Matías Pellegrino

Lactic acid bacteria from raw milk as potentially beneficial strains to prevent bovine mastitis

Bovine mastitis produces a wide variety of problems in the dairy farm. The treatment of this disease is based on the use of antibiotics which are not always effective. These drugs are also responsible for the presence of residues in the milk and the increase of antibiotic-resistant strains. Probiotic products were proposed as a valid alternative to antibiotic therapies and are also useful for the prevention of infectious syndromes. With the aim of designing a probiotic product to prevent bovine mastitis, lactic acid bacteria (LAB) were isolated from foremilk samples from different dairy farms in Córdoba-Argentina. One hundred and seventeen LAB were isolated and their beneficial characteristics such as the production of inhibitory substances, surface properties and production of exopolysaccharides (EPS) were assessed. Most of them displayed low degree of hydrophobicity, autoaggregation, EPS negative phenotype and were identified as Enterococcus hirae and Pediococcus pentosaceus. Nine LAB strains inhibited three indicator bacteria. Some isolates were pre-selected and genetically identified according to the results obtained. Antibiotic resistance and virulence factors were studied for the assessment of the safety of the strains. The results obtained were compared to those reported previously from samples obtained in the North-western area of the country and some differences were found.

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.

Effects of intramammary inoculation of Lactobacillus perolens CRL1724 in lactating cows' udders

Bovine mastitis is the most important infectious disease on dairy farms. Conventional antibiotic therapy is often unsatisfactory and alternative treatments are continually under investigation. Lactobacillus (Lb.) perolens CRL 1724 and Lactobacillus plantarum CRL 1716 were previously isolated from milk of dairy cows and selected according to their potential probiotic properties. In the present work the in-vitro capacity of Lactobacillus strains to adhere to bovine teat canal epithelial cells (BTCEC) and to inhibit and co-aggregate 14 mastitis-causing pathogens (MCPs) was investigated. The effect of Lb. perolens CRL 1724 after intramammary inoculation in lactating cows was evaluated through determination of clinical signs of mastitis, milk appearance, somatic cell counts and Lb. perolens CRL 1724 recovery from milk. Lb. perolens CRL 1724 was able to inhibit 12 of 14 MCPs (85·7%) in vitro, especially those considered to be major pathogens. In addition, Lb. perolens CRL 1724 co-aggregated with all of them. Lb. plantarum CRL 1716 was able to inhibit 7 of 14 MCPs (50%) in vitro and showed co-aggregation ability similar to Lb. perolens CRL 1724. Lb. perolens CRL 1724 showed a higher efficacy of adhesion to BTCEC (values of percentage of adhesion and adhesion index of 75% and 14·4, respectively) than Lb. plantarum CRL 1716 (37% and 7·4, respectively). Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after the inoculation of 106 cfu/ml. The udders presented a normal aspect and there were no changes in the appearance of the milk. The results obtained will serve as the basis for further trials to evaluate the potential of Lb. perolens CRL 1724 to be included in a non-antibiotic formulation for the prevention of bovine mastitis.

Histological examination of non-lactating bovine udders inoculated with Lactobacillus perolens CRL 1724

The effect of intramammary inoculation of Lactobacillus perolens CRL 1724 on bovine udders at drying off was evaluated through histological examination of the canal and cistern tissues. The persistence of the strain in the udder 7 d post inoculation was also determined. Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after inoculation of 10(6) cfu/ml. The udders showed a normal structural aspect and there were no modifications of the milk appearance. Lb. perolens CRL 1724 cells were evidenced on the surface of the epithelial cells of the cistern without causing any morphological modifications or cell alterations. Lb. perolens CRL 1724 produces a mild inflammatory reaction, characterized by recruitment of neutrophils to the epithelial zone and a slight hyperaemia into blood vessels. This preliminary study provides important information for further studies directed towards the inclusion of Lb. perolens CRL 1724 in the design of probiotic products for preventing bovine mastitis in non-lactating dairy cows.

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.

Efficacy of corn silage inoculants on the fermentation quality under farm conditions and their influence on Aspergillus parasitucus, A. flavus and A. fumigatus determined by q-PCR

Laboratory-scale silos were prepared to evaluate the efficacy of two different lactic acid bacteria (LAB) on the fermentation quality and mycobiota of corn silage. Their influence on Aspergillus species’ variability by using the q-PCR technique was studied. Silage inoculated with Lactobacillus rhamnosus RC007 or L. plantarum RC009 were compared with uninoculated silage. Silos were opened after 1, 7, 45, 90 and 120 days after ensiling. At the end of the ensiling period, silos were left open for 7 days to evaluate aerobic stability. Rapid lactic acid production and decline in pH values were seen in the early stages of fermentation in silage inoculated with L. rhamnosus RC007. After aerobic exposure, a significant decline in lactic acid content was observed in untreated and L. plantarum RC009-inoculated silages. Counts for yeasted and toxigenic fungus remained lower, after aerobic exposure, in L. rhamnosus RC007-inoculated silage, in comparison with L. plantarum RC009 and uninoculated silages. Comparing the influence exerted by both BAL, it was observed that L. rhamnosus RC007 was more efficient at inhibiting the three fungal species tested whose DNA concentrations, determined by q-PCR, oscillated near the initial value (pre-ensiling maize). The ability of L. rhamnosus RC007 to produce lactic acid rapidly and the decline in pH values in the early stages of the fermentation along with the reduction of yeast and mycotoxicogenic fungus after aerobic exposure shows its potential as a bio-control inoculant agent in animal feed. Graphical Abstract

Staphylococcus aureus avirulent mutant vaccine induces humoral and cellular immune responses on pregnant heifers

Bovine mastitis produces economic losses, attributable to the decrease in milk production, reduced milk quality, costs of treatment and replacement of animals. A successful prophylactic vaccine against Staphylococcus aureus should elicit both humoral and cellular immune responses. In a previous report we evaluated the effectiveness of a live vaccine to protect heifers against challenge with a virulent strain. In the present study the immunological response of heifers after combined immunization schedule was investigated. In a first experimental trial, heifers were vaccinated with 3 subcutaneous doses of avirulent mutant S. aureus RC122 before calving and one intramammary dose (IMD) after calving. Antibodies concentration in blood, bactericidal effect of serum from vaccinated animals and lymphocyte proliferation was determined. The levels of total IgG, IgG1 and IgG2 in colostrum and the lymphocyte proliferation index were significantly higher in vaccinated respect to non-vaccinated group throughout the experiment. The second trial, where animals were inoculated with different vaccination schedules, was carried out to determine the effect of the IMD on the level of antibodies in blood and milk, cytokines (IL-13 and IFN-γ) concentration and milks SCC and bacteriology. The bacterial growth of the S. aureus strains was totally inhibited at 1-3×10(6) and 1-3×10(3)cfu/ml, when the strains were mixed with pooled serum diluted 1/40. The results shown that IMD has not a significant effect on the features determinate. In conclusion, a vaccination schedule involving three SC doses before calving would be enough to stimulate antibodies production in milk without an IMD. Furthermore, the results showed a bactericidal effect of serum from vaccinated animals and this provides further evidence about serum functionality. Immune responses, humoral (antigen-specific antibodies and Th2 type cytokines) and cellular (T-lymphocyte proliferation responses and Th1 type cytokines), were augmented by administration of the avirulent mutant which represent an antigenic pool.

In Vitro Characterization of Lactic Acid Bacteria Isolated from Bovine Milk as Potential Probiotic Strains to Prevent Bovine Mastitis

Bovine mastitis causes economic losses on dairy farms worldwide. Lactic acid bacteria (LAB) in animal health are an alternative tool to avoid antibiotic therapy on the prevention of bovine mastitis. In previous studies, 12 LAB isolated from bovine milk were selected taking into account some of the following characteristics: hydrophobicity, auto aggregative capability, inhibition of indicator pathogens, hydrogen peroxide, and capsular polysaccharide production. These LAB were considered because of their beneficial properties. In this work, we also analyzed the antimicrobial activity and the co-aggregation against mastitis causing bacteria, auto-inhibition, adhesion to bovine teat canal epithelial cells (BTCEC), and growth kinetic curves for the 12 LAB. Two of them, Lactococcus lactis subsp. lactis CRL 1655 and Lactobacillus perolens CRL 1724, were selected because they had an interesting pattern of adhesion to BTEC, the inhibition of pathogens and the co-aggregation with the 100% of the assayed pathogens. They showed a predictable difference in the PFGE genomic pattern bands. The kinetic growth of these two strains was similar between them and with the rest of the assayed LAB. The strains selected in the present study showed indispensable characteristics for their inclusion in a probiotic formulation to be used at dry-off period for the prevention of bovine mastitis.

Bovine mastitis prevention: humoral and cellular response of dairy cows inoculated with lactic acid bacteria at the dry-off period

The use of lactic acid bacteria (LAB) in animal feed, constitute an alternative tool for bovine mastitis prevention. Previously, two LAB strains were isolated from bovine milk and selected for their probiotics properties. So far, immune response of inoculating LAB in bovine udders at dry-off period has not been investigated. The immunoglobulin isotype levels and memory cell proliferation in blood and milk of animals inoculated with Lactobacillus lactis subsp. lactis CRL1655 and Lactobacillus perolens CRL1724 at dry-off period was studied. Ten animals were inoculated intramammarily with 106 cells of each LAB (IG) and 2 animals used as control (NIG). Milk and blood samples were taken before inoculation and 1, 2, 4, 6, 12 and 24 h and 7 and 14 days after inoculation. Somatic cell count (SCC) in milk, the presence of bovine mastitis pathogens, the levels of antibodies and lymphocyte proliferation were determined. In the IG, the SCC was <250,000 cells/ml up to 4 h after intramammary inoculation. Six and 12 h after inoculation, the SCC increased up to 600,000 and 2,000,000 cells/ml, respectively. In the NIG, the SCC reached the maximum value 7 days after inoculation. Microbiological analysis showed that all samples were negative for major bovine mastitis pathogens after 24-48 h of incubation. In general, LAB inoculation increased the amount of IgG isotypes in blood and milk, and these antibodies were able to recognise Staphylococcus aureus epitopes. Lymphocytes proliferation was significantly higher in the IG at all time points assayed, following LAB or S. aureus stimulation. The lymphocytes of animals inoculated with LAB do not react in vitro to the presence of S. aureus antigen.. The results showed that probiotic microorganisms could be a natural and effective alternative in the prevention of bovine mastitis at dry-off period and act as immunomodulatory stimulating local and systemic defence lines.

Pathogenesis and inflammatory response in experimental caprine mastitis due to Staphylococcus chromogenes

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in cases of subclinical mastitis in dairy cows. CNS species may differ in their pathogenicity, but very little is known about their virulence factors or their immune response in intramammary infections. To our knowledge, no experimental studies into the mastitis pathogenesis caused by CNS have been described in lactating goats. The aim of this study was to induce an experimentally Staphylococcus chromogenes mastitis in lactating goats aimed at verifying if the model can be used to evaluate the inflammatory response, the dynamics of infection and the pathological findings within the first hours of intramammary inoculation. Six Saanen goats in mid-lactation were inoculated with 1 × 107 colony forming units of S. chromogenes. Bacterial growth peaked in milk from the challenged right halves of the mammary glands (RMG) at 4 h post inoculation (PI). Shedding of viable bacteria showed a marked decrease at 12 h PI. An increase in mean somatic cell counts was observed in the milk samples from 8 h PI onwards. Mild clinical signs were evoked by intramammary inoculation. Staphylococcus chromogenes could be isolated in tissue from all RMG. Histological examination of specimens of the RMG and lymph nodes of the goats showed an increased inflammatory response throughout the experiment with respect to control halves. In conclusion, the experimental inoculation of S. chromogenes in lactating goats is capable of eliciting an inflammatory response and capable of causing pathological changes. This research represents a preliminary study for a better knowledge of the mastitis pathogenesis caused by S. chromogenes.