Liliana Odierno

Efficacy of immunization against bovine mastitis using a Staphylococcus aureus avirulent mutant vaccine

Bovine mastitis is responsible of major economic losses on dairy farms worldwide. In Argentine dairy herds, Staphylococcus aureus is the main causative agent of the disease. The ineffectiveness of some current practices to control S. aureus infections, often leads to a chronic and recurrent infection with persistent bacterial reservoir within a herd. Vaccination against S. aureus seems to be a rational approach for the control of the disease. In the present study, we investigate the response of dairy heifers after a combined immunization schedule with the avirulent mutant RC122 S. aureus vaccine. Vaccinated and non-vaccinated heifers were challenged 40 days after calving with the parental virulent strain. After challenge, and during the study period, milk bacterial recovery was significantly higher in non-vaccinated heifers than vaccinates. Importantly, inoculated bacteria could not be isolated from the milk of vaccinated heifers until 72 h after challenge, and the overall percentage of infected quarters in these animals was significantly lower. An increase in the level of specific IgG was observed in blood and milk of vaccinated heifers during the trial. At calving, IgG(2) was the main antibody isotype found in blood. Immune sera from vaccinated heifers increased phagocytosis over sera from non-vaccinated heifers and were able to opsonize heterologous S. aureus strains. Results demonstrated that immunization of dairy heifers with strain RC122 was able to elicit a significant opsonic antibody production in blood and milk and provides protection by a significant reduction in bacterial shedding after challenge.

Distribution of virulence-associated genes in Streptococcus uberis isolated from bovine mastitis

Streptococcus uberis is an important pathogen that has been implicated in bovine mastitis but the virulence factors associated with pathogenesis are not well understood. The aim of this work was to examine 11 putative and known virulence-associated genes by PCR in 78 S. uberis strains isolated from infected animals in Argentina. Additionally, the distribution of virulence patterns over various herds was determined. Not all genes were present in the strains but all of the detected virulence-associated genes were present in combination. Forty-seven (60.3%) isolates carried seven to 10 virulence-associated genes. Further analysis revealed 58 virulence patterns. Different patterns were found within the same herd and among herds, demonstrating that strains with different virulence patterns were able to cause mastitis. Despite the large number of strains with different virulence patterns, strains with identical patterns was found. Detection of virulence-associated genes in individual S. uberis strains isolated from infected animals revealed one to 10 virulence genes. This may indicate that other virulence factors could be involved. The present study reveals the occurrence and distribution of 11 virulence-associated genes among S. uberis isolates from bovine mastitis in various herds and contributes to a better understanding of the pathogenicity of this bacterium.

Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms

The aim of this study was to investigate the phenotypic and genotypic characteristics of Streptococcus uberis isolated from subclinical mastitis (SCM) cases, and to examine the possible association between both characteristics. A total of 32 S. uberis were isolated from 772 quarter milk samples (SCM > 250,000 cells/ml) collected from 195 cows selected randomly from 18 dairy farms located in Argentina. The S. uberis strains were characterized phenotypically by the presence of virulence factors as plasminogen activator factor (PAF), hyaluronidase (HYA), capsule (CAP) and CAMP factor, and were further characterized genotypically by pulsed-field gel electrophoresis (PFGE). S. uberis strains expressed plasminogen activator factor, hyaluronidase or capsule (65.5 %, 56.3 %, 59.4 %, respectively), but only 25 % of isolates were CAMP factor positive. Thirteen different virulence profiles were identified on the basis of the combination of virulence factors. Eighteen PFGE patterns with 90% of similarity were identified among 32 S. uberis. A great diversity of virulence profiles and PFGE patterns were present among dairy farms. S. uberis strains with the same PFGE pattern showed different virulence profiles. Bovine S. uberis strains causing SCM included in the present study showed heterogeneity in regard to their phenotypic and genotypic characteristics, and the PFGE patterns are not associated with the virulence profiles.

Development of an experimentally induced Streptococcus uberis subclinical mastitis in goats.

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

Prevalence and antibiotic susceptibility of coagulase-negative Staphylococcus species from bovine subclinical mastitis in dairy herds in the central region of Argentina

Coagulase-negative staphylococci (CNS) are a common cause of bovine subclinical mastitis (SCM). The prevalence of CNS species causing SCM identified by genotyping varies among countries. Overall, the antimicrobial resistance in this group of organisms is increasing worldwide; however, little information exists about a CNS species resistant to antibiotics. The aim of the present study was to genotypically characterize CNS at species level and to determine the prevalence and antibiotic resistance profiles of CNS species isolated from bovine SCM in 51 dairy herds located in the central region of the province of Cordoba, Argentina. In this study, we identified 219 CNS isolates at species level by PCR-restriction fragment length polymorphism of the groEL gene. Staphylococcus chromogenes (46.6%) and Staphylococcus haemolyticus (32%) were the most prevalent species. A minimum of three different CNS species were present in 41.2% of the herds. S. chromogenes was isolated from most of the herds (86.3%), whereas S. haemolyticus was isolated from 66.7% of them. The broth microdilution method was used to test in vitro antimicrobial susceptibility. Resistance to a single compound or two related compounds was expressed in 43.8% of the isolates. S. chromogenes and S. haemolyticus showed a very high proportion of isolates resistant to penicillin. Resistance to two or more non-related antimicrobials was found in 30.6% of all CNS. S. haemolyticus exhibited a higher frequency of resistance to two or more non-related antimicrobials than S. chromogenes.

Accuracy of the composite somatic cell count to detect intra-mammary infection in dairy cows using latent class analysis.

The somatic cell count (SCC) is considered an important indicator of intra-mammary infection (IMI). The purpose of this study was to determine the accuracy of both SCC and culture to detect IMI and their conditional dependence by means of latent class methods. This study involved 175 dairy cows from 2 herds with different udder infection prevalences. Quarter and composite milk samples were collected for SCC and bacteriological culture. Latent-class models using Bayesian methods were used to estimate test sensitivity (Se) and specificity (Sp) and population prevalence. The models ran involved only major mastitis pathogens and composite SCC (CSCC). Five thresholds between 100,000 and 300,000 cells/mL were evaluated and the receiver operating characteristics (ROC) curve analysis was performed. Fifty-five percent of the cows had CSCC ≥200,000 cells/mL and 95.4% of the cows had at least one infected quarter either with minor or major pathogens. Considering a threshold of 150,000 cells/mL, the estimated Se and Sp for the CSCC were, 0.80 (95% CrI 0.71-0.88) and 0.57 (95% CrI 0.44-0.71), respectively. The estimated culture Se and Sp were 0.83 (95% CrI 0.73-0.93) and 0.89 (95% CrI 0.74-0.98), respectively. There was no evidence of dependence between CSCC and culture. The area under curve for CSCC was 0.72. To the best of our knowledge, this is the first report of the CSCC accuracy to detect IMI for major pathogens considering the effect of culture misclassification. The estimates provided here could help to examine the performance of sampling schemes based on CSCC to manage udder health.

Phenotyping and genotyping of streptococci in bovine milk in Argentinean dairy herds.

Most veterinary and milk hygiene laboratories identify streptococci and enterococci based on serological and biochemical tests. The analysis of 16S rDNA was suggested to be used for more exact identification; however, its use has not been considered so far in monitoring studies. The objective of the present study was to compare a conventional phenotypic method with restriction fragment length polymorphism analysis of 16S rDNA (16S rDNA RFLP) for identification of streptococci isolated from composite milk samples collected in connection with intramammary infection (IMI) in six Argentinean dairy farms. Composite milk samples (n = 1223) from cows belonging to six herds were collected for bacteriological analysis. Twelve reference strains and fifty streptococci or streptococcuslike isolates were identified to species level by the API 20 Strep system, conventional biochemical tests and 16S rDNA RFLP in a blind assay. The remaining streptococci or streptococcus-like isolates (n = 40) were identified to the species level both by 16S rDNA RFLP and conventional biochemical tests. As indicated by Kappa values, agreement between the 16S rDNA RFLP and the conventional scheme for identification of Streptococcus agalactiae, S. dysgalactiae, S. uberis, S. equinus and Enterococcus faecalis was 0.91, 0.73, 0.92, 0.81 and 0.85, respectively. Together with the less frequently isolated streptococcal species, the conventional scheme correctly identified 77 out of 90 isolates (85.5%). Thus, the use of 16S rDNA RFLP is considered valuable for monitoring studies due to its affordable cost for standard laboratories.

Calidad microbiológica del agua utilizada en establecimientos lecheros de la zona de Villa María (Córdoba)

Resumen es: Se evaluo la calidad bacteriologica del agua de pozo y del agua de lavado en una muestra aleatoria de 50 tambos distribuidos en la cuenca lechera de Vill...

Streptococcus uberis: In vitro biofilm production in response to carbohydrates and skim milk

Streptococcus uberis has become one of the most important environmental pathogens associated with clinical and subclinical bovine mastitis. Biofilm confers to bacteria more resistance to physical and chemical agents as well as to different mechanisms of the innate immune system. The aim of this work was to evaluate the ability of in vitro biofilm production in 32 S. uberis isolates from bovine mastitis and identified by biochemical tests and subsequently confirmed by the amplification of the pauA gene. The isolates were cultivated in TMP broth and TMP broth with the addition of 0.5% glucose, 1% sucrose, 1% lactose or 0.5% skim milk in microtiter plates stained with crystal violet. We demonstrated that S. uberis isolated from bovine mastitis are able to produce biofilms in TMP broth and, also that biofilm formation by S. uberis can be significantly enhanced by the addition of 0.5% glucose or 1% sucrose to TMP broth. This may suggest that the carbohydrates in milk or within the ruminant gut might affect the growth mode of S. uberis. In addition, our results showed that in vitro biofilm production under different conditions of supplementation displays variation among the isolates and that each isolate shows a particular profile of biofilm production. This phenotypic heterogeneity in biofilm production exhibited by S. uberis could at least partly explain why this bacterium has the ability to adapt to different niches facilitating survival to diverse and stressful conditions.

Genetic patterns of Streptococcus uberis isolated from bovine mastitis

The aim of this study was to evaluate the genotypic relationships among 40 Streptococcus uberis isolated from bovine mastitis by using pulsed-field gel electrophoresis (PFGE). Additionally, the association between PFGE patterns and virulence profiles was investigated. The isolates exhibited 17 PFGE patterns. Different strains were found within and among herds; however, a low number of isolates within the same herd shared an identical PFGE type. No association between PFGE patterns and virulence profiles was found. However, the detection of specific strains in some herds could indicate that some strains are more virulent than others. Further research needs to be undertaken to elucidate new virulence-associated genes that might contribute to the capability of these strains to produce infection.