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We optimized a combination of microbiological and molecular methods to quickly identify the presence of the O157 and the six non-O157 serogroups (O26, O45, O103, O111, O121 and O145) most frequently associated with VTEC status, at herd level. The lower detection limit of this methodology is 10 1 CFU/ml for each of the serogroups tested. We tested 67 bulk tank milk (BTM) and raw milk filters (RMF) derived from dairy herds located in Lombardy and Trentino Alto Adige. We identified 3 positive samples and 20 positive samples out of 67 respectively in the BTM and RMF. Interestingly, several samples showed positivity for more than one serogroups at the same time. We also identified the presence of E. coli O45 and O121 for the first time in raw milk and raw milk filters. Once screened the seven serogroups of interest in our samples, we evaluated the real pathogenicity of our positive, non-O157 samples through two parallel molecular biology methods: virulence gene research by PCR, and HRMA and sequencing. The most frequently isolated serogroups in milk were O157 (2.64%), O103 (2.11%), and O145 (1.06%), while in RMF the frequencies were, respectively 14.92%, 4.48%, and 2.98%. Moreover, this is the first published report in Italy of positive recovery of O45 and O121 serogroups in milk and milk filters. The new diagnostic approach proposed investigate the presence of the O157 and big six non-O157 serogroups at farm level and not to identify VTEC hazard only once the product is processed and/or is ready to be consumed.

In this Research Communication we report the results of a controlled study conducted under field conditions in which we analysed milk ejection curves in cows with chronic mastitis, and assessed the influence of antinflammatory treatment with ketoprofen. Total milking time was reduced in chronic mastitis cows, irrespective of ketoprofen treatment, and the proportion of bimodal flow curves was increased. This latter effect was partially reversed by ketoprofen. To our knowledge, this is the first study showing that chronic mastitis has a significant effect on the milk ejection curve. Antinflammatory treatment with ketoprofen was shown to be efficacious in reducing these negative effects, re-establishing a pattern close to the one observed in healthy cows.

Data on the prevalence of major contagious pathogens in bulk tank milk (BTM) in Italy are generally not available. The availability of Real-Time PCR procedures (qPCR) to perform BTM analysis by represents an important step to define herd health status. Therefore, a cross-sectional epidemiological study was designed to assess the prevalence of contagious pathogens and Prototheca spp in BTM samples. The study was performed on 581 herds from four districts in the west Lombardy region of Italy. Additionally, the relationship between pathogens in BTM and SCC or milk yield; the presence of an association between four risk factors (district, herd size, average milk yield and SCC) with pathogens in BTM were assessed. The overall data showed that S. aureus was recovered in 42% of the herds, Str. agalactiae in 10%, Prototheca spp in 11% and M. bovis in 1.5% of the herds. The GLM model applied showed a significant influence of BTM results, district, herd size and their interactions on SCC and on milk yield variance. Particularly, S. aureus or Str. agalactiae have a significant effect on milk yield variability and, in a lesser extent, on SCC. The very high prevalence of contagious pathogens significantly affects milk characteristics and yield, thus affecting economic sustainability of the herds, and suggests the need to implement control programmes to decrease the prevalence of contagious pathogens, This will also allow to decrease the use of antimicrobials and to improve cow welfare.HighlightsFirst study on a large sample of Italian dairy herds on the prevalence of contagious pathogens in bulk tank milk samples. The prevalence value observed exceeded 50%.First study estimating the prevalence of M. bovis in bulk tank milk in a large sample of Italian dairy herds, and the prevalence observed was 1.5%.Prevalence of contagious pathogens has a significant influence on milk yield and SCC.Bulk tank milk SCC confirmed to have a low accuracy to identify infected herds. First study on a large sample of Italian dairy herds on the prevalence of contagious pathogens in bulk tank milk samples. The prevalence value observed exceeded 50%. First study estimating the prevalence of M. bovis in bulk tank milk in a large sample of Italian dairy herds, and the prevalence observed was 1.5%. Prevalence of contagious pathogens has a significant influence on milk yield and SCC. Bulk tank milk SCC confirmed to have a low accuracy to identify infected herds.

Data on the presence of pathogenic Escherichia coli in bulk tank milk (BTM) and raw milk filters (RMF) are not available in Italy and there are few studies worldwide. Therefore, a study under field condition was conducted to assess the presence of E.coli pathogenic and commensal (CoEC) strains in BTM and RMF samples and their associated AMR pattern. One hundred forty-nine E.coli isolates were characterized. Among all the isolates, 53 (35.6%) were classified as pathogenic while the other ones were classified as CoEC. Among the pathogenic ones, 23 (54.7%) were classified as enterotoxigenic E.coli (ETEC), 6 (11.3%) as enteroinvasive E.coli (EIEC), 2 (3.8%) as enteroaggregative E.coli (EAEC), 12 (22.6%) harboured virulence factors (VF) common to ETEC+EIEC, and 2 (3.8%) common to ETEC+EAEC. To our knowledge, it is the first time that ETEC isolates harboring VF associated with EAEC or EIEC are observed in raw milk. These data support the presence of transmission of VFs genes among isolates. None of the isolates showed resistance to three or more antimicrobials. The CoEC role as a vector of AMR was confirmed by the presence of 18% ampicillin- and cephalexin-resistant isolates. The presence of AMR in CoEC supports the role of these bacteria as source of resistance genes. Monitoring raw milk by either BTM or RMF analysis, and the relatively cheap procedure applied to identify E.coli pathotypes can be useful to identify hazards related to the spread of enteric diseases and antimicrobial resistance.

Staphylococcus aureus isolated from mammary gland are characterized by different genetic patterns. Ninety four isolates from 33 dairy herds were analyzed by the means of a microarray to investigate S. aureus virulence patterns and the distribution of genes believed to be involved in immune evasion. None of the 94 isolates considered were MRSA. However, 50% of the isolates belonged to complexes related to MRSA and to human diseases, while only about 25% of them can be considered as exclusively of bovine origin. The distribution of clonal complexes and the different gene patterns observed confirmed the presence of an influence of geographical localization. The assessment of the influence of genes related to immune evasion on quarter milk cell count showed as four of them showed to be significantly associated to an increase quarter milk SCC. These genes could be potential target for developing new vaccines against S. aureus.

Staphylococcus aureus represents one of the leading causes of mastitis in dairy cows worldwide. S. aureus IMI have variable outcomes due to virulence of the strain involved, immune defenses of the host, and by antibiotic resistance. The difficulty in eradication and the increasing concerns on antibiotics usages underscore the interest in developing new tools to control S. aureus mastitis. Vaccination represents one of the most studied of these tools but, so far, no vaccine seems to provide reliable protection. This review summarizes current knowledge on the major vaccine targets, including surface proteins, capsular polysaccharides, biofilm, and toxins. Finally, the present status of vaccination against S. aureus and the future of vaccines design were discussed,includi ng how differences among in vivo models may influence vaccines development. Fil: Scali, F.. Universita Degli Studi Di Milano; Italia Fil: Camussone, Cecilia María. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Calvinho, L. F.. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Cipolla, M.. Universita Degli Studi Di Milano; Italia Fil: Zecconi, A.. Universita Degli Studi Di Milano; Italia

The need for antibiotic treatments in dairy cattle farms can be reduced by a combined intervention scheme based on: (1) timely clinical inspections, (2) the assessment of animal-based welfare parameters, and (3) the use of predictive laboratory tests. These can provide greater insight into environmental adaptation of dairy cows and define animals at risk of contracting disease. In the long-term, an improved disease control justifies the adoption of such a combined strategy. Many antibiotic treatments for chronic disease cases are often not justified with a cost/benefit analysis, because the repeated drug administration does not give rise to the expected outcome in terms of animal health. In particular, compared with untreated cases, antibiotics may not lead to greater cure rates for some forms of mastitis. Lastly, a substantial reduction of antibiotic usage in dairy farms can be achieved through the proper use of immunomodulators, aimed at increasing immunocompetence and disease resistance of cows.

This study analysed the usefulness of near infrared spectroscopy (NIRS), combined with volatile compound (VOC) and fatty acid (FA) analyses, for the authentication of the unique Italian Valle d'Aosta Arnad Protected Designation of Origin (PDO) lard. Ensuring the authenticity of high value meat products remains an emerging topic within the food sector. This study validated a FA, VOC and NIRS model for use in the authentication of Arnad PDO lard. The model showed a high potential rate to recognize patterns in lard samples. In particular the sensitivity and specificity calibration values were both 100%, and cross-validation models were performed using FAs and VOCs separately. The NIRS model obtained sensitivity and specificity values of 98.2% in the calibration data set, and 94.4% in the cross-validation step. This analytical approach may represent an effective tool to prevent food fraud, which is crucial for meat derived products with a high commercial value.

In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at approximately 60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 +/- 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.

For its characteristics, donkey milk has been proposed as an alternative to goat or artificial milk to feed allergic infants. Therefore, it is important to increase our knowledge on health and immunological characteristics of donkey milk. Ten donkeys, bred as companion animals, were enrolled in this study and sampled once a month, for eight months. Milk (10 ml) was collected from each half udder for somatic cell count (SCC), bacteriological analysis and total bacteria count (TBC). The major pathogens were tested for antimicrobial susceptibility, and Staphylococcus aureus isolates were further genotyped by nanoarray analysis. Whey lysozyme and NAGase (NAG) activities were also assessed. Overall, 101 half-udder milk samples were taken. They showed very low values of TBC (Staph. aureus was isolated only from 5 milk samples (3 animals), Streptococcus equi from 2 samples and Str. equisimilis from a single sample. All the isolates were sensitive to all antibiotic classes used in veterinary medicine. None of the Staph. aureus isolates were shown to harbour genes coding for any enterotoxin, toxic-shock syndrome toxin or antibiotic resistance. Lysozyme levels were always very high (4000–5000 U/ml), while NAG values were mostly low (

To investigate the relationships between milk flow traits and udder health status in primiparous cows, 74 primiparous Holstein cows were randomly selected in 5 herds and monitored monthly throughout the whole lactation. A total of 2902 quarter milk samples were collected for bacteriological analyses and the determination of lysozyme, N-acetyl-β-glucosaminidase (NAGase) and somatic cell count (SCC). Milk flow curves of the whole udder of each cow were registered with continuous electronic milk flow meters. Teat conditions and teat thickness changes during milking were assessed monthly. Quarters, udders and cows were classified as healthy, latent, inflamed and subclinical depending on SCC and the results of bacteriological analyses. Lysozyme in milk, teat apex score and teat thickness change did not vary with udder health status while NAGase in milk significantly increased as udder health status worsened (PPPPv. 3·48 kg/min; PP0·8 min) there was an increased risk of poor udder health status. These milk flow traits can be predictive indicators of udder health status; time of plateau phase, expressed as percentage of time of milk flow, can also be a useful parameter for animal selection.

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. Studies of the molecular epidemiology of S. aureus strongly suggest that some genetic subsets of strains are particularly well adapted for causing infections in cattle. This communication reports the setup of experimental protocols to identify the immunogenic proteins expressed by one of the most common field isolated strain of S. aureus responsible for sub-clinical mastitis cases. The serological proteome analysis (SERPA) approach applied consists of three main steps: two-dimensional electrophoresis-based separation of the proteins contained in field isolated S. aureus extracts enriched for surface proteins, detection of immunogenic spots using anti-serum collected from sub-clinical mastitis cases and identification of antigens by mass spectrometric-based methodologies. The study allowed to identify three immunogenic proteins: DNAase translocase FtsK, ribosomal proteins S1 and a Tell-like protein.

Despite the fact that health communication is a discipline developed only recently, its importance in human medicine is well recognized. However, it is less considered in veterinary medicine, even if it has the potential to improve public health because of the role of veterinary medicine in public health. For this reason, an One Health approach is useful for communication as well. This approach leads to a “One Communication” concept, which is the result of the synergy in communicative efforts both in human and in veterinary medicine. Our analysis explores the potential of communication in several veterinary fields: institutions, food safety, companion animal and food-producing animal practice, pharmacology and drugs, wildlife fauna and environment. In almost all the areas of veterinary activity communication can contribute to human health. It takes many forms and use several channels, and this variety of communicative opportunities represent a challenge for veterinarians. For this reason, the communication course should be included in the curricula of Veterinary Medicine Schools. As One Health, One Communication is a strategy for expanding collaborations in health communication and it will enhance public health.

Staphylococcus aureus causes many serious diseases in humans and animals, and it is the most common aetiologic agent of contagious bovine mastitis. The bacteria produce several virulence factors and the importance of evaluating the combination of these virulence factors has been recently emphasized. In study, the combination of several virulence factors: coagulase gene (coa), protein A gene (spa), collagen-binding protein gene (cna), fibrinogen-binding protein gene (efb), Panton-Valentin leukocydin gene (pvl) and enterotoxins (sea,seb, sec, sed, see, seg, seh, sei, sej) was considered. The analysis of the relationship between presence/absence of the different genes and the udder inflammatory response measured by milk somatic cell counts was performed by general linear models and logistic regression. The classification of isolates in clusters by virulence genes combinations showed that at least one cluster induced a higher inflammatory response. Moreover, the analysis of the association between virulence genes and the presence of a subclinical mastitis showed the role of spa and sej gene as risk factors. These results support that the development of subclinical mastitis could be related to strains characteristics and to the expression of specific combinations of the virulence factors.

Summary Different protocols to evaluate teat skin status under field conditions based on scoring and on the measurement of pH and hydration of teat skin were evaluated. After determination of the teat score for all eligible animals in a commercial dairy herd, 50 of them were chosen, based on their pre-trial average teat score, and separated in two groups. Two different post-dipping products with the same amount of disinfectant, but with a different concentration of emollient were applied. All four teats were assessed with the corneometer and pH probes. A digital picture of each teat skin and teat orifice was taken with a digital camera. Hydration and pH data were analysed by anova with repeated-measurement factors, while teat skin and apex score patterns, were assessed by the means of Mann–Whitney test; between- and within-group changes were assessed by the mean of Mantel–Haenzel chi-square statistic. Data showed that teat skin pH and hydration could be assessed under field conditions and they were influenced by teat conditioning. The approach based on evaluating odds for scores, stratified for sampling and for treatment, was found to be a sensitive and informative way to compare the changes between and within treatment groups.

Summary Changes in prevalence in intramammary infection, by pathogen type, in herds applying a stringent contagious mastitis control programme was studied. Enrollment of 1651 lactating cows and collection of milk samples was made in this ancillary study to a cohort study of the dynamics of mastitis prevalence after adoption of a strict contagious mastitis control programme that targeted the elimination of mastitis caused by Staphylococcus aureus. Nine commercial dairies in Italy were used. Aseptic collection of milk samples from all lactating cows was performed at the time of enrollment, from all cows within 7–14 days of entering the lactating herd after the date of enrollment, and from all lactating cows at 2, 4, 7, 10 and 14 months after the date of enrollment. Prevalence of intramammary infection by pathogen type was determined from culture of milk samples. Application of the strict contagious mastitis programme did not lead to an increased risk of non-contagious mastitis. The risk of coliform, environmental streptococcal and coagulase-negative staphylococcal intramammary infections decreased after adoption of the programme. The data reported herein indicate that the overall risk for any intramammary infections decreases with adoption of a strict contagious mastitis programme, and that such a programme therefore does not necessarily lead to an increase in environmental mastitis.

To assess the impact of automatic milking systems (AMS) on the different aspects of milk production a research projectinvolving both commercial and experimental dairy farms with different AMS and different management was started. Thispaper reports the results of a follow-up study on primiparous cows focused on assessing some markers to be used tomonitor udder and teat health. Heifers were included after calving and sampled for at least 12 months. Quarter milk samplesand teat measurements were taken to assess: intramammary infection (IMI) frequencies, somatic cell counts (SCC),teat thickness changes, teat skin and apex conditions. The study included 28 cows in herd A and 27 in herd B for a totalnumber of 2344 samples. Overall, teat apex and skin conditions were maintained along the lactation. Teat skin conditionstended to decrease because of the accumulated number of milkings while lactation proceeded, but at a largelyacceptable level in both herds. Teat apex conditions showed a decrease. Teat thickness changes displayed different patternsin the two herds, probably because of the different type of AMS, but in both cases a trend to decrease in thicknesscould be observed. The application of AMS in herd B, free from contagious pathogens, did not influence the frequency ofIMI and the SCC. In herd A, characterized by the presence of Staphylococcus aureus IMI, the frequency of IMI showeda progressive increase, very likely as a consequence of the spread of infections during milking. Teat skin had no associationwith the frequency of IMI. Teat thickness changes outside values considered as physiological proved to be associatedwith decreased conditions in the teat apex score in herd A, but not in herd B. However, a decrease in teat apex scoreproved to be associated with an increase in IMI frequency in both herds. The results of this field trial confirm that AMShave no negative impact on IMI incidence, SCC and teat tissue conditions when the initial cow health status and overallherd management are good. In the presence of contagious pathogens, and when cows have more than 300 days in milk(DIM) the frequency of negative outcomes significantly increases and the proper control measures should be taken.

Effective communication in dairy farms improves management and herd health, and it is also pivotal for public health in a “from farm to fork” perspective. This paper reports the results of a descriptive study on dairy farmers' perception of veterinarian and other consultants' communication skills. Perceived communication skills showed to be significantly lower than desired ones for all the professional figures considered. Despite these unsatisfactory results, veterinarian were the most appreciated and skilled consultants. The observed farmers' dissatisfaction increases farmers' difficulties in identifying proper targets and proper consultant. An increase in the skill of veterinarian to deliver effective and tailored messages could help to overcome the problem.

Nowadays food wrapping assures attractive presentation and simplifies self-service shopping. Polyvinylchloride (PVC)- and polyethylene (PE)-based cling-films are widely used worldwide for wrapping cheeses. For this purpose, films used in retail possess suitable technical properties such as clinginess and unrolling capacity, that are achieved by using specific plasticizers during their manufacturing process. In the present study, the main VOCs of three cling-films (either PVC-based or PE-based) for retail use were characterized by means of Solid-Phase Micro-Extraction and GC/MS. In addition, the effects of cling film type and contact time on the migration of VOCs from the films to four different PDO Italian cheeses during cold storage under light or dark were also investigated. Among the VOCs isolated from cling-films, PVC released 2-ethylhexanol and triacetin. These compounds can likely be considered as a “non-intentionally added substance”. These same compounds were also detected in cheeses wrapped in PVC films with the highest concentration found after 20 days storage. The PE cling-film was shown to possess a simpler VOC profile, lacking some molecules peculiar to PVC films. The same conclusions can be drawn for cheeses wrapped in the PE cling-film. Other VOCs found in wrapped cheeses were likely to have been released either by direct transfer from the materials used for the manufacture of cling-films or from contamination of the films. Overall, HS-SPME is shown to be a rapid and solvent free technique to screen the VOCs profile of cling-films, and to detect VOCs migration from cling-films to cheese under real retail storage conditions.

Two strains of Bovine viral diarrhea virus 2 (BVDV-2) were isolated from calves in northern Italy. Variations in the 5'-untranslated region (UTR) of the genome were studied by primary structure alignment and neighbor-joining method based phylogenetic tree analyses and by palindromic nucleotide substitutions at the three variable loci in the 5'-UTR. Genetic analysis indicated their appurtenance to genovar BVDV-2a. Nucleotide sequence at the 5'-UTR of strain BS-95-II, one of the Italian isolates from healthy calves, showed 98% homology to that of the Japanese isolate OY89, a cytopathic strain derived from cattle with mucosal disease.

The distribution of Staphylococcus aureus within herds seems to be related to interactions among the shedding characteristics of the bacteria, their pathogenicity and mammary gland immune status. The aim of the present study was to investigate the relationships between selected mammary gland immune factors and intramammary infections associated with Staph. aureus. Overall, 70 cows from five commercial dairy herds were included in the study and quarter milk samples were assessed using bacteriological and cytological tests. We evaluated differential cell count, lysozyme concentration, N-acetyl-β-glucosaminidase (NAGase) activity, cell viability and respiratory burst activity in randomly chosen quarter milk samples from each cow. Staph. aureus intramammary infection elicited different responses in the mammary gland immune defences investigated. Polymorphonuclear leucocytes (PMN) as a proportion of total somatic cells in milk, cell viability and NAGase activity were higher in infected quarters, while the proportions of macrophages and lymphocytes, respiratory burst activity and lysozyme levels were lower. Mean values differed among herds, but the differences were not significant. These changes were associated with Staph. aureus infection. The reduced respiratory burst activity together with the increase in the proportion of PMN suggests that both the number and activity of PMN could influence the susceptibility of the mammary gland to pathogens. Indeed, the logistic model adopted suggests that impairment of milk immune factors could be concurrent with the development of an infection.

An epidemiological survey on prevalence distribution of antibodies to BVDV was carried out in dairy cattle herds during 1995-1996 in northern Italy. A total of 704 serum samples from 29 non-vaccinated herds reported to have reproductive problems were tested for serum neutralising antibodies. In each herd, sampling was based on the stratification by age into five classes (< 6 months old calves, 6-12 months old calves, pregnant heifers, uniparous, pluriparous). Overall, 53.3% of samples were serologically positive, with the lowest ratio in 6-12 months old calves (37.9%) and the highest in pluriparous cows (71.2%).

Milk samples from 40 primiparous Sardinian ewes were examined during a whole lactation period in order to evaluate the dynamics of somatic cell counts (SCC), differential cell counts (DCC) and bacteriological status of the mammary gland. Only four samples out of 240 were bacteriologically positive and this was confirmed by the geometrical mean of SCC being always below 200 000 cells per ml. DCC during lactation showed two different dynamics: one concerning polymorphonuclear neutrophil leucocytes (PMN) and macrophages, and the other one lymphocytes and epithelial cells. Macrophages and PMNs in particular showed a progressive increase as lactation went on, while lymphocytes and epithelial cells showed a uniform decrease during lactation. The relationship between SCC and DCC indicated that PMN was the only cell type increasing when SCC rose. The progressive increase of SCC during lactation in absence of infection was probably an effect of cell concentration owing to the physiological reduction of milk yield, while the increase of macrophages and PMN was very likely a consequence of immune mechanisms approaching the dry period. The results confirmed that DCC and particularly PMN rate could provide a more sensible method of identifying mammary inflammations, particularly in well managed herds with low levels of infections.

Staphylococcus aureus (S. aureus) is one of the most widespread agent of diseases in humans and animals. In dairy cows, S. aureus is the most frequently isolated contagious pathogens in mastitis cases and vaccines are one of the potential tools to control the infections, thus decreasing the use of antibiotics. Among all the virulence factors produced by S. aureus, extra cellular fibrinogen binding protein (Efb) is an important one in the pathogenesis of mastitis. Plants are useful bioreactors to produce antigens and the aim of the study was the production of Efb in two cultivars of Nicotiana tabacum as a mean to produce vaccine against S. aureus in plants. A matrix attachment region (MAR) sequence was inserted near the two borders of transfer-DNA in the transformation vector in the two possible orientations. The presence of MAR elements in the transformation system significantly improved transformation efficiency and Efb protein yield up to a 2% level on total soluble protein (TSP). Mice orally immunized with transgenic lyophilized leaves produced an antigen-specific immune response.

Gene expression in early stage embryos relies mostly on post-transcriptional control of maternal transcripts accumulated during oocyte maturation. However, while the building process to obtain a competent oocyte is now better understood, it is still not clear what transcriptome blueprint composes a competent oocyte. The aim of the study was to compare the mRNA expression pattern between oocytes collected from fertile heifers and repeat breeders by using RNAseq. Oocytes were collected by ovum pickup from 3 heifers that were 11–15 months of age and became pregnant at the following oestrus and from 4 adult cows with an age of 4 to 7 years, classified as repeat breeders after they failed to become pregnant for a minimum of 3 consecutive AI. To obtain oocytes from follicles with the same degree of development, at time 0 all follicles visible through transrectal ultrasound examination were removed by transvaginal aspiration. Five days later oocytes were collected by ovum pick up from the newly formed follicles with diameters >5 mm. Oocytes from each animal were pooled and sequenced as a single sample. Total RNA was extracted by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (Nugen Technologies, San Carlos, CA, USA). After library preparation with TruSeq DNA Sample Prep kit (Illumina, Madison, WI, USA), sequencing was performed on an Illumina HiSEqn 2000. Galaxy and Chipster open web-based platforms were used to analyse the data. We identified 49 differentially expressed genes. Heifers’ oocytes mRNA pattern indicated greater potential to sustain cell division. In particular, oocytes expressed more Keratin 14 (a gene involved in cell proliferation) and kinesin family member 20B (a protein involved in cytokinesis). More competent oocytes also have a greater ability to repair single-strand breaks due to the high levels of endo/exonuclease (5′-3′), endonuclease G-like. This may reflect greater capacity to neutralise DNA damage and, therefore, greater ability to preserve and transmit high-quality DNA. Repeat breeders portray a different landscape; their greater expression of Jun oncogene, Heat shock protein 1, Stimulated by retinoic acid gene6, arylhydrocarbon receptor nuclear translocator, fibromodulin, and aryl-hydrocarbon receptor repressor suggest that these oocytes have been subjected to environmental stress during oocyte maturation. Their greater expressions of inhibin α, stearoyl-CoA desaturase, junctional adhesion molecule 2 have been previously shown to correlate with a reduced oocyte developmental potential. Furthermore, the Cannabinoid receptor protein 1 expression suggests a compromised ion function that can lead to a failed activation of the development program. Finally, the greater expression of Ubiquilin3 and Heat shock protein 1 led to high protein and mRNA degradation, respectively, suggesting that these oocytes are deprived of essential components to sustain embryo growth. In conclusion our data provide the first detailed snapshot of the mRNA pattern defining the differences between a competent oocyte and an incompetent oocyte in vivo. Study supported by PRIN 2008, 2009 and EU-Quantomics.

In the last decades, Staphylococcus aureus acquired a dramatic relevance in human and veterinary medicine for different reasons, one of them represented by the increasing prevalence of antibiotic resistant strains. However, antibiotic resistance is not the only weapon in the arsenal of S. aureus. Indeed, these bacteria have plenty of virulence factors, including a vast ability to evade host immune defenses. The innate immune system represents the first line of defense against invading pathogens. This system consists of three major effector mechanisms: antimicrobial peptides and enzymes, the complement system and phagocytes. In this review, we focused on S. aureus virulence factors involved in the immune evasion in the first phases of infection: TLR recognition avoidance, adhesins affecting immune response and resistance to host defenses peptides and polypeptides. Studies of innate immune defenses and their role against S. aureus are important in human and veterinary medicine given the problems related to S. aureus antimicrobial resistance. Moreover, due to the pathogen ability to manipulate the immune response, these data are needed to develop efficacious vaccines or molecules against S. aureus.

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3 mm and 4 to 7 mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8 mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.

Non-specific (innate) immune response plays a major role in defending the udder from bacterial invasion. Moreover, recent investigations suggest that mammary gland epithelial cells (MGEC) could have a large and important role as a source of soluble components of immune defences. Despite many attempts to find other ways to control/prevent mastitis (i.e. vaccine) antimicrobial therapy is still the most used and effective means of curing clinical and subclinical mastitis. However, drug concentrations and therapy durations are far from the optimal in order to reduce costs. Therefore, efficacy of antimicrobial therapy is dependent not only on the substance activity but also on the positive interactions with the host innate immune response. Surprisingly, information on these interactions is rather scarce in the mastitis field. A simple experimental model was developed based on BME-UV cell line,Staphylococcus aureusas a challenge and a macrolide as an antimicrobial to assess the interactions among epithelial cells,Staph. aureusand the potential effects of antimicrobials on the immune system. The results of this study confirmed that tylosin has good antimicrobial activity against both intracellular and extracellularStaph. aureusin bovine MGEC without affecting cell functions. In this study, a significant down-regulation of IL-1 and IL-6 was observed, while TNF and IL-8 expression rate numerically increased, but differences were not significant. To our knowledge, this is the first paper assessing the concentration of two lysosomal enzymes, lysozyme and N-acetyl-β-d-glucosaminidase (NAGase), inStaph. aureus-stimulated MGEC. The results of this study confirmed that tylosin could have a significant effect on the release of these enzymes. Moreover, even if both enzymes have a similar substrate as a target, the results suggest different secretion mechanisms and an influence of antimicrobial treatment on these mechanisms. Successful mastitis cure is the result of achieving the optimal efficiency of both innate immune defences and therapeutical activities, by means of killing bacteria without eliciting an excessive inflammatory response. Therefore, antimicrobials for mastitis therapy should be selected not only on bacterial sensitivity, but also for their positive interactions with the innate immune response of the mammary gland. This study showed that an in-vitro model based onStaph. aureuschallenge on MGEC could be helpful in assessing both the intracellular and extracellular activity of antimicrobials and their influence on epithelial cell immune and inflammatory response.

Staphylococcus aureusis the most common aetiologic agent of contagious bovine mastitis. It is characterized by a wide array of virulence factors. The differences among strains jeopardize the development of effective vaccines againstStaph. aureusmastitis. We tested the immunogenicity of a peptide subunit vaccine coding for three different adhesion factors, fibrinogen-binding protein (Efb), fibronectin-binding protein A (FnbpA) and clumping factor A (ClfA). Then we evaluated the influence of some virulence factors on the ability of specific anti-adhesin antibodies to react with sixteenStaph. aureusstrains isolated from bovine subclinical mastitis. Immunization with the recombinant adhesins stimulated a strong humoural (IgG and IgA) and mucosal IgA immune response in all animals tested. Hyperimmune serum recognized with diverse efficiency the sixteenStaph. aureusstrains and this circumstance correlated well with the level of expression of adhesins. Among the different virulence factors considered to classify strains,spagene polymorphisms showed the strongest influence on isolate reactions to hyperimmune serum. Our results indicate the importance of a disease- and environment-specific analysis of isolates. Thus, as opposed to other pathogens to obtain an effective vaccine we should characterize multiple strains and identify the prevalent virulence factors expressed.

The importance to evaluate Staphylococcus aureus virulence analysing the combination of virulence genes is largely recognised, and the recent availability of simplified microarray tools allows performing these analyses also in the dairy field. The combined availability of herd-specific S. aureus mastitis prevalence data, isolates from these herds, and microarray technology offered the opportunity to investigate the relationship between S. aureus genetic pattern and their prevalence among dairy herds. Eleven commercial dairy herds following a S. aureus control programme were enrolled in the study, and 33 S. aureus isolates were collected from these herds. Diagnostic DNA microarrays based on the array-tube platform were used for genotyping of staphylococcal DNA. The genetic analysis of the 157-genes microarray showed as only 19 genes were present in all the isolates considered, and among them the genes coding for the leukocidin subunits (LukF, LukS and LukY), haemolysins (hla, hld and an unnamed haemolysin) and enterotoxin X. Several genes considered in the arrays were absent in all the isolates, including the ones encoding the resistance to most of the antimicrobials, except for tetracycline. In our isolates, some agr alleles were never identified (B-III, C-III, D-III, C-IV and D-IV). The comparison of epidemiological data with the genetic pattern suggests that agr type II is associated to the most diffusive isolates, being recovered from the largest number of herds and with the highest frequency. Microarray technique showed to be a useful method to assess the characteristics of virulence of S. aureus isolated in dairy herds and to investigate the relationship with the prevalence of the microorganism. These results support previous evidence that specific gene patterns could be associated to S. aureus mastitis.

Knowledge of the epidemiological pattern and the potential sources of infections is important to controlStaphylococcus aureusin dairy herds. This paper reports the results of a study applying both pulse field gel electrophoresis (PGFE) and the assessment of a selected number of virulence genes to investigate the role of teat skin onStaph. aureustransmission among cows and on the contamination of milk. Overall 61 isolates were considered, 23 from teat skin, 33 from milk samples and 5 from curd samples. Teat swabs were taken in five herds, but in only three of them couldStaph. aureusbe isolated. Curd was sampled in three herds, butStaph. aureuscould be isolated in only two herds. The distribution of isolates among herds confirmed the presence of herd-specificStaph. aureusstrain in most of the herds. The same pattern was observed in teat skin samples, in quarter milk samples, and in the curd samples. Our findings are consistent with other studies showing the role of teat skin as a potential reservoir. Moreover,Staph. aureuswas isolated from teat skin of confirmedStaph. aureus-negative cows that were segregated from infected ones. Our findings also suggest that some strains have higher chances to survive on teat skin and therefore to increase the risk for contamination of milk and milk products due to the persistence of intramammary infections.

This study evaluated the in vivo adjuvant activity of two peptides derived from Clostridium difficile: a fragment of the receptor-binding domain of toxin A (TxAC314) and a fragment of the 36 kDa surface-layer protein (SLP-36kDa) from strain C253. Their ability to affect the magnitude, distribution and polarization of the immune response against fibronectin-binding protein A (FnbpA), a protective vaccine antigen against Staphylococcus aureus, was evaluated using two different routes of immunization: intranasal and subcutaneous. It was shown that (i) the route of immunization affected the magnitude of the immune response; (ii) both peptides enhanced the production of circulating anti-FnbpA IgG and IgA; (iii) following mucosal immunization TxAC314 was more effective than SLP-36kDa at inducing antibody in the gastrointestinal tract; (iv) the adjuvant influenced the Th1/Th2 balance; and (v) TxAC314 was more effective than SLP-36kDa in inducing a cell-mediated response. These studies provide insight into the ability of different C. difficile-derived peptides to differentially affect and polarize the activity of the immune system and on their potential use as adjuvants in newly developed vaccines.

In this study, we evaluated two biomolecular techniques for discriminating between strains of Escherichia coli isolated form a variety of sources. The DNA of 211 strains of E. coli collected from dairy farms, calves, feces, pigs, primates, humans, and food products was analyzed by pulsed-field gel electrophoresis (PFGE) and repetitive-element polymerase chain reaction using the BOXA1 primer (BOX-PCR). Objectives of the present study were to compare PFGE and BOX-PCR for discriminating among strains of E. coli and investigate their capability in clustering E. coli strains according to the origin of bacterial isolation. Our results showed that PFGE and BOX-PCR were both able to distinguish closely related strains of E. coli; however, PFGE was able to discriminate between isolates indistinguishable by BOX-PCR and interpretation of PFGE data was easier. BOX-PCR proved to have good discrimination power, was less expensive, and could be performed in a PCR thermocycler. Neither of the methods used were effective in clustering E. coli strains according to the source of the organism.

To support a voluntary disease control program, this study aimed to develop an integrated scoring system for the risk assessment of bovine viral diarrhoea virus (BVDV) infection in dairy herds in Northern Italy. Sixty-two dairy herds were classified according to their BVDV serological status. Farmers were interviewed using a questionnaire on potential BVDV risk factors. Scores were used to define risk levels for factors related to (1) BVDV introduction (through livestock trade, attendance of animals at shows/exhibitions and grazing common pasture), (2) within-herd spread of BVDV and (3) the results of initial serological testing. The calculated odds ratios were significant for all categories, except for livestock trade. The application of the screening test, the questionnaire and the related risk assessment showed this to be a practical approach to predicting BVDV herd status.

Several milk components related to immune defences (lysozyme, lactoferrin and gamma-globulins, gamma-G) and to inflammation (somatic cell counts, SCC; N-acetyl-beta-glucosaminidase, NAGase; albumin) were considered. Forty-one quarters and 685 samples of 24 cows were included in the study; among them 534 samples were defined as negative (78.0%), 93 as diseased (13.5%) and 58 (8.5%) as subclinical. The pattern of each milk component in quarters always negative during the follow-up period was evaluated by a mixed model. Statistical analysis showed that days in milk (DIM), age (primiparous, pluriparous), herd and the interaction between herd and days in milk significantly influenced all the markers, with very few exceptions. A subset of samples including the negative quarters before the first outcome of an infection or a subclinical mastitis and the samples from quarters always negative was also selected. The analysis showed that herd, DIM and health status had a significant influence on most markers. Overall, primiparous cows were confirmed to have higher levels of most of the markers than pluriparous cows. The presence of a herd effect on non specific immune defences in fully negative quarters implies that when the mechanisms behind their release are fully elucidated, it might be possible to modulate them. Udder tissues were confirmed as an important source of some immune components, as supported by the inconsistency between SCC mean values and NAGase, lysozyme and lactoferrin values. Overall, quarters with high levels of NAGase, lysozyme and gamma-G, exposed to bacteria, did not develop subclinical mastitis. Hence, invading pathogens could induce the development of subclinical IMI when these components and gammaG are in low concentration.

Staphylococcus aureus causes different pathologies in humans and animals. In particular, it is involved in intramammary infections in cows, causing economic losses and milk-safety problems. Although it is well-known that surface components (proteins and capsular polysaccharides) and exotoxins are virulence factors involved in the pathogenesis of bovine mastitis, less is known about the precise biochemical identity of such molecules. Therefore, mapping of surface proteins using specific disease- and environment-isolates provides a benchmark for strain comparison of pathogens with different pathogenic characteristics and antibiotic resistance mechanism and can aid in defining specific vaccine and therapeutic targets. In this study, we used a proteomic approach on protein extracts of lysostaphin-treated S. aureus in isotonic conditions, to produce a reproducible and well resolved 2-D electrophoresis (2-DE) reference map of surface associated proteins of isolated S. aureus from a case of bovine mastitis. The most abundant protein components were identified by Matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry.

Staphylococcus aureus is the most common etiologic agent of mastitis in bovines and a major cause of economic losses in the dairy industry. Since adhesins, that anchor bacteria to the extracellular matrix (ECM), are among the most important S. aureus virulence factors, we used four adhesion factors (fibrinogen binding protein, fibronectin binding protein A, clumping factor A and collagen adhesin) as target in a DNA vaccine. Intranasal immunization with a pDNA mixture coding the four adhesins, triggered significant levels of specific serum and mucosal Ig that inhibited S. aureus adhesion to cow mammary gland epithelial cells in vitro. Splenocytes of immunized mice challenged in vitro with S. aureus extracts showed a strong proliferative response. Finally, immunized mice were significantly protected against intramammary challenge with S. aureus. Our data confirm the feasibility of a genetic mucosal vaccine targeting S. aureus adhesins and prompt further study to test the efficacy in preventing cow mastitis.

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intramammary infections in dairy herds when collecting data from the field.

International audience; A practical protocol to study udder immune status in field conditions was planned with the aim to assess different non-specific immune parameters in milk samples from dairy heifers during the periparturient period. Five herds located in northern Italy were selected and overall 39 heifers were enrolled in the trial. Milk samples were taken at 7, 14, 21, 28, 45, 60, and 75 days after calving. The parameters assessed were N-acetyl-$\beta$-glucosaminidase (NAGase), lysozyme, respiratory burst (RB), somatic cell counts (SCC) and serum protein profile. SCC and NAGase were higher in the first sampling after calving, while lysozyme showed large variations during the observation period without a definite trend. The levels of RB observed in the first two weeks after calving, even if lower, were not statistically different from the values observed in samples taken over the following weeks. This study confirmed that the levels of immune components in milk are different from what is observed at blood level in the same cow. A significant decrease in RB in milk polymorphonuclear leukocytes (PMN) post-calving was not observed; milk PMN from healthy cows showed low RB levels, while the values from infected quarters were significantly higher. Significant differences between healthy and infected animals were also observed for milk NAG, lactoglobulin and albumin. These data suggest that udder immune response could be influenced both by the cow immune status and by external factors such as pathogens and management. Therefore, the reduction in immune defences, particularly in heifers, is not unavoidable and methods to boost PMN activity should be explored.

The aim of this research was to evaluate the effect of the modification of diet starch content on milk composition and onits nitrogen and mineral fractions. Ten Italian Friesian primiparous cows were randomly assigned to two groups and feda basal total mixed ration, (BSD, basal starch diet, 24.9% starch/DM) until 42 days in milking (DIM). At 43 DIM, 5 animals(control group, CTR) continued to receive the same ration and the remaining 5 cows (experimental group, EXP)were fed a low starch diet (LSD, 21.0% starch/DM) until 65 DIM, followed by a high starch diet (HSD, 28.3% starch/DM)66 to 85 DIM. From 86 DIM until 94 DIM, cows of the EXP group returned to the BSD. Milk samples were collected at 37,50, 60, 70, 80, 94 DIM. Starch intake was lower for EXP at DIM 50, 60 and higher at 70 and 80 DIM (P < 0.01). Milkyield and fat corrected milk (FCM) did not vary between groups and times of sampling, but HSD caused a significant (P< 0.05) reduction of milk fat in the EXP animals. NPN and MUN contents were affected by dietary treatments at 80 DIM(P < 0.05), being higher in CTR and lower in EXP group in comparison to their basal values (37 DIM). The αs1-caseinpercentages at 80 and 94 DIM increased in the EXP but not in the CTR group, and a marked decrease of αs2-casein percentagefor the EXP group at 94 DIM was observed (P < 0.05). Milk sodium content decreased at 80 and 94 DIM and thesodium to potassium ratio was reduced after the return to the basal diet in the EXP group (94 DIM), indicating that dietarystarch variations can be involved in the control of epithelium integrity of mammary gland in early lactation.

During the periparturient period, some impairment of immune defences were observed. Reference values for the different non-specific immune parameters in cows are not generally available, thus limiting the application of these parameters in dairy practice. This paper reports the data on the measurements of different parameters in the blood, and explores the possible influence of the herd on the non-specific immune status of the cow. Five herds located in Northern Italy were selected and overall 39 heifers were enrolled in the trial. Blood samples were taken 14 and 7 days before the expected date of calving, then at 7, 14, 21, 28, 45, 60, and 75 days after calving. The parameters assessed were N-acetyl-β-glucosaminidase (NAGase), lysozyme, nitric oxide, superoxide dismutase, haptoglobin, respiratory burst, and serum protein profile. After calving, a significant decrease of respiratory burst and nitric oxide concentration were observed in comparison with the pre-calving values but not with the post-calving samplings. Total proteins, β- and γ- globulins showed a progressive and significant increase in concentration after calving, in comparison with pre-calving values. The results of the study confirmed that a decrease of immune functions can be observed in commercial dairy herds in the first four weeks after calving. The amplitude of this phenomenon is not common to all animals and all herds, suggesting the possibility to reduce the impairment by improved management and genetic selection. heifers / periparturient period / blood / non-specific immunity

Objective—To determine the epidemiologic pattern of intramammary infections (IMIs) with Staphylococcus aureus during implementation of a control program in 9 commercial dairy herds. Design—Cohort study. Animals—1,651 lactating cows and 53,098 quarter milk samples. Procedures—Nine herds located in different regions of Italy were enrolled. Control of S aureus infections followed the general principles of contagious mastitis control and was based on precise diagnostic procedures and strict control and segregation of infected cows. All lactating cows in each herd were tested, and those free of S aureus IMI were enrolled as the cohorts. Further additions to the cohort group were cows and heifers free of S aureus IMI, as determined from aseptically collected milk samples taken approximately 7 and 14 days after calving. Results—After the ninth month of the program, incidence decreased to < 2 new IMIs/100 cow-months in 7 of the herds. At the end of the study, 8 of 9 herds had an incidence of ≤ 1 new IMI/100 cow-months. Heifers were most at risk of developing an IMI. Conclusions and Clinical Relevance—Results suggest that control of S aureus IMIs can be achieved by use of a control program based on segregation and use of hygienic and therapeutic protocols. Analysis of incidence and identification of risk factors in a herd could avoid the possible shortcomings of the program, maximizing the probability of success. (J Am Vet Med Assoc 2003;223:684–688)

The teat is the main entrance for pathogens into the mammary gland. It also acts as a sensory, motor and primary defence organ. This latter function is important in preventing intramammary infections while efficiency in preventing new infections is determined by teat tissue integrity. Machine milking may evoke mechanical and circulatory impairment in teat tissues. These local metabolic disorders may decrease the efficiency of the local immune defence mechanisms. Teat tissue changes can be estimated by measuring teat thickness before and after milking. Experimental and field studies showed a high correlation between changes in thickness and infection risk. Teats with >5% change in thickness have significantly increased teat duct colonisation rates and intramammary infection rates. The link between changes in teat thickness and infections should be found in changes in local immune defences and measurable changes in cytological and biochemical immune factors are expected. Indeed, the application of experimental milking conditions (i.e. no pulsation milking and positive pressure milking) showed to have a significant influence on some non specific immune factors in teat secretion. Positive pressure milking increases PMNs content and decreases macrophages content of teat secretion. Some enzymes such as NAGase and lysozyme were decreased by positive pressure milking, the concentration of the same enzymes were higher after no pulsation milking. A better knowledge on the interaction between the teat apex immune defense mechanisms and the machine milking process is necessary to reduce the new infection rate of the bovine mammary gland.

The genetic variation of bovine viral diarrhoea virus (BVDV) was studied by comparative nucleotide sequence analysis of 26 Italian field strains collected during the period 1995–2000 in 18 cattle herds. A fragment within the 5′-untranslated region (UTR) was sequenced directly from gel-purified products obtained by reverse transcription polymerase chain reaction. BVDV-1b (n=14), -1c (n=1), -1d (n=1) and BVDV-2 (n=2) strains have been isolated. Most herds were infected by BVDV-1b. Pairwise similarity and cluster analysis of the remaining BVDV-1 isolates (n=8) did not provide a clear-cut assignation to defined BVDV-1 groups. This is the first time that a BVDV-2 isolation was reported in Italy. Among BVDV-2 reference strains, Italian BVDV-2 isolates showed the highest sequence similarity with the CD87 strain. Both BVDV-2 strains were isolated in two healthy animals from different herds. The 5′-UTR sequence of one of the two BVDV-2 strains was identical to a German BVDV field strain. Complete nucleotide homology was found only among BVDV strains isolated from the same herd, showing a herd-specific clustering. Moreover, 99.6% homology was observed between strains from herds linked by livestock trade. Despite the small number of BVDV isolates analysed, it revealed a high level of genetic diversity among Italian field BVDV strains.

The identification of cows that are positive for mastitis caused by Staphylococcus aureus is difficult under field conditions. The frequency of isolation of S. aureus from quarter milk samples was compared with the frequency of recovery of S. aureus from sediment after centrifugation of those same samples. Overall, 776 quarter milk samples from 194 cows were studied. Cultures that were positive for S. aureus were obtained from 82 samples; 153 sediments from quarter milk samples were also positive for S. aureus. The results of this investigation showed that cultures of the sediment of quarter milk samples increased the number of positive outcomes up to 145.5%, depending on the herd. Using a different group of samples, including samples taken 1 to 5 d or 7 to 10 d after calving and samples taken after intramammary therapy, a 94% increase in cultures that were positive for S. aureus after centrifugation was found compared with cultures of the same quarter milk samples that were not centrifuged. Sedimented cultures may be useful in S. aureus control programs that require the segregation, selective treatment, or culling of cows that are positive for S. aureus.

Maternal transcripts are accumulated during oocyte growth and drive early embryonic development; therefore, their characterisation is a relevant factor for predicting fertility. DNA microarrays have been the method of choice for transcriptional profiling, but this method has some limitations when applied to domestic species because it relies upon existing knowledge about genome sequence and offers a limited quantitative evaluation. These limits are overcome by next-generation sequencing technology. The aim of the work was to define a reference standard for bovine fertility determining the list and the level of transcripts stored in fully grown oocytes collected from heifers (H) and to compare this pattern with that of adult repeat breeders (RB). Oocytes were collected by ovum pick-up (OPU) from 5 Italian Dappled Red heifers of 11 to 15 months of age that became pregnant at the following oestrus and from 4 adult cows of the same breed with an age of 4 to 7 years, classified as repeat breeders after they failed to become pregnant for a minimum of 3 consecutive AI. In both groups, oocytes were aspirated from follicles of 4 to 6 mm in diameter. Each oocyte was carefully denuded and immediately snap frozen in liquid nitrogen. Oocytes from each animal were pooled together (range 4 to 11) and analysed as a single sample. Total RNA extraction was performed by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, from both mRNA and non-polyadenylated transcripts, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (NuGEN Inc., San Carlos, CA, USA). Purified cDNA was ligated directly into an Illumina sequencing library using TruSeq DNA Sample Prep kit (Illumina Inc., San Diego, CA, USA). Sequencing was performed on Illumina HiSEqn 2000 in the 50-bp long single-read set-up, at a 4-plex of multiplexing level, producing 30 to 40 million reads per sample. Data were annotated using the cDNA ENSEMBL UMD 3.1.67 database. On average, the number of transcripts present in each sample was 15 438 ± 766 in H and 15 624 ± 768 in RB oocytes. Nineteen thousand one hundred sixty-one transcripts were detected at least in one sample, and 12 174 were detected in all samples. The comparison between H and RB showed that 598 transcripts out of 19 161 (3.12%) and 437 out of 12 174 (3.59%) are expressed at a significantly different level (P

Recent studies have suggested an association between reproductive failure and mastitis in lactating dairy cows, but our understanding of how mastitis affects reproduction is still limited. In the present study we investigated the effects of naturally occurring chronic mastitis on the population dynamics of ovarian follicles. Ovaries and milk samples were collected from 74 cows at slaughter. Milk samples from each quarter, were analyzed following National Mastitis Council procedures. Based on the presence of major pathogens and somatic cell count results, animals were sorted in 9 groups, but only the 2 extremes were considered for further analysis: uninfected (n = 8) and affected by chronic mastitis (n = 9). Primordial, primary, and secondary follicles were counted and scored on similar surface area of ovary sections for each animal (mean ± SD = 5.65 ± 0.25 cm2). They were analyzed with Fisher’s exact test, and the association between health status and follicle number was estimated by odds ratios ± confidence limits. Vasculature area in the ovarian cortex of healthy and mastitic animals was identified using Bandeiraea simplicifolia-I lectin (BSL-I). Results were quantified with the dedicated software MacBiophotonics image J, NIH, USA, and subsequently analyzed with t-test for statistical significance. Follicles were further characterized by immunostaining with a GDF-9-specific antibody. The intensity of the staining was semi-quantified using a relative scale: 0, 1, and 2 for no, weak, and strong staining, respectively. Our results indicate no (P > 0.05) difference between the numbers of primordial and primary follicles in healthy and affected animals. In contrast, the number of secondary follicles was significantly lower in sick animals (odds ratio 10.50*; P

SummaryMachine-induced changes in teat thickness were measured in a randomly selected group of 22 cows from a commercial dairy herd consisting of 110 cows during an experiment lasting 1 year. Half the cows used were free from mastitis whereas the remainder had at least one quarter infected by environmental pathogens. Teats were classified according to a threshold change in teat end thickness of 5%. The relationships between quarter infection or teat duct colonization and teat end thickness changes induced by machine milking were investigated. Teats with > 5% change in thickness had significantly increased teat duct colonization and a slight, non-significant, increase in quarter infection. We conclude that machine-induced changes in teat end thickness are predisposing factors for teat duct colonization by environmental pathogens. This may provid: an increased new infection risk, especially when hygiene is poor.