Genomic Characterization of Mycoplasma arginini Isolated from a Housefly on a Dairy Farm and Comparison with Isolates from Bovine Milk and Lung Tissue.

Mycoplasma mastitis can be highly contagious, unresponsive to treatment, and cause severe economic problems in affected herds. Notable routes of Mycoplasma spp. transmissions are contaminated milking equipment and animal contact through respiratory secretions. Only a few studies report the environment as a possible source of infection. Our group studied the presence of pathogens in houseflies (Musca domestica) in a New York State dairy in the United States. Among others, a Mycoplasma spp. was found in the gut of a housefly captured in the sick pen and identified as M. arginini. Here, we characterized its genome and investigated its relatedness with eight isolates from milk, one isolate from lung tissue collected in the same dairy, and five other dairies in New York State. We applied whole-genome sequencing and phylogenetic analysis based on the sequences of the 16S rRNA gene and 76 conserved proteins. We also assessed an in silico virulence profile by considering a panel of 94 putative virulence genes. As a result of the genome analysis, the housefly M. arginini isolate was highly similar to the milk isolates; interestingly, the similarity was highest with M. arginini isolated from milk on the same dairy farm where the housefly was captured. The housefly and milk M. arginini isolates possessed 54 of the 94 pathogenicity genes considered. Our data support the hypothesis that houseflies are carriers of Mycoplasma spp. and can be considered within the possible roots of environmental transmission of infection in dairy cows. Nevertheless, M. arginini pathogenicity will need to be investigated with dedicated studies. IMPORTANCE It is critical to control the spread of bovine mastitis caused by Mycoplasma spp., as this disease can be highly contagious and have a severe economic impact on affected dairies. A better understanding of possible transmission routes is crucial for infection control and prevention. Based on our data, the composite milk isolates are genetically similar to the housefly isolate. This provides evidence that the same Mycoplasma species found in milk and associated with mastitis can also be isolated from houseflies captured in the dairy environment.

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.

Effect of using mycotoxin-detoxifying agents in dairy cattle feed on natural whey starter biodiversity.

Natural whey cultures (NWC) are undefined multiple-strain bacterial starter communities that can be affected by even small changes along the entire dairy chain. We applied a multidisciplinary approach to investigate how the addition of 2 mycotoxin-detoxifying agents [sodium smectite and lignocellulose-based material (B1); leonardite and betaine (B2)] to cow diets modified the microbiota of the NWC in manufacture of a Grana-like cheese. Microbiological and flow cytometry analyses showed that the content and viability of lactic acid bacteria (LAB) and the total whey microbiota were not affected by the detoxifying agents, and Streptococcus thermophilus, Lactobacillus helveticus, and Limosilactobacillus fermentum were the dominant taxa. Random amplified polymorphic DNA-PCR fingerprinting and metagenomic analysis highlighted differences in the bacterial community of the NWC and in the relative abundance of Bacteroidetes that increased when B1 and B2 were included in the diet. Two of 6 St. thermophilus biotypes were detected only in control samples; conversely, none of the Lb. helveticus biotypes found in control samples were isolated from B1 and B2. In vitro tests showed that the 2 binders did not significantly affect the development of St. thermophilus, but they stimulated the growth of Lb. helveticus strains recovered only from B1 and B2 NWC. The addition of binders in cow feed can affect the LAB biotypes present in NWC.

Inoculation of mother's own milk could personalize pasteurized donor human milk used for feeding preterm infants.

BACKGROUND: Human milk is a vehicle for bioactive compounds and beneficial bacteria which promote the establishment of a healthy gut microbiome of newborns, especially of preterm infants. Pasteurized donor human milk (PDHM) is the second-best option when preterm mother's own milk is unavailable. Since pasteurization affect the microbiological quality of donor milk, PDHM was inoculated with different preterm milk samples and then incubated, in order to evaluate the effect in terms of bacterial growth, human milk microbiome and proteolytic phenomena. METHODS: In an in-vitro study PDHM was inoculated at 10% v/v using ten preterm milk samples. Microbiological, metataxonomic and peptidomic analyses, on preterm milk samples at the baseline (T0), on PDHM and on inoculated milk (IM) samples at T0, after 2 h (T1) and 4 h (T2) of incubation at 37 °C, were conducted. RESULTS: IM samples at T2 showed a Total Bacterial Count not significantly different (p > 0.01) compared to preterm milk samples. At T2 lactic acid bacteria level was restored in all IM. After inoculation, metataxonomic analysis in IM samples showed that Proteobacteria remained the predominant phylum while Firmicutes moved from 3% at T1 to 9.4% at T2. Peptidomic profile of IM resembled that of PDHM, incubated for the same time, in terms of number and type of peptides. CONCLUSION: The study demonstrated that inoculation of PDHM with mother's own milk could restore bacterial growth and personalize human milk microbiome in PDHM. This effect could be beneficial because of the presence of maternal probiotic bacteria which make PDHM more similar to mother's own milk.

Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries.

Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.

Staphylococcus aureus intra-mammary infection affects the expression pattern of IL-R8 in goat.

IL-1R8 is a member of Interleukin-1 receptor family acting as a negative regulator of inflammation reliant on ILRs and TLRs activation. IL-1R8 role has never been evaluated in acute bacterial mastitis. We first investigated IL-1R8 sequence conservation among different species and its pattern of expression in a wide panel of organs from healthy goats. Then, modulation of IL-1R8 during natural and experimental mammary infection was evaluated and compared in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected goats. IL-1R8 has a highly conserved sequence among vertebrates. Goat IL-1R8 was ubiquitously expressed in epithelial and lymphoid tissues with highest levels in pancreas. IL-1R8 was down-regulated in epithelial mammary cells following S. aureus infection. Interestingly it was up-regulated in leukocytes infiltrating the infected mammary tissues suggesting that it could represent a target of S. aureus immune evasion.

Evaluation of the effects of different diets on microbiome diversity and fatty acid composition of rumen liquor in dairy goat.

Fat supplementation plays an important role in defining milk fatty acids (FA) composition of ruminant products. The use of sources rich in linoleic and α-linolenic acid favors the accumulation of conjugated linoleic acids isomers, increasing the healthy properties of milk. Ruminal microbiota plays a pivotal role in defining milk FA composition, and its profile is affected by diet composition. The aim of this study was to investigate the responses of rumen FA production and microbial structure to hemp or linseed supplementation in diets of dairy goats. Ruminal microbiota composition was determined by 16S amplicon sequencing, whereas FA composition was obtained by gas-chromatography technique. In all, 18 pluriparous Alpine goats fed the same pre-treatment diet for 40±7 days were, then, arranged to three dietary treatments consisting of control, linseed and hemp seeds supplemented diets. Independently from sampling time and diets, bacterial community of ruminal fluid was dominated by Bacteroidetes (about 61.2%) and Firmicutes (24.2%) with a high abundance of Prevotellaceae (41.0%) and Veillonellaceae (9.4%) and a low presence of Ruminococcaceae (5.0%) and Lachnospiraceae (4.3%). Linseed supplementation affected ruminal bacteria population, with a significant reduction of biodiversity; in particular, relative abundance of Prevotella was reduced (-12.0%), whereas that of Succinivibrio and Fibrobacter was increased (+50.0% and +75.0%, respectively). No statistically significant differences were found among the average relative abundance of archaeal genera between each dietary group. Moreover, the addition of linseed and hemp seed induced significant changes in FA concentration in the rumen, as a consequence of shift from C18 : 2n-6 to C18 : 3n-3 biohydrogenation pathway. Furthermore, dimethylacetal composition was affected by fat supplementation, as consequence of ruminal bacteria population modification. Finally, the association study between the rumen FA profile and the bacterial microbiome revealed that Fibrobacteriaceae is the bacterial family showing the highest and significant correlation with FA involved in the biohydrogenation pathway of C18 : 3n-3.

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.

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds, related swine farms, and humans in contact with herds.

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms' owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval=0-10.2%) and 60% (33/55; 95% confidence interval=47.05-72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.

Short communication: Methicillin-resistant Staphylococcus aureus in bulk tank milk of dairy cows and effect of swine population density.

The methicillin-resistant Staphylococcus aureus (MRSA) has recently frequently been reported in dairy cattle, usually with low prevalence. The livestock-associated MRSA (LA-MRSA) ST398 is especially involved in cases of subclinical and clinical mastitis. Swine carry LA-MRSA without clinical symptoms and are considered its reservoir and shedder. People exposed to swine are particularly at risk of LA-MRSA colonization. Environments with relevant livestock density are a demonstrated risk factor for humans to be carriers of a LA-MRSA. This work investigated dairy farms located in an area with a high livestock density, mainly represented by swine. Bulk tank milk samples from 224 dairy farms were collected, and their status was defined as MRSA-positive or MRSA-negative based on culture on chromogenic medium. The number of fattening swine and of fattening swine herds was calculated in an area of 3 km around each dairy farm through georeferencing. The probability of a Staphylococcus aureus-positive dairy farm to be MRSA positive based on the extent of potential infective pressure due to swine density was calculated. Both the number of swine herds and the number of swine were associated with the MRSA status of dairy herds. The 9 MRSA isolated were typed by multi-locus sequence typing and spa-typing, and characterized for their virulence factors and antimicrobial resistance profiles. The ST and spa-types detected are consistent with those present in the Italian swine population. Virulence and resistance profiles are mostly consistent with the types detected. This work provides the first evidence of the epidemiological challenge exerted by the density of the swine population on MRSA in dairy cows.

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.

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.

Detection of food spoilage and pathogenic bacteria based on ligation detection reaction coupled to flow-through hybridization on membranes.

Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells.

Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants.

Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n=40) and from udder tissue (n=7) and foremilk (n=24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology.

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.

Transcriptome analysis to identify differential gene expression affecting meat quality in heavy Italian pigs.

Suppressive subtractive hybridization (SSH) was used to analyse the muscle transcriptome and identify genes affecting meat quality within an Italian pig population of Large White and Landrace purebred individuals. Seven phenotypes were recorded at slaughter: dorsal fat thickness, ham fat thickness, ham fat coverage, muscle compactness, marbling, meat colour and colour uniformity. Two subtractive libraries were created from longissimus dorsi tissue of selected pigs with extreme phenotypes for meat quality. Eighty-four differentially expressed ESTs were identified, which showed homology to expressed pig sequences and/or to genomic pig sequences produced within the pig genome project. Sixty-eight sequences were mapped on the pig genome, and most of these sequences co-localized with the same chromosomal positions as QTLs that have been previously identified for meat quality. Thirty sequences, including eight matching known genes previously related to muscle metabolic pathways, were selected to statistically validate their differential expression. Association analysis and t-test results indicated that 28 ESTs of the 30 analysed were associated with phenotypes investigated here and have significant differential expression levels (P≤ 0.05) between the two tails of the phenotypic distribution.