Authentication of Italian PDO lard using NIR spectroscopy, volatile profile and fatty acid composition combined with chemometrics.

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.

Which are important targets in development of S. aureus mastitis vaccine?

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 vaccine design were discussed, including how differences among in vivo models may influence vaccines development.

Short communication: study on veterinarian communication skills preferred and perceived by dairy farmers.

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.

Chronic mastitis is associated with altered ovarian follicle development in dairy cattle.

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.

Newborn pig ovarian tissue xenografted into Severe Combined Immunodeficient (SCID) mice acquires limited responsiveness to gonadotropins.

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.

Discriminating between strains of Escherichia coli using pulsed-field gel electrophoresis and BOX-PCR.

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.

Comparison of blood non-specific immune parameters in Bovine virus diarrhoea virus (BVDV) persistently infected and in immune heifers.

Several data from different authors show that Bovine virus diarrhoea virus (BVDV) could be a key component in multiple-etiology diseases, indeed a lower leukocytes number and their impaired functions decrease the resistance to infections. However, most of the information on the impairment of immune function during BVDV infections arise from circumstantial evidence and from experimental infection studies, and few from field data. To assess the effects of BVDV on blood cells parameters, cellular and humoral functions under field conditions, we designed a controlled study in commercial dairy herds, comparing persistent infected (PI) and healthy heifers. A total of 45 heifers were considered, the PI animals were nine, the control animals were 34, while two controls were considered as acute infected animals. The comparison of the mean values in PI calves showed a significant decrease for leukocytes and granulocytes, while platelets showed a significant increase, when compared with control animals. The total number of lymphocytes decreased not significantly in PI animals, while the proportion significantly increased. The number and proportion of monocytes was significantly reduced in PI animals, when compared with controls. The data collected on markers of cellular immunity during our study cannot be compared with the literature because there are no reference values. The presence of a persistent infection affected the cellular enzymes: NAGase, lysozyme and respiratory burst showed a large statistically significant decrease in PI animals when compared with controls. The presence of a persistent infection with BVD virus influenced blood cells number and impaired some blood cell functions. Such impairment confirms that PI animals represent a threat to the herd not only because they could spread BVDV, but also because they are more susceptible to other infectious diseases.

Field study on protocols for evaluation of teat skin conditions.

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.

Epidemiological study of non-contagious intramammary infections in nine commercial dairy herds following a Staphylococcus aureus control programme.

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.

Genotypic characteristics of bovine viral diarrhea virus 2 strains isolated in northern Italy.

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.

Distribution pattern of bovine viral diarrhoea virus strains in intensive cattle herds in Italy.

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.

Relationship among plasmids recovered from Staphylococcus aureus, milk leukocytes, and antimicrobial resistance.

The efficacy and efficiency of mastitis control schemes in reducing the incidence of Staphylococcus aureus mastitis could be improved if the different strains of Staph. aureus were characterized by their pathogenicity. Plasmid finger printing of Staph. aureus strains could be a rather simple and efficient method to characterize the strains and their interaction with host cellular immune defenses. The aim of this study was to investigate the presence and distribution of plasmids in strains of Staph. aureus isolated from bovine mastitis and their relationship with mammary gland immune defenses and antimicrobial resistance. Overall, nine different plasmids were identified by their molecular weight. One or more plasmids were isolated from 293 strains (96.4%), and only 11 strains did not show the presence of any plasmid. A significant difference in polymorphonuclear neutrophil proportions was observed in association with 23-kb, 9416-bp, 2027-bp, or 1353-bp plasmids, and for macrophage proportions, significant differences were observed for 9416-bp, 6557-bp, or 1353-bp plasmids. Penicillin G minimum inhibitory concentration values showed significant differences when 6557-bp, 4361-bp, 2322-bp, or 2027-bp plasmids were present, and weak or no differences were observed for cephacetrile, rifamycin, and norfloxacin. The presence of 2027-bp plasmid was associated with a reduced neutrophil response and an increased resistance against beta-lactam antimicrobials; therefore, the strains carrying this plasmid have a higher probability to invade and colonize mammary gland, to survive to antimicrobial therapy, and to remain undetected. Results confirmed that plasmid fingerprinting is a simple and effective way to characterize Staph. aureus strains.

Relationship between teat tissue immune defences and intramammary infections.

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.

Study on the relationship between milk immune factors and Staphylococcus aureus intramammary infections in dairy cows.

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-beta-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.

Efficacy of a biological response modifier in preventing Staphylococcus aureus intramammary infections after calving.

A change in the epidemiology of mastitis in recent years has emphasized the role of the udder immune system in the pathogenesis of Staphylococcus aureus. Therefore, if the bovine or udder immune capability could be enhanced, susceptibility to Staph. aureus could be reduced and antibiotic efficacy could be increased. Immune system defense mechanisms could be enhanced by vaccination and by biological response modifiers. Within this latter group, a biological response modifier obtained from Parapox ovis that was attenuated over 200 tissue culture passages was recently developed and commercialized in some European countries. This study reports the results of a field trial on the efficacy of this biological response modifier in reducing Staph. aureus intramammary infection (IMI) after calving in primiparous and pluriparous cows. The trial included 106 cows sampled six times (55 cows from herd A and 51 from herd B) for a total of 2544 quarter milk samples. The analysis of IMI prevalence showed that 25.09% of samples were bacteriologically positive in the placebo group, and 23.17% of the positive samples were observed in the biological response modifier group. Staphylococcus aureus IMI had a frequency of 11.44% in the placebo group and 6.00% in the biological response modifier group. The dynamic of the hazards showed significantly lower rates in the biological response modifier group than in the placebo group (risk ratio = 0.47). Treatment with the parapox-containing biological response modifier showed significant reduction of Staph. aureus IMI around calving, and this reduction was attributed to an increase in immune defenses.

Study on prevalence of bovine viral diarrhoea virus (BVDV) antibodies in 29 Italian dairy herds with reproductive problems.

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%).

Recovery of Staphylococcus aureus from centrifuged quarter milk samples.

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.

Field study on the relationship between teat thickness changes and intramammary infections.

The aim of this study was to describe the results of teat thickness measurement applied routinely in three commercial dairy herds and to evaluate the influence of machine-induced teat thickness changes on intramammary infection and the frequency of new infection. A total of 1018 fore milk samples and the same number of teat apex measurements have been evaluated. Overall, relative teat thickness changes were normally distributed (mean -0.16%, SD 10.15%), while a specific pattern could be observed within herds. Increases in teat thickness of > 5% were significantly associated with infection and new infection (odds ratio > 1), but the association was not significant when teat thickness decreased by more than 5%. When results were classified according to aetiology, analysis showed that coagulase-negative staphylococcal infections were significantly associated with both increases and decreases in teat thickness numerically greater than 5%.

Phagocytic activity of bovine polymorphonuclear neutrophil leucocytes.

Two different investigations were conducted on the chemiluminescent activity of bovine milk polymorphonuclear neutrophil leucocytes (PMN) activated by different stimuli: zymosan, derived from the wall of Saccharomyces cerevisiae (Experiment A), and Streptococcus uberis and Escherichia coli (Experiment B). In Experiment A, a quarter with a phagocytic activity of PMN < 20 mV/1000 PMN following stimulation with zymosan was found to be 23 times more likely to be a clinical mastitis case than a quarter with phagocytic activity above this threshold value. In Experiment B, calculation of the odds ratio showed similar results following stimulation with Str. uberis or Esch. coli. These results provide evidence that immunocompromisation of mammary gland defences could predispose to clinical mastitis. They also support the need to challenge phagocytic cells with appropriate stimuli, and the Esch. coli test seems to be the most sensitive.

Seroepidemiological and clinical survey of feline immunodeficiency virus infection in northern Italy.

Four hundred and thirty-nine feline serum samples from cats with different living conditions in the north of Italy were tested for antibodies to feline immunodeficiency virus (FIV) and for antigen of Feline Leukemia Virus by enzyme-linked immunosorbent assay. A Western blot technique was also used on the positive sera in order to confirm the presence of specific antibodies to FIV. The Western blot enabled the detection of a false positive serum. The prevalence of FIV infection in this population was 12.5% and among the seropositive cats a greater proportion was male (74.5%) than female (25.5%). A correlation between the clinical status and the evolution of the pathology is described together with a score based on the severity of the stomatitis in infected cats. The Western blot patterns of positive samples were then compared with the stage of the pathology. Statistical analysis on the distribution of FIV in stray cats, cats with garden and courtyard access and strictly house-confined cats showed a highly significant risk of the infection in the first group.