Circulation of Streptococcus agalactiae ST103 in a Free Stall Italian Dairy Farm.

We report here on an outbreak of mastitis caused by Streptococcus agalactiae, or group B Streptococcus, in a northern Italy (Lombardy Region) free stall dairy farm. This outbreak was unusual because it occurred in a closed dairy herd and proved to be extremely difficult to resolve even after the application of the classical control procedures, which are specifically focused on the contagious nature of S. agalactiae. In order to better understand the potential origins of the pathogen and the critical points that could impair the eradication program and to investigate the possible presence of S. agalactiae in sources outside the mammary gland, we collected 656 individual composite milk samples, 577 samples from extramammary body sites (289 rectal, 284 vaginal, and four throat samples from milking cows, dry cows, heifers, and calves), and 81 samples from the cattle environment, including the milking parlor and the barn. Twenty-two S. agalactiae isolates were obtained from lactating cows or their environment. Of these, nine were isolated from milk, two were from rectal swabs, and two were from vaginal swabs, while nine were isolated from environmental samples. Based on molecular serotyping, pilus island (PI) typing and multilocus sequence typing, all isolates belonged to serotype III, pilus type PI-1/2b, and sequence type 103 (ST103), a type previously described to have an environmental transmission cycle and a potential human origin. Once the classical mastitis control measures were supplemented with environmental hygiene measures, herd monitoring using bulk tank milk revealed no further positive results for S. agalactiae, and the outbreak was considered resolved. IMPORTANCE Streptococcus agalactiae is an important pathogen in humans and cattle. Bovine mastitis caused by this bacterium and its control are generally associated with contagious transmission between animals. More recently, the presence of a fecal-oral transmission cycle in cattle has been proposed, linked to the ability of some S. agalactiae strains to survive in the bovine gastrointestinal tract and environment. Based on analysis of 1,316 specimens from cattle and their environment on a single dairy farm, we demonstrate the presence of sequence type 103 (ST103), which may have an environmental mode of transmission. This possibility was supported by the fact that the mastitis outbreak could not be controlled through measures to prevent contagious transmission alone and required additional environmental hygiene measures to be brought to a halt. This case study highlights that measures to control animal disease need to evolve alongside the microorganisms that cause them.

Comparison of the response of mammary gland tissue from two divergent lines of goat with high and low milk somatic cell scores to an experimental Staphylococcus aureus infection.

Mastitis represents one of the major economic and health threats to the livestock sector associated with reduction in milk quality, loss of production and is a major reason for culling. Somatic cell score (SCS) is used as a criterion in breeding programmes to select cows genetically less susceptible to mastitis. The relevance of SCS as a predictor of udder health and susceptibility to mastitis is still untested in goats. In this study, two lines of French Alpine goats selected for extreme breeding values for somatic cell scores, one line with high SCS (HSCS) and the other with low SCS (LSCS), were used to test the hypothesis that the mammary response and function differed between the lines. The aim of the present study was to investigate differences in the early immune response in caprine mammary gland tissues challenged with Staphylococcus aureus, one of the main pathogens responsible for the intra-mammary infection in small ruminants, using transcriptomic and histopathology analyses. The comparison between HSCS and LSCS goat lines, showed differences in the response at the histological level for inflammation, presence of neutrophils and micro-abscess formation, and at the molecular level in the expression of CXCL8, IL-6, NFKBIZ and IL-1ß. CXCL8 and CXCL2 genes, which showed a higher level of expression in the experimentally infected HSCS line. The molecular data and histopathology both suggested that following S. aureus infection, mobilization, recruitment, infiltration, and chemotaxis of neutrophil, leads to a more severe inflammation in the HSCS compared to LSCS animals. Our results represent an initial basis for further studies to unravel the genetic basis of early mastitis inflammatory responses and the selection of dairy animals more resistant to bacterial mastitis.

Host range of mammalian orthoreovirus type 3 widening to alpine chamois.

Mammalian orthoreoviruses (MRV) type 3 have been recently identified in human and several animal hosts, highlighting the apparent lack of species barriers. Here we report the identification and genetic characterization of MRVs strains in alpine chamois, one of the most abundant wild ungulate in the Alps. Serological survey was also performed by MRV neutralization test in chamois population during five consecutive years (2008-2012). Three novel MRVs were isolated on cell culture from chamois lung tissues. No respiratory or other clinical symptoms neither lung macroscopic lesions were observed in the chamois population. MRV strains were classified as MRV-3 within the lineage III, based on S1 phylogeny, and were closely related to Italian strains identified in dog, bat and diarrheic pig. The full genome sequence was obtained by next-generation sequencing and phylogenetic analyses showed that other segments were more similar to MRVs of different geographic locations, serotypes and hosts, including human, highlighting genome reassortment and lack of host specific barriers. By using serum neutralization test, a high prevalence of MRV-3 antibodies was observed in chamois population throughout the monitored period, showing an endemic level of infection and suggesting a self-maintenance of MRV and/or a continuous spill-over of infection from other animal species.

Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates.

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.

Contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection to Staphylococcus aureus.

To differentiate between the contribution of mammary epithelial cells (MEC) and infiltrating immune cells to gene expression profiles of mammary tissue during early stage mastitis, we investigated in goats the in vivo transcriptional response of MEC to an experimental intra mammary infection (IMI) with Staphylococcus aureus, using a non-invasive RNA sampling method from milk fat globules (MFG). Microarrays were used to record gene expression patterns during the first 24 hours post-infection (hpi). This approach was combined with laser capture microdissection of MEC from frozen slides of mammary tissue to analyze some relevant genes at 30 hpi. During the early stages post-inoculation, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signalling pathway and initiate a sharp induction of innate immune genes predominantly associated with the pro-inflammatory response. At 30 hpi, MEC express genes encoding different acute phase proteins, including SAA3, SERPINA1 and PTX3 and factors, such as S100A12, that contribute directly to fighting the infection. No significant change in the expression of genes encoding caseins was observed until 24 hpi, thus validating our experimental model to study early stages of infection before the occurrence of tissue damage, since the milk synthesis function is still operative. This is to our knowledge the first report showing in vivo, in goats, how MEC orchestrate the innate immune response to an IMI challenge with S. aureus. Moreover, the non-invasive sampling method of mammary representative RNA from MFG provides a valuable tool to easily follow the dynamics of gene expression in MEC to search for sensitive biomarkers in milk for early detection of mastitis and therefore, to successfully improve the treatment and thus animal welfare.

Response of the goat mammary gland to infection with Staphylococcus aureus revealed by gene expression profiling in milk somatic and white blood cells.

BACKGROUND: S. aureus is one of the main pathogens responsible for the intra-mammary infection in dairy ruminants. Although much work has been carried out to understand the complex physiological and cellular events that occur in the mammary gland in response to S. aureus, the protective mechanisms are still poorly understood. The objectives of the present study were to investigate gene expression during the early response of the goat mammary gland to an experimental challenge with S. aureus, in order to better understand the local and systemic response and to compare them in two divergent lines of goat selected for high and low milk somatic cell scores. RESULTS: No differences in gene expression were found between high and low SCS (Somatic Cells Score) selection lines. Analysing the two groups together, an expression of 300 genes were found to change from T0 before infection, and T4 at 24 hours and T5 at 30 hours following challenge. In blood derived white blood cells 8 genes showed increased expression between T0 and T5 and 1 gene has reduced expression. The genes showing the greatest increase in expression following challenge (5.65 to 3.16 fold change) play an important role in (i) immune and inflammatory response (NFKB1, TNFAIP6, BASP1, IRF1, PLEK, BATF3); (ii) the regulation of innate resistance to pathogens (PTX3); and (iii) the regulation of cell metabolism (CYTH4, SLC2A6, ARG2). The genes with reduced expression (-1.5 to -2.5 fold) included genes involved in (i) lipid metabolism (ABCG2, FASN), (ii) chemokine, cytokine and intracellular signalling (SPPI), and (iii) cell cytoskeleton and extracellular matrix (KRT19). CONCLUSIONS: Analysis of genes with differential expression following infection showed an inverse relationship between immune response and lipid metabolism in the early response of the mammary gland to the S. aureus challenge. PTX3 showed a large change in expression in both milk and blood, and is therefore a candidate for further studies on immune response associated with mastitis.

Molecular characterization and phylogenetic analysis of small ruminant lentiviruses isolated from Canadian sheep and goats.

BACKGROUND: Small Ruminant Lentiviruses (SRLV) are widespread in Canadian sheep and goats and represent an important health issue in these animals. There is however no data about the genetic diversity of Caprine Arthritis Encephalitis Virus (CAEV) or Maedi Visna Virus (MVV) in this country. FINDINGS: We performed a molecular and phylogenetic analysis of sheep and goat lentiviruses from a small geographic area in Canada using long sequences from the gag region of 30 infected sheep and 36 infected goats originating from 14 different flocks. Pairwise DNA distance and phylogenetic analyses revealed that all SRLV sequences obtained from sheep clustered tightly with prototypical Maedi visna sequences from America. Similarly, all SRLV strains obtained from goats clustered tightly with prototypical US CAEV-Cork strain. CONCLUSIONS: The data reported in this study suggests that Canadian and US SRLV strains share common origins. In addition, the molecular data failed to bring to light any evidence of past cross species transmission between sheep and goats, which is consistent with the type of farming practiced in this part of the country where single species flocks predominate and where opportunities of cross species transmissions are proportionately low.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources.

BACKGROUND: Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. RESULTS: Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. CONCLUSIONS: This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

Multinucleated giant cells with an osteoclast phenotype derived from caprine peripheral blood mononuclear cells.

Formation of multinucleated giant cells (MGCs) by macrophage fusion is a typical cytopathic effect of lentiviral replication in caprine monocytes and MGC formation from cultured caprine peripheral blood mononuclear cells (PBMCs) has been considered to be diagnostic for small ruminant lentivirus (SRLV) infection. In this study, formation of MGCs was observed after 7-14 days when PBMCs were cultured from healthy goats free from SRLV infection. These MGCs expressed tartrate-resistant acid phosphatase, calcitonin receptor, integrin αVß3, cathepsin K and matrix metalloproteinase 9 and were able to resorb bone in vitro in the absence of RANKL and macrophage colony stimulating factor, consistent with an osteoclast phenotype.

Genetic analysis of small ruminant lentiviruses following lactogenic transmission.

Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission.

Suppurative adenitis of preputial glands associated with Corynebacterium mastitidis infection in mice.

Suppuration of the preputial gland in mice occurs as a septic complication of fight wounds around the external genitalia. Currently reported bacterial isolates from these lesions are limited to Staphylococcus aureus, Pasteurella pneumotropica, and Klebsiella oxytoca. In the context of a pilot experiment aimed at defining the aging phenotype of estrogen receptor beta knockout (BERKO) mice, 2 male mice (1 of the BERKO line and the other from the age- and sex-matched wild-type control group) were discovered at necropsy to have preputial gland lesions. In both cases, histopathologic examination confirmed severe suppuration and abscesses of the preputial glands associated with systemic reactive (secondary) amyloidosis. Both Gram staining and Bacillus Calmette-Guérin immunohistochemistry highlighted the presence of numerous bacillary to rod-shaped bacteria within the preputial lesions. Subsequent PCR analysis coupled with denaturing gradient gel electrophoresis identified Corynebacterium mastitidis in the preputial gland abscesses. This organism is isolated infrequently from the milk of sheep with subclinical mastitis and was identified as part of the normal microflora of the human ocular surface. No information regarding the epidemiology and pathogenesis of C. mastitidis infection in laboratory animals is currently available, and to our knowledge this report is the first description of C. mastitidis infection in mice.

Molecular biological characterization of avian poxvirus strains isolated from different avian species.

Fifteen strains of Avipoxvirus from different avian species were isolated and molecular biologically characterized. Most strains did not produce evident pocks on the chorioallantoic membranes of commercial and specific-pathogen free embryonated chicken eggs where, on the contrary, microscopic signs of viral growth were always detected. Polymerase chain reaction of highly conserved P4b gene was positive for all cases confirming to be a reliable diagnostic method for Avipoxvirus. Sequencing of these amplicons confirmed most strains clustered either with Fowlpox virus or with Canarypox virus whereas a possible new clade could be hypothesized for one strain from Japanese quail. Classification of Avipoxvirus strains by amplification of the newly identified locus fpv140 revealed major limitations as only five samples were positive. These results underline the importance to undertake similar studies on higher numbers of Avipoxvirus isolates and on wider genomic regions of this large viral group.

Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node.

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I(f) current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.

Systemic and in vitro expression of goat alpha(1)-acid glycoprotein during Caprine Arthritis-Encephalitis Virus infection.

The present study was carried out in order to investigate the systemic and local expression of the acute-phase protein alpha(1)-acid glycoprotein (AGP, Orosomucoid) during Caprine Arthritis-Encephalitis Virus (CAEV) natural infection. The aminoacid sequence of goat AGP (gAGP), which was unknown, was determined by cDNA sequencing of the gene. AGP serum concentration was analyzed from 40 healthy and 36 CAEV-induced arthritis-affected goats. The mean concentration of AGP in healthy goats was of 219.8 microg/ml (+/-178.6 s.d.) and did not statistically differ from that of arthritis-affected goats (157 microg/ml, +/-137.8 s.d.). In a second set of experiments, AGP was purified to homogeneity from the serum of healthy and unhealthy goats, and the glycan pattern modifications were analyzed by means of specific binding with lectins. In particular, branching, fucosylation and alpha(1-6)- and alpha(1-3)-linked sialylation were analyzed. Goat AGP is not fucosylated in neither physiological nor pathological status. On the contrary, both major (branching) and minor (sialylation) microheterogeneities increase during arthritis. Finally, the possible local synovial origin of gAGP was determined by means of in vitro expression studies (real-time PCR) which used goat synovial membrane (GSM) as cellular model. It was found that gAGP's mRNA can be constitutively produced by GSM cells, but real-time PCR experiments revealed that the expression of AGP was not influenced by in vitro infection with CAEV.

Avian mycobacteriosis in companion birds: 20-year survey.

The causative agents of avian mycobacteriosis in pet birds are rarely identified. The aim of this study is to add information about the etiology of avian mycobacteriosis. The identification of mycobacterium species in 27 cases of avian mycobacteriosis in pet birds was investigated by polymerase chain reaction (PCR) and sequencing of a rRNA hypervariable region. Avian mycobacteriosis appeared to be an infrequent diagnosis. Interestingly, a few cases of avian mycobacteriosis were recorded in very young birds. The most commonly affected species were the canary (Serinus canarius), the Eurasian goldfinch (Carduelis carduelis) and the red siskin (Spinus cucullatus). All but one bird were infected with Mycobacterium genavense. Mycobacterium avium was identified only in one case.

Seroprevalence, clinical incidence, and molecular and epidemiological characterisation of small ruminant lentivirus in the indigenous Passirian goat in northern Italy.

Eight dairy flocks, comprising a total of 323 indigenous Passirian goats from northern Italy, were examined to determine the seroprevalence and clinical incidence of small ruminant lentivirus (SRLV) infections and to identify the SRLV subtypes. The seroprevalence was 81.5% (55-95%). The clinical incidence was 2.5% (0-8.3%) and was apparently low due to the practice of culling clinically affected animals. Phylogenetic analysis of eight PCR fragments (one sample from each flock) revealed that all proviruses belonged to the SRLV subtype B1, which suggests a common source of infection. Subtype B1 being the only circulating SRLV, coupled with the fact that mixed herd systems are very rare in South Tyrol, gives hope that an eradication programme in goats can be successful even without including sheep as long as sheep are kept strictly and permanently isolated.

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats.

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.