Pentraxin 3 is up-regulated in epithelial mammary cells during Staphylococcus aureus intra-mammary infection in goat.

Pentraxin 3 is the prototypic long pentraxin and is produced by different cell populations (dendritic cells, monocytes/macrophages, endothelial cells, and fibroblasts) after pro-inflammatory stimulation. Different studies demonstrated the up-regulation of PTX3 during mastitis in ruminants, but its role is still unknown. We first investigated the conservation of PTX3 sequence among different species and its pattern of expression in a wide panel of organs from healthy goats. We studied the modulation of PTX3 during natural and experimental mammary infection, comparing its expression in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected animals. We confirmed the high conservation of the molecule among different species. Goat PTX3 was expressed at high levels in bone marrow, mammary gland, aorta, rectum, pancreas, skin and lungs. PTX3 was up-regulated in epithelial mammary cells and in milk cells after S. aureus infection, suggesting that it represents a first line of defense in goat udder.

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.

Effect on quarter milk somatic cell count and antimicrobial susceptibility of Staphylococcus rostri causing intramammary infection in dairy water buffaloes.

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant cause of intramammary infections (IMI) in dairy cows and in water buffaloes, as well. A longitudinal field study was carried out on one well-managed dairy water buffalo herd to determine the prevalence and distribution of CNS and a recently described CNS-species, Staphylococcus rostri, in milk samples to explore its relevance for buffaloes' udder health throughout lactation, and to gain insight into the susceptibility of the latter species toward commonly used antimicrobials. Twice weekly quarter milk samples from a cohort of 11 lactating water buffaloes were collected over an 8-mo period. The CNS (n=109; 76.2% of all culture-positive samples) were the predominant pathogens causing IMI, followed by Corynebacterium bovis (n=11; 7.6%) and Streptococcus spp. (n=9; 6.2%) other than Stretococcus uberis (n=2; 1.4%). Thirty-seven hemolytic staphylococci suspected to be Staphylococcus aureus were further differentiated using transfer DNA-intergenic spacer-PCR and rpoB-gene sequencing because they were coagulase-negative. Thirty-three of those isolates were identified as Staph. rostri, whereas 2 others were identified as Staphylococcus epidermidis. None of the Staph. rostri isolates displayed resistance to the antimicrobial agents tested. Mean quarter milk somatic cell count (qSCC) of all samples collected throughout lactation was 20,970 cells/mL. The qSCC at sampling of quarters infected with Staph. rostri (34,466 cells/mL) and CNS other than Staph. rostri (34,813 cells/mL) were significantly higher than the qSCC of noninfected quarters (20,287 cells/mL), yet not significantly different from each other. These findings provide novel insight into the prevalence and distribution, antimicrobial susceptibility, and relevance of Staph. rostri compared with other CNS species causing IMI in water buffaloes. Further studies are needed to pinpoint the relevance, niches, and transmission routes of Staph. rostri, as well as other CNS in water buffaloes.

Short communication: Epidemiology and genotyping of Candida rugosa strains responsible for persistent intramammary infections in dairy cows.

The present study was undertaken during an outbreak of clinical and subclinical mastitis in 14 dairy cows caused by Candida rugosa, in which high somatic cell counts were seen and cases did not respond to antibiotic treatment. Intramammary infection cured spontaneously in 10 cows, whereas 4 cows were culled as a result of persistent infections. Repeated sampling of these cows and biomolecular analysis of the isolates showed that the infections were caused by the same genotype, even over a period of 2 lactations. Random amplification of the genome of C. rugosa milk isolates gave 3 different DNA banding patterns (genotypes G1, G2, and G3). Viable cells of C. rugosa were also isolated from various environmental sources and were present in high concentrations in total mixed ration samples, which could be considered the primary source of diffusion of viable yeast cells in the environment, as demonstrated by genotyping. The proven capacity of these microorganisms to survive in the environment of the cow, such as the total mixed ration, bedding, water, and cow skin, and to cause persistent intramammary infections highlights the importance of mycotic spread in dairy herds.

Differentially expressed genes associated with Staphylococcus aureus mastitis in dairy goats.

To study gene expression within the mammary glands of dairy goats with mastitis, mRNA was collected from milk somatic cells (MSCs) of left udder halves challenged with Staphylococcus aureus and right udder halves infused with PBS, as control, at different time points (0, 12, 24 and 48h post-infection). Transcriptional profiles were investigated using bovine cDNA microarrays; of the total 288 differentially expressed genes identified with ANOVA analysis (False Discovery Rate=0.05, 1.5-fold change), 26, 36 and 16 genes were down-regulated at 12, 24 and 48h post-infection, respectively, while 60, 141 and 9 genes were up-regulated at the same corresponding time points. The expression profiles clearly changed at 24h post-infection with 177 genes significantly altered, corresponding to a 10-fold increase of S. aureus bacterial count in milk from infected udders. Differential expression of selected genes (CD2BP2, BCAP31, MHCII, FOSL2, MAPK13, ILT5 and JUNB) was also confirmed by real-time PCR at the different time points considered, showing high correlation with the microarray measurements and high reliability of the microarray analyses. The most readily inducible classes of genes in caprine MSCs infected with S. aureus were pro-inflammatory cytokines, chemokines and their receptors; IL-1alpha, lymphotoxin alpha, granulocyte chemotactic protein (CXCL6), and IL-2 receptor gamma were all up-regulated in infected udders versus healthy controls. This study identified a number of differentially expressed genes induced by S. aureus intramammary infection and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.

Pathogen detection in milk samples by ligation detection reaction-mediated universal array method.

This paper describes a new DNA chip, based on the use of a ligation detection reaction coupled to a universal array, developed to detect and analyze, directly from milk samples, microbial pathogens known to cause bovine, ovine, and caprine mastitis or to be responsible for foodborne intoxication or infection, or both. Probes were designed for the identification of 15 different bacterial groups: Staphylococcus aureus, Streptococcus agalactiae, nonaureus staphylococci, Streptococcus bovis, Streptococcus equi, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus uberis, Streptococcus pyogenes, Mycoplasma spp., Salmonella spp., Bacillus spp., Campylobacter spp., and Escherichia coli and related species. These groups were identified based on the 16S rRNA gene. For microarray validation, 22 strains from the American Type Culture Collection or other culture collections and 50 milk samples were tested. The results demonstrated high specificity, with sensitivity as low as 6 fmol. Moreover, the ligation detection reaction-universal array assay allowed for the identification of Mycoplasma spp. in a few hours, avoiding the long incubation times of traditional microbiological identification methods. The universal array described here is a versatile tool able to identify milk pathogens efficiently and rapidly.

Epidemiological investigation of Streptococcus equi subspecies zooepidemicus involved in clinical mastitis in dairy goats.

An outbreak of clinical mastitis was observed in dairy goats due to the zoonotic pathogen Streptococcus equi ssp. zooepidemicus. Affected goats were culled to prevent transmission of infection to other animals or humans. The objective of the study was to determine whether horses on the same farm were the source of the pathogen. Streptococcus equi ssp. zooepidemicus was obtained from milk of 10% of goats in the herd and from feces of 3 of 7 healthy horses that shared pasture and housing with the goats. Isolates of caprine and equine origin had identical biochemical profiles, including the ability to ferment sorbitol and lactose, which distinguishes S. equi ssp. zooepidemicus from S. equi ssp. equi. Sequencing of the 16S-23S intergenic spacer region and results from sodA-seeI multiplex PCR supported identification of isolates as S. equi ssp. zooepidemicus. Based on random amplified polymorphic DNA typing and rpoB and sodA sequencing, caprine isolates were indistinguishable from each other, but distinct from equine isolates. Further analysis of equine fecal samples showed that multiple strains of S. equi ssp. zooepidemicus can be present in a single sample or in sequential samples obtained from a single horse. Failure to detect the mastitis-causing strain in equine feces may indicate that horses were not the source of the mastitis outbreak in goats. Alternatively, the outbreak may be due to presence of multiple S. equi ssp. zooepidemicus strains in equine feces and a failure to detect all strains when analyzing a limited number of isolates per sample.

Short communication: isolation of Prototheca species strains from environmental sources in dairy herds.

Composite milk samples from 548 cows, and samples from feces, feed, bedding, water, liners (before and after milking), and the postdipping product were aseptically collected from 2 Italian dairy herds from February to November of 2006. Prototheca zopfii was isolated from 11.9% of milk samples, 15% of feces, and 33.3% of bedding samples. No viable cells of P. zopfii were observed in water before washing procedures, whereas 25 to 28.6% of samples from water used for washing both refrigeration tanks and milking equipment were contaminated with this yeast-like microalga. Analogously, the presence of P. zopfii was detected only on swabs collected from the liners after milking. Interestingly, in 1 of the 2 herds, water from the drinking trough was contaminated by viable cells of both P. zopfii and the related environmental species Prototheca stagnora. No viable cells were observed in cow feed. On the basis of the results presented herein, P. zopfii seemed to be widespread throughout the environments of dairy herds where outbreaks of bovine mastitis had occurred.

Rhabdomyosarcoma of the pectoral muscles of a free-living European robin (Erithacus rubecula).

An adult free-living European robin (Erithacus rubecula) with a large, firm, subcutaneous mass on the pectoral muscle was examined. The bird was unable to fly and died spontaneously. Necropsy revealed a yellowish, bilobate mass almost completely replacing the pectoral muscles with extensive osteolysis of the keel bone. Histopathology revealed a poorly demarcated, highly cellular sarcomatous tumour with metastases to the lungs, pulmonary blood vessels and heart. Immunohistochemistry was negative for neuron-specific enolase, S-100 protein and the p-27 major capsid protein of avian leukosis viruses. The homogeneously positive immunolabelling for vimentin and scattered positivity for myoglobin and desmin suggested a diagnosis of rhabdomyosarcoma. A retrospective examination of the records for 194 birds of the thrush family, including 64 robins submitted over a 20-year period, showed no diagnoses of neoplasia.

Short communication: Outbreak of Nocardia neocaledoniensis mastitis in an Italian dairy herd.

Nocardia spp. are an uncommon cause of mastitis, and outbreaks have typically been reported in dairy farms with poor hygienic and management conditions. The outbreak described herein involved a dairy farm with 43 lactating cows that, after a long period with low bulk milk somatic cell counts (<180,000 cells/mL), experienced an increasing incidence of clinical mastitis with bulk milk somatic cell counts greater than 300,000 cells/mL. Fifteen mastitic quarters milk samples from 9 dairy cows were found to be infected by a member of the genus Nocardia, as identified on the basis of selected phenotypic and chemotaxonomic characteristics. The isolates were confirmed as Nocardia neocaledoniensis by 16S rDNA gene sequencing. Average quarter milk somatic cell count for infected udders was 863,057 cells/mL, significantly greater than the average value in noninfected quarters (189,710 cells/mL).

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese.

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.

Mycobacterium genavense and avian polyomavirus co-infection in a European goldfinch (Carduelis carduelis).

Systemic mycobacteriosis associated with avian polyomavirus infection was diagnosed histologically in an 8-year-old, captive European goldfinch with a history of nervous signs. Severe mycobacterial lesions were observed in the central nervous system, lungs, cervical air sacs and adrenal glands, without involvement of the gastrointestinal tract. In addition to mycobacteriosis, intranuclear inclusions, typical of polyomavirus, were identified in the adrenal glands. Polymerase chain reaction assays were used to identify Mycobacterium genavense and finch polyomavirus as the causative agents. The absence of involvement of the gastrointestinal tract and the severity of the lesions in the respiratory tract suggested that inhalation may have been the primary route of infection with M. genavense.

Epornitic of avian pox in common buzzards (Buteo buteo): virus isolation and molecular biological characterization.

Six common buzzards from a bird rescue centre showed wart-like lesions on their toes. The lesions consisted of multiple crusty and proliferative nodules surrounded by skin swelling. Histologically, epithelial cell hypertrophy and hyperplasia with ballooning degeneration and large intracytoplasmic inclusion bodies consistent with avipoxvirus infection were seen. The virus was isolated in embryonated chicken eggs. Positive chorioallantoic membranes and samples of skin lesions were submitted for polymerase chain reaction. Molecular characterization based on the 4b core protein indicates a 100% homology of the isolated poxvirus with avian poxviruses belonging to subclade A2. However, analysis of fpv139 locus does not reveal similarities of the isolate with other avian poxviruses.

Influence of estrus of dairy goats on somatic cell count, milk traits, and sex steroid receptors in the mammary gland.

Two experiments were conducted to study the effect of the stage of a spontaneous estrus cycle on milk yield and constituents [somatic cell count (SCC), fat, protein, caseins, lactose, and urea content] and on estrogen receptor-alpha (ERalpha ) and progesterone receptor (PR) immunostaining in the mammary gland. In experiment I, the major components of milk and SCC were monitored weekly in 80 lactating Saanen goats for 6 wk, whereas detection of estrus was daily. In experiment II, milk samples were collected daily for SCC determination during 1 spontaneous estrus (d 0) until the second spontaneous estrus in 14 Saanen goats. The day of the estrous cycle was confirmed by plasma progesterone and 17beta-estradiol levels. Immunoreactivity of ERalpha and PR was analyzed in mammary gland samples of 8 Saanen goats (d 0, n = 4; d 10, n = 4) and the number of positive nuclei and intensity of the staining were evaluated in 1,000 cells. In experiment I, milk casein and protein percentages were significantly affected by the stage of estrous cycle; during proestrus and estrus, these variables were higher (3.32 +/- 0.06 and 4.44 +/- 0.08) than during metestrus (3.03 +/- 0.07 and 4.07 +/- 0.10), but not higher than during diestrus (3.23 +/- 0.06 and 4.35 +/- 0.09, respectively). In experiment II, daily measurement of SCC revealed higher levels at estrus (7,195 +/- 672 x 10(3) cells/mL) and a decline toward the luteal phase (1,694 +/- 672 +/- 10(3) cells/mL). Estrogen receptor-alpha and PR immunostaining were exclusively detected on epithelial cells. The percentage of positive nuclei to ERalpha was higher on d 0 than on d 10 (75.4 +/- 8.8 vs. 68.3 +/- 8.8%), but no change was observed for PR (4.0 +/- 0.3 vs. 3.5 +/- 0.4%). The average immunostaining intensity for both receptors was greater on d 0 than on d 10 (ERalpha : 1.44 +/- 0.02 vs. 1.35 +/- 0.02; PR: 0.079 +/- 0.008 vs. 0.057 +/- 0.008). The high SCC at estrus in experiment II was associated with high plasma estradiol and low progesterone, suggesting that the increased SCC could be brought about by the estrogen-induced proliferation and exfoliation of epithelial cells. In addition, this action may be supported by the higher sensitivity to estrogens (ERalpha content) found at d 0.

Molecular typing of Staphylococcus aureus isolated from cows, goats and sheep with intramammary infections on the basis of gene polymorphisms and toxins genes.

We investigated 116 Staphylococcus aureus isolates from cows, goats and sheep with intramammary infections (IMI) in Italy to provide information about the spread of enterotoxigenic strains and to compare strains isolated from different ruminant species. The isolates were typed by restriction fragment length polymorphism (RFLP) analysis of the coagulase (coa) gene, by analysis of polymorphisms of the X region of protein A (spa) gene and by detection of genes encoding enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej and sel). Seven different coa types and 12 different spa types were distinguished. On the basis of polymerase chain reaction-RFLP, 29 different coa subtypes were identified. Two different coa subtypes accounted for 49% and 67% of bovine and ovine isolates respectively. Only seven coa subtypes were observed in isolates from more than one host species and no coa subtype was present in isolates from all three ruminant species. Furthermore, 85 of the isolates (73%) harboured at least one enterotoxin gene (se) with a predominance of sea, sed and sej among isolates from bovine IMI, and sec and sel among isolates from caprine and ovine IMI. Comparing the S. aureus isolates on the basis of gene polymorphisms and presence of se genes, significant differences were found in distributions of genotypes among isolates from cows, goats and sheep.