they were penicillin sensitive except in those strains where an additional, bla operon-independent resistance mechanism was observed. Further, WGS analyses showed that penicillin resistance is truly assessed by the MIC assay. In contrast, caution is required when interpreting disk diffusion and PCR results.

Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy).

Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates.

Animal-based measures on fattening heavy pigs at the slaughterhouse and the association with animal welfare at the farm level: a preliminary study.

Monitoring animal welfare (AW) in pig farms requires both proper indicators and a feasible approach. Animal-based measures (ABMs) are well-established AW indicators. Furthermore, AW screening at the slaughterhouses could be useful for identifying problems on farm. The aim of this study was to evaluate ABMs at the slaughterhouse and, when possible, to compare these ABMs with those collected on the farm. The study was carried out in northern Italy in a commercial abattoir and in a sample of farms. Animal-based measures were recorded on pigs from 62 batches of 54 farms, during ante-mortem (n=10 085 pigs) and post-mortem (n=7952 pigs) inspections. Sixteen of 54 farms were selected to compare ABMs collected at the slaughterhouse with ABMs collected on the farm. Overall, 2295 pigs (mean pigs examined per farm 119±45) were inspected at the slaughterhouse (group S) and 420 pigs (mean pigs per farm 26±5) on the farm (group F). Non-animal-based measures were also collected at the 16 farms. Differences between groups S and F, at the animal level, were assessed by a two-tailed paired Wilcoxon-Mann-Whitney test. Differences at the site of observation level (farm and slaughterhouse) were assessed by Fisher's exact test using a hierarchical log-linear modelling for contingency tables. The most frequent ABMs at the slaughterhouse were manure on the body (47.7%), followed by dermatitis (28.0%), white spot (25.4%) and bursitis (24.7%). Recording ABMs at the slaughterhouse and on the farm usually yielded similar results; however, there were some exceptions. In particular, significant differences were found for non-uniformity of size (P<0.05) and dermatitis (P<0.001), which were higher at the slaughterhouse than on the farm. Results of log-linear modelling underlined the effect of the farm of origin on the percentage of pigs with bursitis, manure on the body and ear injuries that were observed at the slaughterhouse. In group S, significant associations between manure on the body and insufficient presence of clean and dry areas in the corresponding farm were found (P<0.05). Although these results should be interpreted with care due to the limited sample of farms, the slaughterhouse could be a feasible site of observation of ABMs, which could then be integrated in monitoring of AW on farm. Considering the number of slaughtered batches per farm, it would be possible to repeat assessments several times throughout the year for each farm, which could help provide an index for the continuous monitoring of AW.

Genomic and transcriptomic comparison between Staphylococcus aureus strains associated with high and low within herd prevalence of intra-mammary infection.

BACKGROUND: Staphylococcus aureus (Staph. aureus) is one of the major pathogens causing mastitis in dairy ruminants worldwide. The chronic nature of Staph. aureus infection enhances the contagiousness risk and diffusion in herds. In order to identify the factors involved in intra-mammary infection (IMI) and diffusion in dairy cows, we investigated the molecular characteristics of two groups of Staph. aureus strains belonging to ST8 and ST398, differing in clinical properties, through comparison of whole genome and whole transcriptome sequencing. RESULTS: The two groups of strains, one originated from high IMI prevalence herds and the other from low IMI prevalence herds, present a peculiar set of genes and polymorphisms related to phenotypic features, such as bacterial invasion of mammary epithelial cells and host adaptation. Transcriptomic analysis supports the high propensity of ST8 strain to chronicity of infection and to a higher potential cytotoxicity. CONCLUSIONS: Our data are consistent with the invasiveness and host adaptation feature for the strains GTB/ST8 associated to high within-herd prevalence of mastitis. Variation in genes coding for surface exposed proteins and those associated to virulence and defence could constitute good targets for further research.

An alternative experimental case-control design for genetic association studies on bovine mastitis.

The possibility of using genetic control strategies to increase disease resistance to infectious diseases relies on the identification of markers to include in the breeding plans. Possible incomplete exposure of mastitis-free (control) animals, however, is a major issue to find relevant markers in genetic association studies for infectious diseases. Usually, designs based on elite dairy sires are used in association studies, but an epidemiological case-control strategy, based on cows repeatedly field-tested could be an alternative for disease traits. To test this hypothesis, genetic association results obtained in the present work from a cohort of Italian Holstein cows tested for mastitis over time were compared with those from a previous genome-wide scan on Italian Holstein sires genotyped with 50k single nucleotide polymorphisms for de-regressed estimated breeding values for somatic cell counts (SCCs) on Bos taurus autosome (BTA6) and BTA14. A total of 1121 cows were selected for the case-control approach (cases=550, controls=571), on a combination of herd level of SCC incidence and of within herd individual level of SCC. The association study was conducted on nine previously identified markers, six on BTA6 and four on BTA14, using the R statistical environment with the 'qtscore' function of the GenABEL package, on high/low adjusted linear score as a binomial trait. The results obtained in the cow cohort selected on epidemiological information were in agreement with those obtained from the previous sire genome-wide association study (GWAS). Six out of the nine markers showed significant association, four on BTA14 (rs109146371, rs109234250, rs109421300, rs109162116) and two on BTA6 (rs110527224 and rs42766480). Most importantly, using mastitis as a case study, the current work further validated the alternative use of historical field disease data in case-control designs for genetic analysis of infectious diseases in livestock.

Genetic characterization and antimicrobial resistance of Staphylococcus aureus isolated from bovine milk in Tunisia.

Staphylococcus aureus is a major agent of bovine mastitis in dairy herds, causing economic losses in dairy industry worldwide. In addition, milk and milk-products contaminated by Staph. aureus can cause harmful human diseases. The aim of this study was to characterize Staph. aureus strains isolated from dairy farms in Tunisia. Bulk tank milk (n = 32) and individual cow milk (n = 130) samples were collected during the period of 2013-2014. Forty-three Staph. aureus isolates were recovered and typed by spa typing, 16S-23S rRNA intergenic spacer (RS-PCR) and multiplex PCRs for 22 virulence genes. Antimicrobial resistance was also investigated with a disc diffusion test. A selected subsample of 22 strains was additionally genotyped by multilocus sequence typing. Seventeen spa types were recovered, and t2421 (n = 10), t521 (n = 6) and t2112 (n = 5) were the most common. Fourteen different RS-PCR genotypes grouped into 11 clusters were detected in our study, with predominance of the RVI genotype (n = 24). Eight sequence types were identified and Clonal Complex 97, corresponding to RS-PCR cluster R, was the most common (n = 10), followed by CC1 (n = 4), CC15 (n = 3) and other four accounting for one or two strains. Different combinations of virulence genes were reported, and enterotoxin genes were present in few strains (seh, n = 4; sea, n = 2; sea and seh, n = 2; sec and sel, n = 2). The majority of strains were resistant only to penicillin; only one strain was found to be multiresistant and no methicillin-resistant Staph. aureus was demonstrated. Our study reported the isolation of CC97 from bovine milk in Tunisia for the first time and confirmed the relevance of this lineage in intramammary infection in cows. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the characteristics of Staphylococcus aureus isolated from bulk tank and individual cow milk in Tunisia. All strains were genotyped by spa typing and RS-PCR, a method based on the amplification of the 16S-23S rRNA intergenic spacer region, and multiplex PCRs for 22 virulence genes. A selected subsample of strains was also genotyped by multilocus sequence typing. All strains were tested for antimicrobial resistance. Our study evidences a predominance of strains belonging to Clonal Complex 97. Methicillin-resistant strains were not detected, and overall low level of antimicrobial resistance was reported.

Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus clonal complexes in bulk tank milk from dairy cattle herds in Lombardy Region (Northern Italy).

Staphylococcus aureus is the most important causative agent of subclinical mastitis in cattle resulting in reduced milk production and quality. Methicillin-resistant S. aureus (MRSA) strains has a clear zoonotic relevance, especially in the case of occupational exposure. The aim of the study was to evaluate the prevalence of S. aureus and MRSA in bulk tank milk (BTM) from dairy cattle herds in the Lombardy Region (Northern Italy) and to identify the main MRSA circulating genotypes. MRSA strains were characterized by susceptibility testing, multi-locus sequence typing (MLST), spa typing and SCCmec typing. A total 844 BTM samples were analysed and S. aureus and MRSA were detected in 47·2% and 3·8% of dairy herds, respectively. MLST showed that the majority (28/32) of isolates belonged to the typical livestock-associated lineages: ST398, ST97 and ST1. Interestingly, in this study we report for the first time the new ST3211, a single locus variant of ST(CC)22, with the newly described 462 aroE allele. Our study indicates high diffusion of S. aureus mastitis and low, but not negligible, prevalence of MRSA in the considered area, suggesting the need for planning specific control programmes for bovine mastitis caused by S. aureus, especially when MRSA is implicated.

Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer.

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies using ribosomal spacer (RS)-PCR, however, demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infections are genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. Furthermore, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. In addition to RS-PCR, other methods for subtyping Staph. aureus are known, including spa typing and multilocus sequence typing (MLST). They are based on sequencing the spa and various housekeeping genes, respectively. The aim of the present study was to compare the 3 analytic methods using 456 strains of Staph. aureus isolated from milk of bovine intramammary infections and bulk tanks obtained from 12 European countries. Furthermore, the phylogeny of animal Staph. aureus was inferred and the zoonotic transfer of Staph. aureus between cattle and humans was studied. The analyzed strains could be grouped into 6 genotypic clusters, with CLB, CLC, and CLR being the most prominent ones. Comparing the 3 subtyping methods, RS-PCR showed the highest resolution, followed by spa typing and MLST. We found associations among the methods but in many cases they were unsatisfactory except for CLB and CLC. Cluster CLB was positive for clonal complex (CC)8 in 99% of the cases and typically positive for t2953; it is the cattle-adapted form of CC8. Cluster CLC was always positive for tbl 2645 and typically positive for CC705. For CLR and the remaining subtypes, links among the 3 methods were generally poor. Bovine Staph. aureus is highly clonal and a few clones predominate. Animal Staph. aureus always evolve from human strains, such that every human strain may be the ancestor of a novel animal-adapted strain. The zoonotic transfer of IMI- and milk-associated strains of Staph. aureus between cattle and humans seems to be very limited and different hosts are not considered as a source for mutual, spontaneous infections. Spillover events, however, may happen.

Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries.

Staphylococcus aureus is globally one of the most important pathogens causing contagious mastitis in cattle. Previous studies, however, have demonstrated in Swiss cows that Staph. aureus isolated from bovine intramammary infection is genetically heterogeneous, with Staph. aureus genotype B (GTB) and GTC being the most prominent genotypes. In addition, Staph. aureus GTB was found to be contagious, whereas Staph. aureus GTC and all the remaining genotypes were involved in individual cow disease. The aim of this study was to subtype strains of Staph. aureus isolated from bovine mastitic milk and bulk tank milk to obtain a unified view of the presence of bovine staphylococcal subtypes in 12 European countries. A total of 456 strains of Staph. aureus were subjected to different typing methods: ribosomal spacer PCR, detection of enterotoxin genes, and detection of gene polymorphisms (lukE, coa). Major genotypes with their variants were combined into genotypic clusters (CL). This study revealed 5 major CL representing 76% of all strains and comprised CLB, CLC, CLF, CLI, and CLR. The clusters were characterized by the same genetic properties as the Swiss isolates, demonstrating high clonality of bovine Staph. aureus. Interestingly, CLB was situated in central Europe whereas the other CL were widely disseminated. The remaining 24% of the strains comprised 41 genotypes and variants, some of which (GTAM, GTBG) were restricted to certain countries; many others, however, were observed only once.

Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows.

Staphylococcus aureus is one of the most important causes of mastitis in dairy cattle. Based on previous research, Staph. aureus genotypes with different pathogenic and contagious properties can cause intramammary infection (IMI) and coexist in the same herd. Our study aimed to compare Staph. aureus strains from herds that differed in IMI prevalence using different molecular approaches such as ribosomal spacer (RS)-PCR, multilocus sequence typing (MLST), spa typing, ribotyping, pulsed-field gel electrophoresis (PFGE), and multiplex PCR. For this purpose, 31 dairy herds with Staph. aureus IMI were selected, and 16 of these were chosen for a comparison study: the 8 high-prevalence (HP) herds had Staph. aureus IMI prevalence >28% and the 8 low-prevalence (LP) herds had an IMI prevalence <4%. A total of 650 isolates of Staph. aureus from mammary quarters of all positive cows were genotyped with RS-PCR, a technique based on amplification of a portion of the intergenic spacer 16S-23S rRNA, and a subset of 54 strains was also analyzed by multiplex PCR, ribotyping, PFGE, MLST, and spa typing. The RS-PCR analysis revealed 12 different profiles. Staphylococcus aureus strains isolated from 5 out of 8 HP herds showed a profile identical to the genotype B (GTB), described in previous studies as being strongly associated with high within-herd prevalence of Staph. aureus mastitis and the presence of the genes coding for enterotoxins sea, sed, and sej, a long x-region of spa gene, and 3 lukE fragments. Moreover, all strains isolated in the HP herds possessed genes coding for staphylococcal enterotoxins. In LP herds, a limited number of strains of 6 genotypes, different from those isolated in HP herds, were identified and GTB was not found. Within these genotypes, 4 strains were positive for the mecA gene. Preliminary results and comparison with other genotyping methods confirmed that genotyping by RS-PCR is an accurate, rapid, and inexpensive tool for future field studies on Staph. aureus mastitis strains and generates clinically relevant results.

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with low within-herd prevalence of intra-mammary infections in dairy cows: Genotyping of isolates.

Staphylococcus aureus is one of the most common mastitis-causing pathogens worldwide. In the last decade, livestock-associated methicillin-resistant S. aureus (LA-MRSA) infections have been described in several species, included the bovines. Hence, this paper investigates the diffusion of MRSA within Italian dairy herds; the strains were further characterized using a DNA microarray, which detects 330 different sequences, including the methicillin-resistance genes mecA and mecC and SCCmec typing. The analysis of overall patterns allows the assignment to Clonal Complexes (CC). Overall 163 S. aureus isolates, collected from quarter milk samples in 61 herds, were tested. MRSA strains were further processed using spa typing. Fifteen strains (9.2%), isolated in 9 herds (14.75%), carried mecA, but none harboured mecC. MRSA detection was significantly associated (P<0.011) with a within-herd prevalence of S. aureus intra-mammary infections (IMI) ≤5%. Ten MRSA strains were assigned to CC398, the remaining ones to CC97 (n=2), CC1 (n=2) or CC8 (n=1). In 3 herds, MRSA and MSSA co-existed: CC97-MRSA with CC398-MSSA, CC1-MRSA with CC8-MSSA and CC398-MRSA with CC126-MSSA. The results of spa typing showed an overall similar profile of the strains belonging to the same CC: t127-CC1, t1730-CC97, t899 in 8 out of 10 CC398. In the remaining 2 isolates a new spa type, t14644, was identified. The single CC8 was a t3092. The SCCmec cassettes were classified as type IV, type V or type IV/V composite. All or most strains harboured the genes encoding the ß-lactamase operon and the tetracycline resistance. Streptogramin resistance gene was related to CC398. Enterotoxin and leukocidin genes were carried only by CC1, CC8 and CC97-MRSA. The persistence of MRSA clones characterized by broader host range, in epidemiologically unrelated areas and in dairy herds with low prevalence of S. aureus IMI, might enhance the risk for adaptation to human species.

Short communication: Prevalence of Staphylococcus aureus and methicillin-resistant S. aureus in bulk tank milk from dairy goat farms in Northern Italy.

Staphylococcus aureus is regarded as a leading cause of mastitis in goats. However, few data are available on the presence of methicillin-resistant S. aureus (MRSA) in this species. The aim of this study was to assess the prevalence of S. aureus and MRSA in bulk tank milk samples from dairy goat farms in Northern Italy. Eighty-five out of 197 samples (43.1%) tested positive for S. aureus with counts ranging from 10 to more than 1.5 × 10(4) cfu/mL. The MRSA was screened by both direct plating followed by a disk diffusion test to evaluate methicillin resistance and a selective enrichment method. Methicillin-resistance was confirmed by mecA-specific PCR. Methicillin-resistant S. aureus was identified in 4 samples (2.0%) and multilocus sequence typing (MLST) showed the presence of livestock-associated MRSA belonging to lineages ST398 (n = 3) and ST1 (n = 1). In one case we demonstrated that the same MRSA strain was able to persist over time on the farm, being isolated from both bulk tank milk and the udder of 3 goats 1 yr after the first isolation. The high prevalence of S. aureus-positive herds detected in this study and the presence of MRSA strains belonging to livestock-associated genotypes is of concern, and represents a novel finding in the Italian dairy goat production system. The application of stringent measures for the control of S. aureus mastitis at the farm level seems appropriate to reduce the economic losses, and to minimize the risk of foodborne illness and the transmission of MRSA to humans by occupational exposure.

Simultaneous identification by multiplex PCR of major Prototheca spp. isolated from bovine and buffalo intramammary infection and bulk tank.

UNLABELLED: Bovine mastitis caused by Prototheca spp. infection is increasing worldwide, therefore becoming more relevant to the dairy industry. Almost all Prototheca isolates from bovine mammary protothecosis came from P. zopfii genotype 2, with a lower prevalence of infection due to P. blaschkeae and rarely to P. wickerhamii. In this study, we report the development of two multiplex PCR assays able to discriminate among the three species responsible for bovine intramammary infection (IMI). Our assay is based on the specific amplification of new DNA target from mitochondria and chloroplasts partial sequences, of different Prototheca isolates. Both methods were set up using reference strains belonging to all Prototheca species and validated by the analysis of 93 isolates from bovine and buffalo IMI and bulk tank milk samples. The investigation involves 70 isolates from North, 13 from Central and 10 from South Italian regions. Isolates from bovine were most commonly identified as P. zopfii genotype 2, and only in one case as P. blaschkeae, whereas isolates from buffaloes belonged both to P. zopfii genotype 2 and P. wickerhamii. These findings proved the suitability of our multiplex PCRs as a rapid test to discriminate among pathogenic Prototheca strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reports PCR assays based on novel Prototheca spp. mitochondrial and chloroplastic target sequences. The multiplex PCR protocol described in this study is useful for rapid simultaneous detection of P. zopfii, P. wickerhamii and P. blaschkeae.

Intramammary administration of platelet concentrate as an unconventional therapy in bovine mastitis: first clinical application.

Bovine udder infections induce a variety of changes in gene expression of different growth factors that may suggest their possible role in glandular tissue protection or repair processes. Growth factors and also chemokines and cytokines may act synergistically to increase the infiltration of neutrophils and macrophages to promote angiogenesis, fibroplasia, matrix deposition, and, ultimately, re-epithelialization. Considering the vast applications, typically in human medicine, of platelet concentrate (PC) and its ease of preparation, the aim of our study was to evaluate an alternative therapy to stimulate the regeneration of glandular tissue, administering a concentration in excess of the growth factors contained in the PC. In each one of the 3 farms examined in the trial, PC was prepared from donor cows in good health, free from infections, and with no records of medications administered during the previous 2 mo. The platelet produced in one farm was used only for treating the cows of the same farm in a heterologous way. A total of 229 mastitic quarters were divided in 3 groups: antibiotic group (treated with intramammary antibiotic), antibiotic and PC group (treated intramammarily with antibiotics in association with PC), and PC group (treated with intramammary PC alone). The diagnosis of mastitis was based on somatic cell count and bacteriological evaluation of the milk from the affected quarter. Platelet concentrate, alone or in association with antibiotic, was used for 3 consecutive days as an unconventional therapy in bovine acute and chronic mastitis. Our data show that the associated action of antibiotic and PC performed significantly better than the antibiotic alone, either for the recovery of the affected mammary quarters or for somatic cell count reduction. In the same way, the association antibiotic plus PC showed significantly fewer relapses compared with the antibiotic alone, either for acute or chronic mastitis. The treatment with only PC did not show statistically significant differences compared with both antibiotic alone or associated treatment for acute mastitis, and it was better than the use of only antibiotic for chronic mastitis. Our results show that PC alone may be useful for a quick resolution of the inflammatory response, playing a role in limiting the tissue damage to the mammary gland parenchyma and reducing the recurrence rates.

Technical note: Identification of Prototheca species from bovine milk samples by PCR-single strand conformation polymorphism.

We report the development of a PCR-single strand conformation polymorphism (SSCP) method to identify Prototheca spp. responsible for bovine mastitis: P. zopfii and P. blaschkeae. The method was set up using reference strains belonging to P. zopfii genotype 1, P. zopfii genotype 2, and P. blaschkeae as target species and P. stagnora, and P. ulmea as negative controls. The assay was applied on 50 isolates of Prototheca spp. isolated from bovine mastitic milk or bulk-tank milk samples, and all isolates were identified as P. zopfii genotype 2. We conclude that the described PCR-SSCP approach is accurate, inexpensive, and highly suitable for the identification of P. zopfii genotype 2 on field isolates but also directly on milk, if preceded by a specific DNA extraction method.

Enterohepatic Helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and immunohistochemical investigations.

Enterohepatic Helicobacter spp. have been described colonizing the large intestine and liver of healthy and symptomatic subjects and are thought to have a role in the development of inflammatory bowel disease (IBD). The prevalence of enterohepatic Helicobacter spp. infection in dogs is largely unknown and to our knowledge there are no data about their potential pathogenic role. In light of these considerations, the aims of this study were (i) to assess the prevalence of enterohepatic Helicobacter spp. in colonic biopsies of symptomatic pet dogs and (ii) to evaluate a possible association between Helicobacter spp. colonization status (heavily colonized, poorly colonized and uncolonized biopsies) and histological lesions. Colonic biopsies from 27 pet dogs of different ages were evaluated by family Helicobacteraceae and enterohepatic Helicobacter spp. PCR, histology, and immunohistochemistry for the in situ detection of Helicobacter spp. organisms. 85% and 52% of colonic biopsies were positive by Helicobacteraceae and enterohepatic Helicobacter spp. PCR, respectively. Immunohistochemistry revealed Helicobacter spp. were localized both in the superficial mucus (55%) and within intestinal crypts (33%). Dogs with heavy enterohepatic Helicobacter spp. colonization were significantly younger and had a higher level of mucosal fibrosis/atrophy than dogs with uncolonized or poorly colonized biopsies (p<0.05). These findings contribute to widen current knowledge regarding canine enterohepatic Helicobacter spp., suggesting the infection is rather common in dogs and acquired at an early age. Furthermore, heavy colonization of colonic crypts is associated with chronic inflammatory lesions (fibrosis/atrophy), supporting the role of enterohepatic Helicobacter spp. in the development of canine IBD.

Outbreak of bovine clinical mastitis caused by Mycoplasma bovis in a North Italian herd.

This report describes an outbreak of Mycoplasma bovis mastitis affecting 45 cows in a herd of 122 dairy cattle in Northern Italy. Clinically, the outbreak was characterized by agalactia, multiple swollen and painless quarters, high milk somatic cell count and unresponsiveness to conventional antibiotic therapy. M. bovis was isolated from the milk samples of all the 32 affected cows tested and from the mammary tissue of three affected cows that underwent necropsy. No other pathogens were isolated from these samples. Lesions in two of the necropsied cows were characterized by mild chronic suppurative mastitis and galactophoritis. The other necropsied cow showed a chronic necrosuppurative and pyogranulamaous galactophoritis, a condition not previously associated with M. bovis. M. bovis was detected immunohistochemically in the lumen of the affected mammary ducts suggesting that ascending infection via the teat canal was the likely route of transmission. No other intralesional pathogens were demonstrated microscopically.

Quantitative structure-activity relationship modelling of oral acute toxicity and cytotoxic activity of fragrance materials in rodents.

Fragrance materials are used as ingredients in many consumer and personal care products. The wide and daily use of these substances, as well as their mainly uncontrolled discharge through domestic sewage, make fragrance materials both potential indoor and outdoor air pollutants which are also connected to possible toxic effects on humans (asthma, allergies, headaches). Unfortunately, little is known about the environmental fate and toxicity of these substances. However, the use of alternative, predictive approaches, such as quantitative structure-activity relationships (QSARs), can help in filling the data gap and in the characterization of the environmental and toxicological profile of these substances. In the proposed study, ordinary least squares regression-based QSAR models were developed for three toxicological endpoints: mouse oral LD(50), inhibition of NADH-oxidase (EC(50) NADH-Ox) and the effect on mitochondrial membrane potential (EC(50) DeltaPsim). Theoretical molecular descriptors were calculated by using DRAGON software, and the best QSAR models were developed according to the principles defined by the Organization for Economic Co-operation and Development.