Detection of Cathelicidin-1 in the Milk as an Early Indicator of Mastitis in Ewes

The objective of the study was the investigation of the behaviour of cathelicidin-1 in the milk after experimental infection with two prominent bacterial pathogens (experiment 1: Mannheimia haemolytica, experiment 2: M. haemolytica and Staphylococcus chromogenes) as a potential early indicator for diagnosis of mastitis in sheep. In two experiments, after bacterial inoculation into the udder of ewes, bacteriological and cytological examinations of milk samples as well as proteomics examinations [two-dimensional gel electrophoresis analysis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) analysis] were performed sequentially. Cathelicidin-1 was detected and spot densities obtained from PDQuest v.8.0 were recorded. Associations were calculated between cell content and spot densities as well as between presence of mastitis in a mammary gland at a given time-point and detection of cathelicidin-1 in the respective milk sample. All inoculated mammary glands developed mastitis, confirmed by the consistent bacterial isolation from mammary secretion and increased leucocyte content therein. Spot density of cathelicidin-1 in samples from inoculated glands increased 3 h post-inoculation; spot density of cathelicidin-1 in samples from inoculated glands was higher than in samples from uninoculated controls. There was clear evidence of correlation between cell content and cathelicidin-1 spot densities in milk samples. There was significant association between presence of mastitis in the mammary gland and detection of cathelicidin-1 in the respective milk sample; overall accuracy was 0.818—this was significantly greater during the first 24 h post-challenge (0.903) than after the first day (0.704). In conclusion, detection of cathelicidin-1 in milk was significantly associated with presence of mastitis in ewes. The associations were stronger during the first 24 h post-infection than after the first day. Cathelicidin-1 has the advantage that it can be a non-specific biomarker, as simply a “positive”/“negative” assessment would be sufficient

Mammary Defences and Immunity against Mastitis in Sheep

The objectives of this review paper are to present udder defences, including teat of the udder, mammary epithelial cells, leucocytes, immunoglobulins, complement system and chemical antibacterial agents, to describe cooperation and interactions between them and to elaborate on potentials regarding their significance in mammary immunisation strategies. The teat of the udder provides initial protection to the mammary gland. The mammary epithelial cells synthesise antibacterial proteins and the leucocytes produce various inflammation mediators (cytokines or chemokines), phagocytose bacteria and recognise antigenic structures. In the mammary gland, four immunoglobulins (IgG1, IgG2, IgM and IgA) have important roles against bacterial pathogens. The complement system is a collection of proteins, participating in the inflammatory process through various pathways. Other components contributing to humoral mammary defence include lactoferrin, lysozyme and the lactoperoxidase/myeloperoxidase systems, as well as oligosaccharides, gangliosides, reactive oxygen species, acute phase proteins (e.g., haptoglobin and serum amyloid A), ribonucleases and a wide range of antimicrobial peptides. Management practices, genetic variations and nutrition can influence mammary defences and should be taken into account in the formulation of prevention strategies against ovine mastitis

Unique Peptides of Cathelicidin-1 in the Early Detection of Mastitis—In Silico Analysis

Based on the results of previously performed clinical studies, cathelicidin-1 has been proposed as a potential biomarker for the early diagnosis of mastitis in ewes. It has been hypothesized that the detection of unique peptides (defined as a peptide, irrespective of its length, that exists in only one protein of a proteome of interest) and core unique peptides (CUPs) (representing the shortest peptide that is unique) of cathelicidin-1 may potentially improve its identification and consequently the diagnosis of sheep mastitis. Peptides of sizes larger than those of the size of CUPs, which include consecutive or over-lapping CUPs, have been defined as ‘composite core unique peptides’ (CCUPs). The primary objective of the present study was the investigation of the sequence of cathelicidin-1 detected in ewes’ milk in order to identify its unique peptides and core unique peptides, which would reveal potential targets for accurate detection of the protein. An additional objective was the detection of unique sequences among the tryptic digest peptides of cathelicidin-1, which would improve accuracy of identification of the protein when performing targeted MS-based proteomics. The potential uniqueness of each peptide of cathelicidin-1 was investigated using a bioinformatics tool built on a big data algorithm. A set of CUPs was created and CCUPs were also searched. Further, the unique sequences in the tryptic digest peptides of cathelicidin-1 were also detected. Finally, the 3D structure of the protein was analyzed from predicted models of proteins. In total, 59 CUPs and four CCUPs were detected in cathelicidin-1 of sheep origin. Among tryptic digest peptides, there were six peptides that were unique in that protein. After 3D structure analysis of the protein, 35 CUPs were found on the core of cathelicidin-1 of sheep origin and among them, 29 were located on amino acids in regions of the protein with ‘very high’ or ‘confident’ estimates of confidence of the structure. Ultimately, the following six CUPs: QLNEQ, NEQS, EQSSE, QSSEP, EDPD, DPDS, are proposed as potential antigenic targets for cathelicidin-1 of sheep. Moreover, another six unique peptides were detected in tryptic digests and offer novel mass tags to facilitate the detection of cathelicidin-1 during MS-based diagnostics

Growth of Staphylococcus epidermidis on the Surface of Teatcups from Milking Parlours

The growth of two Staphylococcus epidermidis isolates (one biofilm-forming and one not) on teatcups for cattle (made of rubber) or sheep (made of silicone) were assessed in nine multiplicates for 24 h post-smearing on the teatcup surface. Staphylococci were smeared on an area of 0.0003142 m2 on the material and their growth and expansion further on were monitored for 24 h. There were no differences in the frequency of recoveries between the two isolates (p > 0.82 for all comparisons). There were more recoveries from sheep teatcups than from cattle teatcups: 1280/1728 (74.1%) versus 942/1728 (54.5%), for both isolates (p < 0.0001). Significance was observed only 6 h to 15 h after smearing (p < 0.0001 for all comparisons). The median speed of linear dissemination of the isolates was 0.00000021 m s−1 on cattle teatcups and 0.00000033 m s−1 on sheep teatcups (p < 0.0001). The increased growth and faster expansion of staphylococci on silicone teatcups raise important points from a clinical viewpoint. The model could be used in the testing of staphylococcal growth in the material of milking parlours in various conditions

Dataset of milk whey proteins of three indigenous Greek sheep breeds

The importance and unique biological traits, as well as the growing financial value, of milk from small Greek ruminants is continuously attracting interest from both the scientific community and industry. In this regard the construction of a reference dataset of the milk of the Greek sheep breeds is of great interest. In order to obtain such a dataset we employed cutting-edge proteomics methodologies to investigate and characterize, the proteome of milk from the three indigenous Greek sheep breeds Mpoutsko, Karagouniko and Chios. In total, more than 1300 protein groups were identified in milk whey from these breeds, reporting for the first time the most detailed proteome dataset of this precious biological material. The present results are further discussed in the research paper “Milk of Greek sheep and goat breeds; characterization by means of proteomics” (Anagnostopoulos et al. 2016) [1]

Dataset of milk whey proteins of two indigenous greek goat breeds

Due to its rarity and unique biological traits, as well as its growing financial value, milk of dairy Greek small ruminants is continuously attracting interest from both the scientific community and industry. For the construction of the present dataset, cutting-edge proteomics methodologies were employed, in order to investigate and characterize, for the first time, the milk whey proteome from the two indigenous Greek goat breeds, Capra prisca and Skopelos. In total 822 protein groups were identified in milk whey of the two breeds, The present data are further discussed in the research article “Milk of Greek sheep and goat breeds; characterization by means of proteomics” [1]. Keywords: Foodomics, milk whey, Capra prisca breed, Skopelos Breed, LC-MS/MS, Greek goa

Zoonotic Problems Reported by Sheep and Goat Farmers and Factors Potentially Contributing to the Occurrence of Brucellosis among Them

The study aimed to investigate the occurrence of zoonotic problems reported by dairy small ruminant farmers in Greece and to study potential associations with socio-demographic characteristics of the farmers and management practices applied in the farms. A countrywide investigation was performed in 325 sheep and 119 goat farms in the 13 administrative regions of Greece. The selected farms were visited and interviews were conducted with respective farmers. The occurrence of zoonotic problems in the farmers was recorded. A total biosecurity score (0–6) was devised, based on biosecurity practices followed in farms. Sixty-seven farmers (15.10%, 95% confidence intervals (CI): 12.1–18.7%) reported experiencing a zoonotic problem. Most of the farmers (n = 57) (85.1%, 95% CI: 74.76–91.7%, of those with a zoonotic problem) (12.8%, 95% CI: 10.0–16.3%, of all) reported that the zoonotic problem had been brucellosis. Odds ratio for the occurrence of brucellosis in goat farmers was 1.879 (95% CI: 1.051–3.359) compared to the occurrence of the infection in sheep farmers (p = 0.033). For the outcome ‘occurrence of brucellosis’ in sheep farmers, the application of hand-milking, the availability of a separate lambing area and the presence of cats in the farm emerged as significant (p < 0.01); for the same outcome in goat farmers, only the availability of a separate kidding area emerged as significant (p = 0.001). The mean biosecurity score in farms in the continental area of the country was significantly higher than in the islands: 3.45 ± 0.05 versus 2.76 ± 0.28, respectively (p = 0.006), whilst there was also a significantly higher score in farms, where the farmer reported occurrence of brucellosis: 3.68 ± 0.15 versus 3.34 ± 0.06 in farms, where the farmer did not report such an incident (p = 0.042). In farms, where the above predictors prevail, farmers should be warned of an increased potential risk for human infection and biosecurity measures should be implemented and tightened

Experimental Study of the Potential Role of <i>Salmonella enterica</i> subsp. <i>diarizonae</i> in the Diarrhoeic Syndrome of Lambs

The objectives of this experimental work were the evaluation of the potential role of Salmonella enterica subsp. diarizonae in diarrhoeic syndrome in lambs and the investigation of facets of the pathogenesis of the infection. In total, 12 lambs were challenged orally on the first day of life, with a S. enterica subsp. diarizonae isolate from a clinical case of diarrhoeic syndrome. Sequential blood, faecal and buccal samples were collected from lambs and faecal and milk samples were taken from their dams. Lambs were euthanised 1, 2, 4, 7, 10, 14 and 21 days after challenge. Samples were processed for recovery of the challenge organism; they were also subjected to examination by PCR for detection of the invA gene. Tissue samples from lambs were also examined as above and histopathologically. S. enterica subsp. diarizonae was recovered from faecal samples of all lambs, in total, from 45/77 samples (median duration: 2.4 days post-inoculation). It was also recovered from buccal samples (10/77) from seven lambs (median duration: 0.8 days), and from tissue samples (small intestine, abomasum, liver, gallbladder) of nine lambs. It was recovered from two consecutive milk samples from the same ewe, but not from any faecal sample from ewes. The invA gene was detected in samples from all lambs (median duration: 5.5 days in faecal and 1.3 days in buccal samples), as well as in milk samples from three ewes. Histopathological findings included abomasitis with subepithelial presence of eosinophils, lymphocytes and plasma cells, consistently observed in all lambs. In the small intestine, salient lesions initially included distension and oedema of intestinal villi, leucocytic infiltration and hyperplasia of lymphoid nodules with apparent germinal centres; this was followed at later stages by atrophy and/or degeneration of the lymphoid tissue of the intestine with marked subepithelial infiltration of lymphocytes, plasma cells and eosinophils

MLST-Based Analysis and Antimicrobial Resistance of Staphylococcus epidermidis from Cases of Sheep Mastitis in Greece

Staphylococcus epidermidis is an important causal agent of ovine mastitis. A literature search indicated a lack of systematic studies of causal agents of the infection by using multi-locus sequence typing (MLST). The objectives were to analyse MLST-based data and evaluate the antimicrobial resistance of S. epidermidis isolates from ovine mastitis in Greece. The database included 1593 isolates from 46 countries: 1215 of human, 195 of environmental and 134 of animal origin, distributed into 949 sequence types (STs) and cumulatively with 450 alleles therein. Among mastitis isolates, bovine isolates were distributed into 36 different STs and ovine ones into 15 STs. The 33 isolates from ovine mastitis in Greece were in 15 different STs, 6 of these (ST677, ST678, ST700, ST 709, ST710, ST711) assigned for the first time; in addition, 5 alleles (65 for arcC, 59 for aroE, 56 and 57 for gtr and 48 for tpiA) were identified for the first time. The spanning tree of these isolates included 15 nodes and 14 edges (i.e., branches). Among these isolates, 19 showed resistance to antimicrobial agents (tetracycline, penicillin, fucidic adic, erythromycin, clindamycin, cefoxitin). Resistance-related genes (tetK, tetT, msrA, tetM, tetS, ermC, mecA) were detected. There was no association between STs and resistance to antimicrobial agents. Isolates with antimicrobial resistance were recovered more often from flocks where hand-milking was practised

Prevalence, Patterns, Association with Biofilm Formation, Effects on Milk Quality and Risk Factors for Antibiotic Resistance of Staphylococci from Bulk-Tank Milk of Goat Herds

The objectives of this work were to study the prevalence and the patterns of antibiotic resistance of staphylococcal isolates from bulk-tank milk of goat herds across Greece, to assess possible associations of the presence of antibiotic resistance with the quality of milk in these herds and to evaluate herd-related factors potentially associated with the presence of antibiotic resistance among these staphylococcal isolates. A cross-sectional study was performed on 119 goat herds in Greece. Bulk-tank milk samples were collected for bacteriological examination; staphylococcal isolates were evaluated for resistance to 20 antibiotics. Oxacillin-resistant, resistant to at least one antibiotic, and multi-resistant staphylococcal isolates were recovered from 5.0%, 30.3%, and 16.0% of herds, respectively. Of 80 isolates, 7.5% were resistant to oxacillin, 50.0% were resistant to at least one antibiotic and 27.5% were multi-resistant. Resistance was seen more frequently among coagulase-negative staphylococci (59.3%) than among Staphylococcus aureus (23.8%). Resistance was more frequent against penicillin and ampicillin (41.3% of isolates) and fosfomycin (27.5%). No association was found with biofilm formation by staphylococci. For recovery of oxacillin-resistant isolates, the presence of working staff in the herds emerged as a significant factor; respective factors for the isolation of staphylococci resistant to at least one antibiotic were part-time farming and high (&gt;10) number of systemic disinfections in the farm annually. The same three factors concurrently were also identified to be significant for the recovery of multi-resistant isolates