Giulietta Minozzi

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 β-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.

Antibacterial activity and immunomodulatory effects on a bovine mammary epithelial cell line exerted by nisin A-producing Lactococcus lactis strains

Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl-β-d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor-α downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor-α release could be an interesting approach to reduce the symptoms linked to clinical intramammary infections. Due to their antibacterial activity and to the stimulation of lysosomal activity of mammary epithelial cells, the L. lactis strains SL153 and LL11 could be of interest for the development of alternative intramammary treatments to control cow mastitis.

Genome wide association analysis of the 16th QTL- MAS Workshop dataset using the Random Forest machine learning approach

BackgroundGenome wide association studies are now widely used in the livestock sector to estimate the association among single nucleotide polymorphisms (SNPs) distributed across the whole genome and one or more trait. As computational power increases, the use of machine learning techniques to analyze large genome wide datasets becomes possible.MethodsThe objective of this study was to identify SNPs associated with the three traits simulated in the 16th MAS-QTL workshop dataset using the Random Forest (RF) approach. The approach was applied to single and multiple trait estimated breeding values, and on yield deviations and to compare them with the results of the GRAMMAR-CG method.ResultsThe two QTL mapping methods used, GRAMMAR-CG and RF, were successful in identifying the main QTLs for trait 1 on chromosomes 1 and 4, for trait 2 on chromosomes 1, 4 and 5 and for trait 3 on chromosomes 1, 2 and 3.ConclusionsThe results of the RF approach were confirmed by the GRAMMAR-CG method and validated by the effective QTL position, even if their approach to unravel cryptic genetic structure is different. Furthermore, both methods showed complementary findings. However, when the variance explained by the QTL is low, they both failed to detect significant associations.

Development of a Droplet Digital Polymerase Chain Reaction for Rapid and Simultaneous Identification of Common Foodborne Pathogens in Soft Cheese

Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a “third-generation PCR”) provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4 × 106 to 4 × 101 CFU/g) of pure cultures from the American Type Culture Collection. Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay) without the necessity of a standard curve. In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.

Polymorphism of the STAT5A, MTNR1A and TNFα genes and their effect on dairy production in Bubalus bubalis

Abstract The water buffalo is a fundamental resource, especially in developing countries, however, differently from other species, its genetic potential is still poorly investigated. In this work, we performed a candidate gene association study for milk composition in 491 female buffaloes. Animals were from four farms located in Southern Italy, where the Out-of-Breeding-Season-Mating technique is usually performed. We analysed three genes: (1) the signal transducer and activator of transcription 5A (STAT5A), (2) the tumour necrosis factor alpha (TNFα) and (3) the melatonin receptor 1A (MTNR1A). We confirmed the mutation at the MTNR1A gene and we found five novel single nucleotide polymorphisms (SNPs): one in the TNFα and four in the STAT5A. No associations were found for the SNPs in the MTNR1A and TNFα genes, while we identified a marked association with milk protein % for a C > T substitution at the STAT5A gene. At this locus, the TT buffaloes showed significantly higher protein percentage in milk. Conversely, this genotype class was the less frequent in the population. Moreover, an A > G substitution at the STAT5A showed an influence on reproductive seasonality, with the advantageous allele most frequent in the population, suggesting a possible effect of selection for this trait. The C > T substitution on STAT5A detected in present study could be used in marker assisted selection of Mediterranean Italian buffalo, and should be monitored to understand the reasons behind the low frequency of the favourable genotype at this locus and to stop this unfavourable trend in the population.

An explant of heifer mammary gland to study the immune response of the organ

Continuous or primary epithelial cell lines have been extensively used to study the mammary gland immune response, but they are constituted by a single cell population. Our aim was to test whether an explant of heifer gland, where the tissue structure is maintained, might be a valid model to investigate the innate immune response to infection. The study was carried out on 2mm3-sections of heifer udders, in 2 consecutive trials, using LPS or LTA in the first trial and two different concentrations of Staphylococcus aureus (Staph. aureus) in the second. Treated and untreated sections were collected after 1h, 3h and 6h incubation; in the first trial, a final time-point at 18h was considered. The mRNA expression of TNFα, IL-1β, IL-6, IL-8 and LAP was analyzed by quantitative real-time PCR. Histological examination showed well-preserved morphology of the tissue, and apoptosis only showed a slight, not significant increase throughout the experiment. IL-1β and IL-6 were significantly up-regulated, in response to LPS or Staph. aureus, while TNF-α and IL-8 significantly increased only under LPS treatment. LAP expression showed a significant late increase when stimulated by LPS. The immunochemical staining of the sections demonstrated a higher number of T lymphocytes within the alveolar epithelium, in comparison with interstitial localization. Since the explants belonged to pubertal non-pregnant heifers, T cells may be regarded as resident cells, suggesting their participation in the regulation of mammary homeostasis. Therefore, applying our model would give new insights in the investigation of udder pathophysiology.

Economic evaluation of genetic improvement in local breeds: the case of the Verzaschese goat

Abstract The paper analyses expected costs and benefits of closed nucleus selection in 1100 females of local goat breed Verzaschese. Returns are based on income from the sale of milk per unit of genetic gain. Costs include milk and pedigree recording, housing and maintenance of males and their transport from nucleus to commercial herds, semen production and artificial insemination in the nucleus. Discounted profits, under eight economic scenarios, over investment periods of 10, 15 and 20 years are analysed. Discounted profit for the 14 breeding schemes under the ‘best conditions’ economic scenario, taking into account returns from increased milk production in both nucleus and commercial population, ranges from 2517–226,434 Euros (10 years period), from 46,387–564,753 Euros (15 years period), and from 106,73–986,676 Euros (20 years period). When we consider genetic gain returns only from the nucleus, over a period of 10 years no breeding schemes show positive discounted profit. In the 15 years period, three schemes show positive discounted profit and two negative discounted profits but above 10,000 Euros; five schemes have positive discounted profit and two schemes above 10,000 in the 20 years horizon. Sensitivity analysis on profit per year shows the variable cost for recording ranking first, followed by return from milk, and by percentage of pregnancy failure.

First insights in the genetics of caseous lymphadenitis in goats

Abstract Abscesses are a common problem in goat farms worldwide. In most cases, the disease is caused by Corynebacterium pseudotuberculosis that is the aetiological agent of caseous lymphadenitis (CLA). CLA causes considerable economic losses, due to reduced milk production, and to carcase damage. At present, no effective therapy for CLA is available and vaccine use is limited. The current study presents evidence for loci associated to CLA susceptibility identified using the 50K Goat SNP panel in a case-control genome wide association study. The analysis of the genotype data identified four chromosomal regions associated with disease status: on chromosomes 5, 7, 8 and 11. These results provide the first evidence for genetic loci involved in the immunological response to CLA in goat. Knowledge of genetic variations related to susceptibility will facilitate the incorporation of this information for the improvement of health status in the goat species.

MicroRNA expression correlated with hygienic behaviour in honeybees

Honeybees (Apis mellifera) play important roles in modern agriculture regarding zootechnical production and crop pollination. Recently, honeybees have received more attention from the public, beekeepers and researchers due to emerging heath issues. Thus, scientific interest for honeybee health and selection resistance to major pathogens is sharply increasing. Honeybees evolved social immunity mechanisms consisting in the cooperation of individuals to control disease level in the hive, and in particular hygienic behavior (HB), as based on the uncapping and removal of dead, diseased or parasitized brood. HB is affected by heritable and environmental factors, and specific neurogenomic states can be inferred based on the coordinated brain expression of transcription factors and their predicted target genes, including Mblk-1 (transcription factor that function in the mushroom body) and Obp4 (sensitive olfactory detection in the antennae of adult bees). Besides, microRNAs are known to influence neurological status linked to age-related social behaviour in honeybees 7 . In order to investigate the relationship between microRNA expression and HB, the present work performed the expression profile of selected honeybee brain microRNA in individual’s honeybee from field colonies with high HB level compared to low HB level, in comparison with the expression profile of Mblk-1 and Obp4. The genetic information resulting from this project could help to understand the role of microRNAs in HB and to drive honeybee selection schemes for production, health, and behavioral traits favoring pathogen control.