Breed and Feeding System Impact the Bioactive Anti-Inflammatory Properties of Bovine Milk.

In the present study, we aimed at assessing the influence of breed and feeding system on the bovine milk profile of betaines and carnitines and milk capacity in counteracting the inflammatory endothelial cell (EC) damage induced by interleukin (IL)-6. In the first experimental design, two breeds were chosen (Holstein vs. Modicana) to investigate the biomolecule content and antioxidant capacity in milk and dairy products. In the second experimental design, two feeding systems (pasture vs. total mixed ratio) were tested only in Holstein to evaluate the possible effect on the functional profile of milk and dairy products. Finally, the bulk milk from the two experimental designs was used to evaluate the efficacy of preventing IL-6-induced endothelial inflammatory damage. Results showed that Modicana milk and whey had higher biomolecule content and antioxidant activity compared to Holstein milk (p < 0.01). Milk from Holstein fed TMR showed higher concentration of γ-butyrobetaine, δ-valerobetaine (p < 0.01), and l-carnitine (p < 0.05). Similarly, whey from Holstein fed TMR also showed higher content of δ-valerobetaine, glycine betaine, l-carnitine, and acetyl-l-carnitine (p < 0.01) compared to the Holstein fed pasture. Conversely, the antioxidant activity of milk and dairy products was not affected by the feeding system. In ECs, all milk samples reduced the IL-6-induced cytokine release, as well as the accumulation of reactive oxygen species (ROS) and the induction of cell death, with the most robust effect elicited by Modicana milk (p < 0.01). Overall, Modicana milk showed a higher content of biomolecules and antioxidant activity compared to Holstein, suggesting that the breed, more than the feeding system, can positively affect the health-promoting profile of dairy cattle milk.

Subclinical Ketosis in Dairy Herds: Impact of Early Diagnosis and Treatment.

Clinical and subclinical ketosis (SCK) in dairy cows occurs during the lactation period frequently in many herds, causing a reduction in milk yield and alterations in milk quality with significant economic losses for farmers. SCK is defined as a preclinical stage of ketosis characterized by an elevated ketone body level without clinical signs. Often many cows develop an elevated ketone body level during the first weeks of lactation even though it never goes up to a critical point causing clinical signs. This study aimed to evaluate the prevalence of SCK in Sicily and assess the effect of a treatment with propylene glycol (PG) to control the SCK, thus, reducing the negative effect on milk quality yield. This cross-sectional study was carried out on 22 farms located south-east of Sicily and 1,588 cows in lactation. A total of 3,989 individual milk samples were collected from calving to 80 subsequently days to check the ß-hydroxybutyrate (BHB) values in order to establish the SCK status by the Fourier Transform Infrared Spectroscopy. Moreover, the contents of fat, protein, lactose, casein, urea, somatic cell count and acetone were evaluated to identify a correlation between SCK and milk quality. A total of 1,100 cows showed BHB values higher than 0.10 mmol/L. These cows were considered SCK positive, were separated from the rest of the herd, and treated with PG (400 g/head per day), all SCK cows were treated with PG and cows without SCK were not treated. The results showed a prevalence of 41.5% of SCK-positive cows during the first 9 days of lactation. The comparison among the cure rate of treated cows shows that the treatment was most effective in the first 7 days of lactation (76.5% of treated cows) than in the following days. PG positively influenced the milk quality parameters, except for the fat proportion. Moreover, the animals treated with PG showed also an increase in milk yield, supporting the economical sustainability of treatment.

Prevalence of Infectious Diseases on Dairy Farms Classified on The Basis of Their Biosecurity Score.

Introduction: The aim of the study was to investigate the relationship between biosecurity as scored on the Italian National Animal Welfare Reference Centre (Centro di Referenza Nazionale per il Benessere Animale - CReNBA) checklist and the prevalence of Mycobacterium avium subsp. paratuberculosis, Chlamydophila abortus and Neospora caninum on dairy farms located in Ragusa, Italy. Material and Methods: The checklist was used to assign an animal welfare score to 31 dairy farms. Twenty-one farms with a moderate score (>33%, <66%) formed group 1, and 10 farms with a high score (>66%) were group 2. Blood samples were collected from all cows on each farm to investigate the titres of antibodies against the relevant pathogens. Two-way analysis of variance was applied to assess differences between the two experimental groups and the Mann-Whitney test was applied to evaluate prevalence differences in the tested parasites between the groups. Results: All tested farms had a score that classified them as either good or excellent. A higher incidence of Neospora caninum was observed in group 1. The incidences of the other two parasites were no different between the two groups. Conclusion: The CReNBA checklist represents an impartial, reproducible, functional and smart instrument based on risk analysis and assigns a farm a mathematical animal welfare score. Among the parasites tested for, only Neospora caninum had prevalence influenced by biosecurity. Our preliminary results highlighted the positive associations between good animal welfare, high levels of biosecurity, and the prevention of the infectious diseases caused by the parasites in our focus, which are common on dairy farms.

First Molecular Survey to Detect Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry Farms in a Strategic Production District of Sicily (South-Italy).

Mycoplasmas are recognized as avian pathogens, which may cause both respiratory disease and synovial infections in poultry, resulting in severe economic losses. Our study aims to determine the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) among commercial and rural laying hens located in Ragusa province (South Italy), using a duplex real time PCR. Four hundred tracheal swabs were collected from seven commercial (200 swabs) and 25 rural (200 swabs) farms without any clinical disease history. Out of 400 swabs collected, 50 (12.5%) and 93 (23.25%) were positive for MG and MS, respectively. In particular, 9 (18%) and 22 (23.65%) positive swabs for MG and MS, respectively, originated from commercial farms, compared to 41 (82%) and 71 (76.34%) obtained from rural farms. Data obtained show a lower prevalence of MG than MS in the studied farms. Moreover, both pathogens were spread in rural and commercial farms. PCR could be concluded as a rapid and sensitive method for the identification of MG and MS in areas where commercial farms that are declared Mycoplasma-free and rural flocks coexist. These data highlight the importance of surveillance also in rural poultry to monitoring the occurrence of mycoplasmas strains in strategic productive districts.

Antimicrobial Resistance (AMR) of Bacteria Isolated from Dogs with Canine Parvovirus (CPV) Infection: The Need for a Rational Use of Antibiotics in Companion Animal Health.

Canine parvovirus type 2 (CPV-2) represents a major viral threat to dogs. Considering the potential effects of pets on antimicrobial resistance, information on the CPV and associated bacterial co-infections is limited. The aim of this study was to analyze the antimicrobial susceptibility and multidrug-resistance profiles of bacterial species from tissue samples of dogs with canine parvovirus infection. A set of PCR assays and sequence analyses was used for the detection and the molecular characterization of the CPV strains and other enteric viruses. Bacterial isolation, the determination of antimicrobial susceptibility via the disk diffusion method, and the determination of the minimum inhibitory concentration were performed. The detection of ß-lactamase genes and toxin genes for specific bacteria was also carried out. CPV infection was confirmed in 23 dogs. Forty-three bacterial strains were isolated and all showed phenotypic resistance. Seventeen multidrug-resistant bacteria and bacteria with high resistance to third- and fourth-generation cephalosporins and metronidazole were detected. Almost 50% of the isolated Enterobacteriaceae were positive for at least one ß-lactamase gene, with the majority carrying more genes as well. The evidence for multi-resistant bacteria with the potential for intra- or cross-species transmission should be further considered in a One Health approach.

Management Factors Influence Animal Welfare and the Correlation to Infectious Diseases in Dairy Cows.

The present study assessed dairy cow welfare through the application of the Italian National Animal Welfare Reference Center (CReNBA) checklist in 36 dairy farms located in Ragusa (Italy) subjected to two different management conditions, housing with free access to pasture (Group 1, farms n = 17) and indoor housing (Group 2, farms n = 19). Five areas of investigation were considered: Area A, "Farm management and personnel"; Area B, "Facilities and equipment"; Area C, "Animal-based measures"; Area D, "Inspection of microclimatic environmental conditions and alarm systems"; and Area E, "Biosecurity". Blood samples were collected by coccygeal venipuncture from all animals (4081 cows). The specific antibodies against Mycobacterium avium subsp. paratuberculosis, Chlamydiophila abortus, Neospora caninum, bovine viral diarrhea virus, and the bovine herpesvirus were assessed by enzyme linked immunosorbent assay (ELISA) serological test. Group 1 (access to pasture) showed a lower value of percentage score recorded in Area A (p = 0.02) and E (p = 0.01) than Group 2 (indoor housing). Herpesvirus (Infectious bovine rhinotracheitis - IBR - detection of gB antibodies/IBR-gB) blood concentrations were higher in the cows housed indoor versus those with access to pasture (p = 0.01). Farm management and personnel (score A) was correlated with the level of bovine viral diarrhea virus (τ = 0.3754) and bovine-herpesvirus-specific antibodies (IBR-gB) (τ = 0.4159). "Biosecurity" percentage score showed a significant correlation with Chlamydiophila abortus (τ = -0.4621) in the cows with access to pasture and IBR-gB (τ = 0.3435) in the cows housed fully indoors. Group 2 showed a significantly reduced level of antibodies against Neospora caninum. In conclusion, differences in the welfare assessment score were observed in the "Farm management and personnel" and "Biosecurity" between the two management conditions. It had an effect on the prevalence of herpesvirus, which occurred more in cattle with access to pasture. Therefore, an accurate application of the checklist could be an instrument to prevent and control the spread of infections in farms.

Quantification of Some Heavy Metals in Hair of Dairy Cows Housed in Different Areas from Sicily as a Bioindicator of Environmental Exposure-A Preliminary Study.

The objective of this preliminary study was to evaluate the levels of selected heavy metals in hair samples of Holstein dairy cows reared on agricultural soils characterized by grassland subjected to anthropogenic impacts. Ninety Holstein-Friesian cows were enrolled in the study and divided into six groups according to farm origin. From each animal, hair samples were collected in order to determine the content of aluminum, chromium, iron, copper, zinc, arsenic, cadmium, and lead. One-way analysis of variance was applied to assess statistically significant differences in the studied heavy metals among the six groups. A significant effect of groups (p < 0.05) on all tested heavy metals was observed. In this study, the low concentration of heavy metals in the hair of the studied animals led us to think that the cows were subjected to low levels of these compounds, preventing them from bioaccumulating. Although the current study provides only preliminary results, it highlights the importance of investigating the concentration of heavy metals in cow hair to improve the health and welfare of both humans and animals.

Welfare assessment in intensive and semi-intensive dairy cattle management system in Sicily.

The present study aimed to compare the welfare of dairy cows kept in two traditional husbandry systems (semi-intensive and intensive farming) in south-eastern Sicily. A total of 18 dairy farms (nine semi-intensive and nine intensive) were evaluated with a multicriteria system adapted for Sicilian conditions and obtained simplifying the model of the European Food Safety Authority (EFSA). Values of welfare measures, collected by inspections of the farms (general well-being indicators, ventilation system, resting areas [cubicles or bedding], flooring, milking parlours and waiting area, manger and watering equipment), and those of health categories (cases of abortions, hypocalcemia, displacement of abomasum, acidosis/ketosis, enteritis, hoof problems, and mastitis) obtained through the farm records, were compared using Mann-Whitney and Chi-squared tests, respectively. Data showed significant differences (p ≤ .05) about the variables related to welfare categories such as housing ventilation system, resting area, manger, and water equipment that were better in the semi-intensive system than the intensive system. No significant differences were observed about the variables related to health indicators. The results demonstrated that in Sicily the semi-intensive farm is better than the intensive to satisfy the conditions of animal welfare.

Early Prediction of Corpus Luteum Functionality Using an Imaging Software.

The present study aimed to assess the applicability of luteal blood flow data acquired through the use of color Doppler ultrasonography and a post-processing analysis tool (ImageJ) for predicting pregnancy in buffaloes (Bubalus bubalis). The experiment was carried out on 59 multiparous Italian Mediterranean buffaloes that underwent synchronization of estrus and fixed-time artificial insemination (TAI). Corpus luteum features (size: CLS and blood flow: BFA) were taken from Day 5 to 10 after TAI and retrospectively measured with ImageJ. In the same period, blood samples were taken to assess progesterone (P4) concentrations. Pregnancy diagnosis was carried out on Day 45 by ultrasound and confirmed on Day 70 post-TAI. Differences in CLS, BFA, and P4 concentrations from Day 5 to 10 after TAI measured between groups were analyzed by ANOVA repeated measures as were differences within each day of measuring. Buffaloes that established a pregnancy (n = 29; 55%) had larger CLS (2.2 ± 0.1 vs. 1.9 ± 0.1 cm2; P < 0.01), higher BFA (0.6 ± 0.0 vs. 0.4 ± 0.0 cm2; P < 0.01), and higher P4 blood level (1.8 ± 0.1 vs. 1.4 ± 0.1; P < 0.01) during Day 5-10 as compared to not-pregnant buffaloes (n = 22). Throughout the entire period, the first feature that changed between groups was P4 blood concentration at Day 7 (1.7 ± 0.1 vs. 1.2 ± 0.1; P < 0.05) followed by BFA at Day 8 (0.6 ± 0.0 vs. 0.5 ± 0.0; P < 0.05), respectively, in pregnant and not-pregnant animals. The ROC analyses indicated that P4 was able to predict pregnancy since Day 5 (P < 0.05) although a more reliable result could be obtained from Day 8 (P < 0.01). At Day 10, it was possible to set a cutoff value for every parameter taken into account. The logistic regression analysis showed that pregnancy was positively influenced by P4 concentration (odds ratio 534.127; P < 0.01) and BFA (odds ratio 744.893; P < 0.01). In conclusion, the use of color Doppler ultrasonography, together with ImageJ, identified different patterns of BFA between pregnant and not-pregnant buffaloes starting from Day 8 post-TAI.

Morphological and Immunohistochemical Examination of Lymphoproliferative Lesions Caused by Marek's Disease Virus in Breeder Chickens.

Marek's disease is widely controlled by vaccination programs; however, chickens are not totally protected, especially immediately after the vaccination when a strong challenge could interfere with the effectiveness of vaccination in the absence of proper biosecurity practice. This case report describes the occurrence of Marek's disease (MD) observed in a breeder chicken flock reared southeast of Sicily. MD outbreak occurred from 32 to 47 weeks with an increase in weekly mortality rate (+0.4-0.6%). Overall, mortality rate related to Marek's disease was about 6% at the end of the cycle. Carcasses of chickens found during the occurrence of disease underwent necropsy, and tissues were collected to confirm the infection. Gizzard, cecal tonsil, intestine, spleen and tumor mass were collected and analyzed from a carcass of one hen, 32 weeks old and apparently asymptomatic. Multiplex real-time PCR performed on spleen tissues detected the presence of MD virus pathogenic strain. Macroscopic and microscopic evaluation of the rest of the samples confirmed the neoplastic disease. Moreover, the immunophenotype of the tumor cells was identified as CD3 positive by immunohistochemical (IHC) staining. The vaccinated flock had become rapidly infected with the MD virus, which proves that the challenge of the MD virus was too strong in the rearing house at the beginning of the cycle, causing the outbreak.

Inflammatory response in repeat breeder buffaloes.

The aim of this study was to investigate the repeat breeding condition in Italian Mediterranean buffaloes that failed to conceive after at least 300 days in milk. The trial was carried out on 40 pluriparous Italian Mediterranean buffaloes with more than 300 days in milk. All the animals underwent ultrasound examination to assess endometrial thickness and oestrus synchronization by the Ovsynch-TAI Program. On the day of oestrus, blood samples were collected for the haemocytometric cell count and biochemical assay, and the animals were slaughtered in a local abattoir. A post-mortem uterine flushing was performed using sterile saline for microbiological analyses. Furthermore, uterine biopsies were carried out for histopathological assessment. Finally, endometrial samples were used for real-time PCR (RT-PCR) analysis to evaluate the expression of genes involved in innate immune recognition of pathogens and the inflammatory response, such as Toll-like receptor (TLR)1, TLR8, interleukin (IL)-1ß, IL-6, IL-8, COL4A2, connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Statistical analysis was performed by one-way ANOVA. Based on the infiltration of lymphocytes and plasma cells or endometrial gland, lymphatic, and blood vessel ectasia recorded in the histopathological examination, the animals were classified into three groups: healthy (H Group; n = 5), moderate endometritis (M Group; n = 25), and severe endometritis (S Group; n = 10). A significantly greater (P < 0.01) endometrial thickness was recorded in the S Group compared to that in the H and M Group (1.07 ± 0.03 vs. 0.70 ± 0.07 and 0.81 ± 0.04 cm in the S, H, and M Group, respectively). The white blood cell count was lower in the H Group compared to that in the M and S Group (6.3 ± 0.6 vs. 9.3 ± 0.4 and 10.5 ± 0.5 in the H, M, and S Group, respectively). To perform RT-PCR analysis, five animals from groups M and S were randomly selected in order to have balanced results. A higher (P < 0.01) expression of TLR1, together with a lower expression of COL4A2, IL-1ß, IL-6, IL-8, and CYR61, was recorded in the H Group, compared to both the M and S Groups. In conclusion, about 90% of repeat breeder buffaloes show moderate or severe endometritis, associated with an altered histopathological endometrial profile and altered mRNA expression of pro-inflammatory and fibrotic factors.

Short communication: Space allocation in intensive Mediterranean buffalo production influences the profile of functional biomolecules in milk and dairy products.

The aim of the present study was to determine if space allocation influenced the concentration of biomolecules in buffalo milk and dairy products. Intensively housed buffaloes (n = 96) were randomly assigned to 2 groups according to days in milk, parity, and milk yield: group S10 had a space allocation of 10 m2 per buffalo and group S15 had a space allocation of 15 m2 per buffalo. Individual milk yield was recorded daily. Twice a month, a bulk milk sample was collected for each group, as well as whey, ricotta, and mozzarella cheese, to assess cheese yield and to conduct HPLC-electrospray ionization-tandem mass spectrometry, milk antioxidant activity, and cell viability analyses. We tested milk extracts from the 2 groups in vitro to evaluate their efficacy in counteracting endothelial oxidative damage induced by high glucose. We evaluated reproductive function in 28 buffaloes from each group using the Ovsynch-timed artificial insemination program. We observed no differences in milk quantity or quality in terms of fat, protein, or lactose, and reproductive function did not differ between the 2 groups. Compared with group S10, group S15 had higher concentrations of carnitine (56.7 ± 1.1 vs. 39.8 ± 0.7 mg/L in milk and 40.9 ± 0.8 vs. 31.7 ± 0.7 mg/L in whey), acetyl-l-carnitine (51.9 ± 0.3 vs. 39.7 ± 0.7 mg/L in milk and 41.1 ± 1.7 vs. 28.7 ± 2.6 mg/L in whey), propionyl-l-carnitine (34.8 ± 1.0 vs. 21.0 ± 0.9 mg/L in milk and 26.9 ± 0.8 vs. 17.6 ± 1.2 mg/L in whey), glycine betaine (23.1 ± 1.9 vs. 13.5 ± 1.6 mg/L in milk and 10.7 ± 0.4 vs. 7.9 ± 0.5 mg/L in whey), and δ-valerobetaine (24.2 ± 0.5 vs. 16.7 ± 0.5 mg/L in milk and 22.0 ± 0.9 vs. 15.5 ± 0.7 mg/L in whey). Group S15 also had higher total antioxidant activity than group S10 (56.7 ± 1.9 vs. 46.4 ± 1.13 mM Trolox equivalents). Co-incubation of high-glucose-treated endothelial cells with milk extracts from group S15 improved cell viability compared with cells treated with high glucose only; it also reduced intracellular lipid peroxidation (144.3 ± 0.4 vs. 177.5 ± 1.9%), reactive oxygen species (141.3 ± 0.9 vs. 189.3 ± 4.7 optical density units), and cytokine release (tumor necrosis factor-α, IL-1ß, IL-6). Greater space allocation was associated with higher levels of biomolecules in buffalo milk. This could have been the result of improved welfare in buffaloes that were allocated more space.

Effect of live body weight and method of synchronization on ovulation, pregnancy rate and embryo and fetal loss in buffalo heifers.

This study aimed to assess the influence of live body weight (LBW) and age on reproductive performance in buffalo heifers synchronized by different treatments. The study was carried out on 146 Mediterranean buffalo heifers (mean age 25.3±13.4 months, LBW 424±47 kg), divided into 2 homogeneous groups and synchronized by Ovsynch-TAI Program (OVS; n = 72) or double prostaglandin administered 12 days apart (PGF; n = 74). All the buffaloes were inseminated twice and follicle dimensions and ovulation rate (OR) were assessed by ultrasound 24 and 48 h post-insemination. Pregnancy was assessed on day 25, 45 and 90 post-insemination and the incidence of late embryonic (LEM) and fetal (FM) mortality were respectively recorded. Data were analyzed by ANOVA, Chi-square test and multiple logistic regression. The LBW was significantly (P<0.05) higher in inseminated animals, compared to those that did not respond to the treatments (450.0±3.2 vs. 423.2±9.6 kg in inseminated and not inseminated heifers, respectively). Total OR was similar between groups, although OR at 24 h tended to be higher (P = 0.06) in OVS (86.7 vs. 72.9% in OVS and PGF, respectively). A (P<0.01) higher LBW was observed in ovulated heifers of PGF, while no differences were recorded in OVS. LBW affected OR (odds ratio = 1,032; P<0.05) only in PGF, while no effects were recorded in OVS. Total pregnancy rate, LEM and FM were similar between groups. In conclusion, the LBW would be considered before including buffalo heifers in a synchronization program and both synchronization treatments can be useful.

Seroprevalence and occupational risk survey for Coxiella burnetii among exposed workers in Sicily, Southern Italy.

OBJECTIVES: The aim of this survey was to assess the seroprevalence of antibodies against Coxiella burnetii (C. burnetii) in subjects at risk of exposure in Sicily, Southern Italy. MATERIAL AND METHODS: Prevalence of IgG antibodies to C. burnetii phase II antigens was evaluated by ELISA in a group of 140 workers at risk of exposure (38 veterinarians, 38 slaughterhouse workers, 44 livestock handlers, 20 laboratory and technical personnel) included in a medical surveillance program and in 42 control subjects. Positive samples were classified as suggestive of prior exposure to C. burnetii. RESULTS: Antibodies against C. burnetii were detected in 88 out of 140 (62.9%) exposed workers and in 6 out of 42 (14.3%) subjects of the control group. The variables evaluated did not seem to have a significant effect on seropositivity to Coxiella with the exception of symptoms in the last 6 months preceding the survey. CONCLUSIONS: Our study demonstrated a high seroprevalence of C. burnetii in the group of exposed workers in comparison to non-exposed subjects of the control group. Clinical illness appears to be rare; nevertheless, physicians should consider Q fever in patients with compatible symptoms and occupational exposure to animals and their products. As aerosols represent the main route of infection in animals and humans, these workers are strongly advised to wear respiratory masks. In addition, occupational physicians should consider routine serologic evaluation and vaccination of occupationally exposed workers.