Comparison of Lateral and Dorsal Recumbency During Endoscope-Assisted Oophorectomy in Mature Pond Sliders (Trachemys Scripta)

Ovariectomy via the prefemoral fossa is a well-described technique for sterilization in chelonians. The choice between lateral and dorsal recumbency is generally left to the surgeon\u2019s preference, with no data supporting an objective superiority of one over the other. Twenty-four sexually mature female pond sliders (Trachemys scripta) were enrolled for elective prefemoral endoscope-assisted oophorectomy, and were randomly divided in two groups: 12 animals were placed in right lateral recumbency with a left fossa approach (Group A), and 12 in dorsal recumbency with a right fossa approach (Group B). Scoring systems were applied to assess the ease of access to the coelomic cavity, and the ease of identification of the ovary opposite to the surgical incision. A negative correlation was found between the body weight of the animals and the ease of access to the coelom (p = 0.013), making the access easier in smaller animals. No significant dierence was found between the two groups in terms of ease of access to the coelomic cavity, first ovary removal time, ease of identification of the second ovary, second ovary removal time, or total surgery time

Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: New perspectives for monitoring and understanding mastitis in dairy goats

The milk somatic cell count (SCC) is a standard parameter for monitoring intramammary infections (IMI) in dairy ruminants. In goats, however, the physiological increase in SCC occurring in late lactation heavily compromises its reliability. To identify and understand milk protein changes specifically related to IMI, we carried out a shotgun proteomics study comparing high SCC late lactation milk from goats with subclinical Staphylococcus aureus IMI and from healthy goats to low SCC mid-lactation milk from healthy goats. As a result, we detected 52 and 19 differential proteins (DPs) in S. aureus-infected and uninfected late lactation milk, respectively. Unexpectedly, one of the proteins higher in uninfected milk was serum amyloid A. On the other hand, 38 DPs were increased only in S. aureus-infected milk and included haptoglobin and numerous cytoskeletal proteins. Based on STRING analysis, the DPs unique to S. aureus infected milk were mainly involved in defense response, cytoskeleton organization, cell-to-cell, and cell-to-matrix interactions. Being tightly and specifically related to infectious/inflammatory processes, these proteins may hold promise as more reliable markers of IMI than SCC in late lactation goats. Significance: The biological relevance of our results lies in the increased understanding of the changes specifically related to bacterial infection of the goat udder in late lactation. The DPs present only in S. aureus infected milk may find application as markers for improving the specificity of subclinical mastitis monitoring and detection in dairy goats in late lactation, when other widespread tools such as the SCC lose diagnostic value

Proteomic datasets of uninfected and Staphylococcus aureus-infected goat milk

We present a proteomic dataset generated from half-udder Alpine goat milk. The milk samples belonged to 3 groups: i) mid-lactation, low somatic cell count, uninfected milk (MLU, n=3); ii) late lactation, high somatic cell count, uninfected milk (LHU, n=3); and late lactation, high somatic cell count, Staphylococcus aureus subclinically infected milk (LHS, n=3). The detailed description of results is reported in the research article entitled \u201cImpact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats\u201d. After milk defatting, high speed centrifugation and trypsin digestion of milk with the FASP protocol, peptide mixtures were analyzed by LC-MS/MS on a Q-Exactive. Peptide identification was carried out using Sequest-HT in Proteome Discoverer. Then, the Normalized Abundance Spectrum Factor (NSAF) value was calculated by label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation by Uniprot was carried out by reporting biological process, molecular function and cellular component. The MS data have been deposited to the ProteomeXchange via the PRIDE with the dataset identifier PXD017243

Relationship of Late Lactation Milk Somatic Cell Count and Cathelicidin with Intramammary Infection in Small Ruminants

Late lactation is a critical moment for making mastitis management decisions, but in small ruminants the reliability of diagnostic tests is typically lower at this stage. We evaluated somatic cell counts (SCC) and cathelicidins (CATH) in late lactation sheep and goat milk for their relationship with intramammary infections (IMI), as diagnosed by bacteriological culture (BC). A total of 315 sheep and 223 goat half-udder milk samples collected in the last month of lactation were included in the study. IMI prevalence was 10.79% and 15.25%, respectively, and non-aureus staphylococci were the most common finding. Taking BC as a reference, the diagnostic performance of SCC and CATH was quite different in the two species. In sheep, receiver operating characteristic (ROC) analysis produced a higher area under the curve (AUC) value for CATH than SCC (0.9041 versus 0.8829, respectively). Accordingly, CATH demonstrated a higher specificity than SCC (82.92% versus 73.67%, respectively) at comparable sensitivity (91.18%). Therefore, CATH showed a markedly superior diagnostic performance than SCC in late lactation sheep milk. In goats, AUC was <0.67 for both parameters, and CATH was less specific than SCC (61.90% versus 65.08%) at comparable sensitivity (64.71%). Therefore, both CATH and SCC performed poorly in late lactation goats. In conclusion, sheep can be screened for mastitis at the end of lactation, while goats should preferably be tested at peak lactation. In late lactation sheep, CATH should be preferred over SCC for its higher specificity, but careful cost/benefit evaluations will have to be made

Milk cathelicidin and somatic cell counts in dairy goats along the course of lactation

This research communication reports the evaluation of cathelicidin in dairy goat milk for its relationship with the somatic cell count (SCC) and microbial culture results. Considering the limited performances of SCC for mastitis monitoring in goats, there is interest in evaluating alternative diagnostic tools. Cathelicidin is an antimicrobial protein involved in innate immunity of the mammary gland. In this work, half-udder milk was sampled bimonthly from a herd of 37 Alpine goats along an entire lactation and tested with the cathelicidin ELISA together with SCC and bacterial culture. Cathelicidin and SCC showed a strong correlation (r = 0.72; n = 360 milk samples). This was highest in mid-lactation (r = 0.83) and lowest in late lactation (r = 0.61), and was higher in primiparous (0.80, n = 130) than in multiparous goats (0.71, n = 230). Both markers increased with stage of lactation, but cathelicidin increased significantly less than SCC. Inaddition, peak level in late lactation was lower for cathelicidin (5.05-fold increase) than for SCC (7.64-fold increase). Twenty-one (5.8%) samples were positive to bacteriological culture, 20 for coagulase-negative staphylococci and one for Streptococcus spp.; 18 of them were positive to the cathelicidin ELISA (85.71% sensitivity). Sensitivity of SCC >500 000 and of SCC >1 000 000 cells/ml was lower (71.43 and 23.81%, respectively). Therefore, the high correlation of cathelicidin with SCC during the entire lactation, along with its lower increase in late lactation and good sensitivity indetecting intramammary infection (IMI), indicate a potential for monitoring subclinical mastitis in dairy goats. However, based on this preliminary assessment, specificity should be improved (40.41% for cathelicidin vs. 54.57 and 67.85% for SCC >500 000 and >1 000 000 cells/ml, respectively). Therefore, the application of cathelicidin for detecting goat IMI will require further investigation and optimization, especially concerning the definition of diagnostic thresholds

Determination of fatty acids profile in original brown cows dairy products and relationship with alpine pasture farming system

This study aimed to evaluate the relationships between fatty acids and the pattern that most contributes to discriminate between two farming systems, in which the main difference was the practice, or not, of alpine summer-grazing. Milk and cheese were sampled every month in two farms of Original Brown cows identical under geographical location and management during no grazing season point of view in the 2018 season. Fatty acids concentrations were determined by gas chromatography. The principal component analysis extracted three components (PCs). Mammary gland de novo synthetized fatty acids (C14:0, C14:1 n9, and C16:0) and saturated and monosaturated C18 fatty acids (C18:0, C18:1 n9c) were inversely associated in the PC1; PC2 included polyunsaturated C18 fatty acids (C18:2 n6c, C18:3 n3) and C15:0 while conjugated linoleic acid (CLA n9c, n11t) and fatty acids containing 20 or more carbon atoms (C21:0, C20:5 n3) were associated in the PC3. The processes of rumen fermentation and de novo synthesis in mammary gland that are, in turn, influenced by diet, could explain the relationships between fatty acids within each PC. The discriminant analyses showed that the PC2 included the fatty acids profile that best discriminated between the two farming systems, followed by PC3 and, lastly, PC1. This model, if validated, could be an important tool to the dairy industry

Proteomic changes in the milk of water buffaloes (Bubalus bubalis) with subclinical mastitis due to intramammary infection by Staphylococcus aureus and by non-aureus staphylococci

Subclinical mastitis by Staphylococcus aureus (SAU) and by non-aureus staphylococci (NAS) is a major issue in the water buffalo. To understand its impact on milk, 6 quarter samples with >3,000,000 cells/ mL (3 SAU-positive and 3 NAS-positive) and 6 culture-negative quarter samples with <50,000 cells/ mL were investigated by shotgun proteomics and label-free quantitation. A total of 1530 proteins were identified, of which 152 were significantly changed. SAU was more impacting, with 162 vs 127 differential proteins and higher abundance changes (P < 0.0005). The 119 increased proteins had mostly structural (n = 43, 28.29%) or innate immune defence functions (n = 39, 25.66%) and included vimentin, cathelicidins, histones, S100 and neutrophil granule proteins, haptoglobin, and lysozyme. The 33 decreased proteins were mainly involved in lipid metabolism (n = 13, 59.10%) and included butyrophilin, xanthine dehydrogenase/oxidase, and lipid biosynthetic enzymes. The same biological processes were significantly affected also upon STRING analysis. Cathelicidins were the most increased family, as confirmed by western immunoblotting, with a stronger reactivity in SAU mastitis. S100A8 and haptoglobin were also validated by western immunoblotting. In conclusion, we generated a detailed buffalo milk protein dataset and defined the changes occurring in SAU and NAS mastitis, with potential for improving detection (ProteomeXchange identifier PXD012355)

SUSCEPTIBILITY TO MAIN ANTIBIOTICS OF GRAM-NEGATIVE BACTERIA FROM BOVINE MASTITIS IN ITALIAN HERDS

Introduction .The incorrect use of antibiotics in veterinary medicine has contributed to reduce the pathogen susceptibility to antibiotics. Through direct contact or food chain the resistant bacteria can be transferred to humans (2). In dairy cows, differently from other production animals, milk discharge during antibiotic administration and related withdrawal time has a direct economical effect on production. This issue has determined a more responsible use of antibiotics. Moreover, in mastitis field antibiotics are used mainly to treat clinical cases and not for prophylaxis, except when blanket dry-cow therapy is applied. For this reason monitoring susceptibility to antibiotic-resistance in dairy cows could identify different trend respect to other food animals. Gram-negative bacteria are frequently cause of clinical mastitis and, as part of enteric flora, they are subjected to the selective pressure of systemic drug treatments. For this reason they are subjected to specific monitoring program for antibiotic resistance. This paper describes a study about in vitro antimicrobial susceptibility of Gram negative bacteria isolated from bovine clinical mastitis in Northern Italy. Materials and Methods Among Gram negative bacteria isolated from bovine clinical mastitis in Northern Italy during the years 2010/2011, 99 isolates were selected: 53 E.coli, 28 Klebsiella spp, 14 Serratia spp, 2 Enterobacter spp. e 2 Citrobacter spp. Pathogens isolation was carried out in milk samples following the National Mastitis Council guideline (3). Species identification was performed through commercial kits API 20 and API 20 NE (Biomerieux Marcy l\u2019Etoile, France). In vitro antimicrobial susceptibility was evaluated using minimal inhibitory concentrations (MICs) broth dilution test, according to the procedure described in Clinical and Laboratory Standards Institute (CLSI) guideline. CLSI resistance breakpoints were used (1). Sensititre susceptibility plates were used according to supplier instructions (Trek Diagnostics System, East Grinstead, UK). The antibiotics used are listed in table 1. After incubation the plates were manually read recording the last dilution able to inhibit bacterial growth. Results and Discussion The 36% of tested E. coli resulted resistant towards ampicillin, while using penicillins associated to clavulanic acid the percentage of resistant isolates declined to 7,5%. Serratia spp isolates demonstrated a higher resistance level to amoxicillin/clavulanic acid, with susceptibility reduced to 74%. Cephalosporins confirmed a very high efficacy in the regards of E coli, as 94,3% of isolates were susceptible to 1st generation cephalosporins and 100% to 3rd generation (ceftiofur and cefpodoxime). Similar results were obtained for Klebsiella spp, while all Serratia spp tested isolates revealed some resistance to 1st generation cephalosporins, maintaining susceptibility to 3rd generation cephalosporins. Among drugs active specifically towards Gram-negatives bacteria, chloramphenicol and sulfonamide associated with trimethoprim demonstrated high efficacy to all tested species. Average susceptibility was 85% for sulfonamide with trimethoprim, and 95% for chloramphenicol. Finally, only one E. coli isolate resulted resistant to fluoroquinolons with an intermediate MIC for marbofloxacin and enrofloxacin, while all the others 98 Gram-negative isolates tested were susceptible (MIC < 0,5 \uf06dg/mL). Complete data are shown in table 1. Table 1. In vitro susceptibility and resistance of E. coli and Klebsiella spp., Serratia spp., Enterobacter spp. and Citrobacter spp. (joined in \u201cother Gram-negatives\u201d) towards tested antibiotics. Antibiotics Sensible E. coli (%) Resistant E. coli (%) Sensible other Gram-negatives (%) Resistant other Gram-negatives (%) Ampicillin 64,2% 35,8% 30,4% 69,6% Amoxicillin/clavulanic acid 92,5% 7,5% 73,9% 26,1% Amikacin 100,0% 0,0% 100,0% 0,0% Cefpodoxime (3rd gen. Ceph.) 100,0% 0,0% 100,0% 0,0% Ticarcillin/clavulanic acid 94,3% 5,7% 89,1% 10,9% Sulfonamide/thrimethoprim 86,8% 13,2% 89,1% 10,9% Cefazolin (1\ub0st gen. Ceph.) 94,3% 5,7% 63,0% 37,0% Gentamicin 100,0% 0,0% 100,0% 0,0% Imipenem 100,0% 0,0% 100,0% 0,0% Penicillin 0,0% 100,0% 0,0% 100,0% Doxiciclin 75,5% 24,5% 71,7% 28,3% Ceftiofur (3rd gen. Ceph.) 100,0% 0,0% 100,0% 0,0% Marbofloxacin 98,1% 1,9% 100,0% 0,0% Enrofloxacin 98,1% 1,9% 100,0% 0,0% Chloramphenicol 94,3% 5,7% 97,8% 2,2% In conclusion, this study demonstrates that Gram-negative bacteria commonly isolable in case of mastitis are susceptible to most antibiotics, included fluoroquinolons, sulfonamide and chloramphenicol. Reduced susceptibility to some beta-lactams in E.coli are avoided associating clavulanic acid, an inhibitor of beta-lactamases. Serratia spp. is an exception probably due to specific beta-lactamases produced, which are resistant to inhibitors. This findings confirm the importance of milk culture in mastitis cases to achieve an etiological diagnosis, that allows to choose treatment or culling for chronically infected animals. The aims of treatment procedures in farm animal should be avoiding abuse of antibiotic therapy and reducing selection of resistant strains. Periodical survey and detection of resistance phenomenon are recommended. References 1. CLSI. (2008). Performances standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: approved standard \u2013 third edition CLSI document M31-A3. CLSI Wayne, PA, (USA): Clinical and Laboratory Standard Institute. 2. Friedman D.B., Kanwat CP, Headrick ML, Patterson NJ, Neely JC, Smith LU. (2007) Importance of prudent antibiotic use on dairy farms in south carolina: a pilot project on farmers\u2019 knowledge, attitudes and practices. Zoonoses Public Health. 54, 366\u2013375. 3.NATIONAL MASTITIS COUNCIL (1999). Laboratory and field handbook on bovine mastitis. National Mastitis Council, Madison WI

Short communication : Circulating extracellular miR-22, miR-155, and miR-365 as candidate biomarkers to assess transport-related stress in turkeys

MicroRNA (miRNA) have been identified in circulating blood and might have the potential to be used as biomarkers for several pathophysiological conditions. To identify miRNA that are altered following stress events, turkeys (Meleagris gallopavo) were subjected to 2 h of road transportation. The expression levels of five circulating miRNA, namely miR-22, miR-155-5p, miR-181a-3p, miR-204 and miR-365-3p, were detected and assessed by quantitative polymerase chain reaction using TaqMan\uae probes, as potential biomarkers of stress. The areas under the receiver operating characteristic curves were then used to evaluate the diagnostic performance of miRNA. A panel of three stress-responsive miRNA, miR-22, miR-155 and miR-365 were identified; their expression levels were significantly higher after road transportation and the area under the curve (AUC) were 0.763, 0.71 and 0.704, respectively. Combining the three miRNA a specificity similar to the one found for the three miRNA separately was found. The AUC of the weighted average of the three miRNA was 0.763. This preliminary study suggests that the expression levels of circulating miR-22, miR-155 and miR-365 are increased during transport-related stress and that they may have diagnostic value to discriminate between stressed- and unstressed animals

Dietary fatty acids on subcutaneous adipose tissue modulation in transition dairy goats

The goal of the present study was to evaluate the metabolic and immune response of peripartal dairy goats to dietary supplementation with fish oil or stearic acid. 15 multiparous alpine dairy goats were involved in the trial. Starting from the last week of gestation until 3 weeks after kidding date the experimental diets, based on alfalfa and mix hays and a concentrate mix, were added either with protected fish oil (FO) or with stearic acid (ST). Feed intake, body weight, energy balance, milk production and composition were measured weekly. Adipose tissue biopsies were performed on day -7, 7 and 21 relative to kidding date and samples were immediately fixed in formalin, paraffin embedded and Hematoxilin Eosin stained. The results discussed in the present work are relative to a subsample of 8 goats, representative of the two experimental groups. Hematological and histological data were analyzed by a Generalized Estimating Equation (GEE) in IBM SPSS 19.0 was used. Production parameters were analyzed by a MIXED repeated model in SAS 9.2. No differences were observed between FO and ST in milk production, BCS, weight, dry matter intake and milk components except for a higher milk protein percentage in the 7 to 21 d period for ST. BHB serum content was higher in ST overall the experiment, whereas NEFA and ALAT serum content were higher at day 7 in FO compared to ST (P < 0.08). ALAT was higher also at day 21 in FO. Treatment had no effect on blood cellular component except for WBC in FO group, where a significant decrease at 7 d was observed. WBC and HCM parameters were in the physiological range for dairy goats during transition period. Histologic adipose tissue analysis revealed a significant decreased adipocytes surface between -7 and 21 d in ST, whereas in FO the adipocyte surface reduction was related to the -7 to 7 d interval reaching a plateau until day 21. The EB pattern and the NEFA serum content at 7d in particular for FO are well correlated with histologic observations indicating goats were using fat depots to cope their negative energy balance. NEFA levels did not confirm the histological evidence at day 21 for ST suggesting a possible different action on subcutaneous adipose tissue during time. Results suggest a modulation in lipid storage management during peripartal negative energy balance by saturated vs. unsaturated dietary fatty acid supplementation that did not affect production levels of goats