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.

Extensive countrywide field investigation of somatic cell counts and total bacterial counts in bulk tank raw milk in goat herds in Greece.

Dairy goat farming is an important sector of the agricultural industry in Greece, with an annual total milk production exceeding 450 000 l and accounting for over 25% of all goat milk produced in the European Union; this milk is used mainly for cheese production. Despite the importance of goat milk for the agricultural sector in Greece, no systematic countrywide investigations in the bulk-tank milk of goats in Greece have been reported. Objectives were to investigate somatic cell counts (SCC) and total bacterial counts (TBC) in raw bulk-tank milk of goat herds in Greece, study factors influencing SCC and TBC therein and evaluate their possible associations with milk content. Throughout Greece, 119 dairy goat herds were visited for milk sampling for somatic cell counting, microbiological examination and composition measurement. Geometric mean SCC and TBC were 0.838 × 106 cells ml-1 and 581 × 103 cfu ml-1, respectively. Multivariable analyses revealed annual frequency of check-ups of milking system and total milk quantity per goat (among 53 variables) to be significant for increased SCC; no factor emerged (among 58 variables) to be significant for increased TBC. Negative correlation of SCC with total protein was found; mean total protein content in the bulk-tank milk in herds with SCC >0.75 × 106 cells ml-1 was 5.1% lower and in herds with SCC >1.5 × 106 cells ml-1, it was 7.8% lower.

Streptococcus spp. from bulk-tank milk and milking machine teatcups on small ruminant farms, and factors potentially associated with their isolation.

The objectives of this work were (a) to determine the presence of streptococci in samples from small ruminant dairy farms (bulk-tank milk and, where possible, teatcup swabs), (b) to investigate the potential adverse effects of streptococci on milk quality and (c) to investigate the importance of some husbandry factors for the isolation of streptococci. Bulk-tank milk samples and teatcups swab samples were examined bacteriologically for the presence of streptococci. Somatic cell counting and milk composition measurements were also performed. The husbandry factors present in each farm were assessed for potential associations with the isolation of streptococci. Streptococci were isolated from milk samples from 31.4% of sheep and 17.4% of goat farms and from 4.8% of sheep and 5.9% of goat teatcups. Streptococci were isolated more frequently from the upper part than the lower part of teatcups: 5.0% vs. 1.9%. Most isolates (57.9%) were identified as Streptococcus uberis. Most isolates (68.4%) were slime-producing; slime-production was more frequent among isolates from teatcups (83.3%) than from bulk-tank milk (55.0%). Somatic cell counts and milk composition did not differ between farms in which streptococci were or were not isolated. Machine-milking was associated with the isolation of streptococci from bulk-tank milk samples. The initial stage of the milking period (first two months) was found to be associated with the isolation of streptococci from milking machine teatcups in sheep farms only.

Predisposing factors for bacterial mastitis in ewes.

Objective of this review paper is the appraisal of predisposing factors for bacterial mastitis in ewes. Factors that predispose ewes to mastitis can be classified into non-animal-related factors (environmental and climatological factors, housing, nutrition, milking practices) and animal-related factors (anatomic, genetic, litter size-ethological factors, number and stage of lactation period, health problems). There are clear management-environment-animal interactions in mastitis development in ewes, which underline its multifactorial nature. Research and studies regarding risk factors are important, in order to develop strategies for their elimination, control or correction. Control measures for bacterial mastitis, which attempt to eliminate predisposing factors for infection, can thus be developed and implemented.

Role of staphylococci in mastitis in sheep.

Staphylococci have been isolated from various sites of the body of healthy sheep, as well as from many infections of those animals, the main one being mastitis. The objective of this review is to appraise the importance and significance of staphylococci in causing mastitis in ewes. The review includes a brief classification and taxonomy of staphylococci and describes the procedures for their isolation and identification, as well as their virulence determinants and the mechanisms of resistance to antibacterial agents. Various staphylococcal species have been implicated in staphylococcal mastitis and the characteristics of isolates are discussed with regards to potential virulence factors. Staphylococcal mastitis is explicitly described, with reference to sources of infection, the course of the disease and the relevant control measures. Finally, the potential significance of staphylococci present in ewes' milk for public health is discussed briefly.

Association of subclinical mastitis prevalence with sheep breeds in Greece.

The objective of the research described in this Research Communication was to describe potential associations of subclinical mastitis with sheep breeds in Greece. A countrywide survey (2198 ewes in 111 farms) was performed. Prevalence of subclinical mastitis was 0·260. Results did not indicate any difference in the prevalence of subclinical mastitis between farms with pure-bred and farms with cross-bred animals, nor any difference in prevalence between farms with Greek pure-bred animals and farms with imported pure-bred animals. Results indicated that prevalence of subclinical mastitis was smaller in farms with Assaf-breed (0·100) and higher in farms with Frisarta-breed (0·625) (P < 0·02). Prevalence of mastitis was smaller in farms with Greek traditional indigenous breeds (0·221) (P = 0·007). In a model that included sheep breed and management system in farm, breed emerged as a significant factor for prevalence of subclinical mastitis (P = 0·003).

Ultrasonographic findings in the ovine udder during lactogenesis in healthy ewes or ewes with pregnancy toxaemia.

Objective of the study was to record, by means of ultrasonographic examination, changes occurring during lactogenesis in the udder of healthy ewes and of ewes with pregnancy toxaemia. The work was carried out in 28 ewes, 16 with pregnancy toxaemia (group A) and 12 healthy controls (group B). B-mode and Doppler ultrasonographic examination of the udder of ewes was performed. During the last month of pregnancy, grey-scale intensity values of mammary parenchyma in group A were significantly greater than in group B (P = 0.007), as was also the progressive increase in grey-scale intensity values in both groups (P < 0.001). Blood mammary input was significantly greater in ewes of group B than in ewes of group A (P < 0.05), as was also the progressive increase in blood input in both groups (P < 0.001). Further, differences between the two groups were identified in pulsatility index (P = 0.007) and in mean blood velocity (P = 0.036), but only during the last fortnight of pregnancy. After lambing, grey-scale values decreased sharply compared to those in pregnancy (P < 0.01), whilst blood input, pulsatility index and mean blood velocity continued the same trend as at the last stage of pregnancy, with differences between the two groups still prevalent (P < 0.05). There was a reverse correlation between grey-scale intensity values and milk quantities (P < 0.035) and a correlation between blood input and milk quantities (P < 0.07). The progressive increase in the diameter of the external pudendal artery was significant (P < 0.001), but no significant differences were evident between the two groups (P > 0.35). Differences between group A and group B in all other haemodynamic parameters studied were not significant, neither throughout the last month of pregnancy (P > 0.25), nor during the first week of lactation (P > 0.06). However, their progressive changes during the last month of pregnancy were significant (P < 0.02).

Administration of antibiotics to ewes at the beginning of the dry-period.

The objective of the present paper is to review the significance of administration of antibiotics at the end of a lactation period/beginning of the dry-period in ewes. During the stage of active involution, there is an increased risk of new mastitis cases and recrudescence of subclinical infections that had occurred during the previous lactation period. The main pathogens involved in the so-called 'dry-period mastitis' are coagulase-negative staphylococci. The principle of antibiotic administration at the end of a lactation period involves the intramammary infusion of a preparation to both mammary glands of ewes in the flock. Although a variety of products is licensed for administration in ewes, preferably the product for administration should be selected on the results of susceptibility testing of bacteria to be isolated from samples from ewes in the flock. In many clinical studies from around the world, performed in dairy- or mutton-production flocks, administration of antimicrobial agents at the end of a lactation period has been found beneficial in curing intramammary infections present at cessation of a lactation period, as well as in minimising the risk for intramammary infections during the dry-period. In dairy flocks, there are also benefits from increase in milk yield and decrease flock bulk milk mean somatic cell counts during the subsequent lactation period. Antibiotic administration at drying-off may be performed to all animals in a flock ('complete') or only to those considered to be infected ('selective'). In all cases, after administration of the antibiotic, definite and complete cessation of the lactation period is essential for success of the procedure. Moreover, maintenance of the prescribed withdrawal periods is essential to safeguard public health. The procedure should always be applied as part of a strategic udder health management plan in a flock; implementation improves the welfare of animals and affords significant financial benefits to the farmer. A mastitis prevention scheme during lactation will minimise the incidence of the disease; effective treatment of cases of the disease during lactation will decrease the bacterial populations in the flock and limit risk of infection of other animals. Administration of antibiotics at the end of a lactation period will complement the above procedures and will contribute to improved mammary health for the forthcoming lactation period.

Ultrasonographic findings in the ovine udder during involution.

The objective of the study was to record, by means of ultrasonographic examination, changes occurring in the udder of ewes during involution and to compare differences between progressive or abrupt udder drying-off. In group A ewes, udder drying-off took place progressively during a period of 22 d; ewes were milked twice daily for the last time on day(D) 0; then, they were hand-milked once daily for a week (D1-D7), which was followed by another week during which ewes were hand-milked once every 2 d (D9, D11, D13), followed by a third week during which ewes were hand-milked once every 3 d (D16, D19, D22). In group B ewes, the procedure took place abruptly; ewes were milked twice daily for the last time on D0 and no milking was carried out after that. B-mode and Doppler ultrasonographic examination of the udder of all ewes was performed throughout the drying-off procedure. Appropriate data management and analysis were performed. Progressive changes of the various parameters evaluated throughout the study period were significant in both groups (P<0.005). Gray-scale results of mammary parenchyma progressively decreased during the study and differed significantly between group A and group B (P=0.049). A temporary increase in cistern volume was evident after cessation of lactation, but differences were not significant between the two groups (P>0.3). Diameter of the external pudendal artery progressively decreased during the study and differed significantly between the two groups (P=0.037). Both resistance index and pulsatility index progressively increased throughout the study period in both groups; for both parameters, differences between the two groups were significant (P<0.0005). B-mode ultrasonographic examination indicated differences in remodelling of the extracellular matrix in relation to the procedure for udder drying-off. Volume of the gland cistern did not appear to be affected by the procedure for udder drying-off. Doppler ultrasonographic examination confirmed that blood flow during initiated involution was lower than during a progressive procedure for drying-off.

Associations of Socio-Demographic Characteristics of Dairy Goat Farmers in Greece with Biosecurity-Related Practices in the Farms.

Given the significance of goat farming in Greece, our study aimed to explore biosecurity conditions on goat farms and refers to an investigation performed on 119 farms countrywide in Greece. The objective of the present work was to evaluate and report on potential associations between the socio-demographic characteristics of goat farmers and practices that play a role in biosecurity on farms. Data were obtained during a large cross-sectional study performed across Greece in 119 goat herds. The socio-demographic characteristics of farmers, as well as 16 variables related to biosecurity, were recorded; these were practices or events that could potentially enhance or compromise biosecurity on farms. A score based on all the biosecurity-related variables was devised by assigning a '1' or '-1' mark for each variable recorded on a farm that could enhance or compromise, respectively, biosecurity. Among variables potentially enhancing biosecurity on farms, the application of disinfections on the farm and the maintenance of isolation for sick animals were practiced more frequently; among variables potentially compromising biosecurity on farms, the presence of hunters in the area around the farm and grazing practices for animals were recorded more frequently. The median overall score for biosecurity-related practices for all farms in the study was 1 (interquartile range: 2.5; minimum: -4; maximum: 5). Among socio-demographic characteristics, the presence of working staff on a farm was associated with five individual biosecurity-related practices and the length of farming experience with three individual biosecurity-related practices. In the multivariable analysis, female farmers (p = 0.007) and the presence of working staff on the farm (p = 0.025) emerged as the two significant socio-demographic characteristics with an association with the overall biosecurity level on farms. This knowledge can be useful when developing biosecurity programs on goat farms. Recognition of locally applied farm-level practices enhancing biosecurity could form a basis for farmers to apply more rigorous and effective relevant plans.

Stress related to wild canid predators near dairy sheep farms associated with increased somatic cell counts in bulk-tank milk.

We investigated the association between wild canid predators reported near sheep farms throughout Greece and somatic cell counts in bulk-tank milk as a reflection of milk quality. The study included 325 dairy sheep flocks, where bulk-tank milk somatic cell counts and total bacterial counts were measured and staphylococci were isolated. Farms were divided into three groups: Cohort A (farms with no reports of wild canid predators nearby), B (farms with canid predators (golden jackal and grey wolf) nearby yet with no experience of livestock losses to predation) and C (farms with canid predators nearby and livestock losses to predation). Somatic cell counts in bulk-tank milk of Cohort C farms were significantly higher, + 43% and + 29%, compared to those for Cohorts A and B, respectively: 0.617 × 106 cells mL-1 versus 0.433 × 106 or 0.477 × 106 cells mL-1, respectively. The presence of wild canid predators near sheep farms was associated with lower quality milk potentially indicative of stress consistent with the potential effects of a landscape of fear. Increasing biosecurity measures at livestock farms, e.g., fencing, and presence of livestock guard dogs could minimise predation risk, whilst also improving livestock welfare by reducing predator-associated stress.

In Silico Molecular Analysis of Carbapenemase-Negative Carbapenem-Resistant Pseudomonas aeruginosa Strains in Greece.

To date, three carbapenem resistance mechanisms have been identified: carbapenemase released from the pathogen, changes in the expression of the outer membrane OprD porin, and overexpression of the efflux pump MexAB-OprM. Twelve carbapenemase-negative carbapenem-resistant Pseudomonas aeruginosa strains, isolated from patients hospitalized at the University Hospital of Larissa, Central Greece, during 2023, which belonged to various sequence types (STs), were selected and were studied focusing on the characterization of their ß-lactamases, on changes to OprD and its regulator MexT proteins, and on alterations to the MexAB-OprM regulator proteins encoded by the mexR, nalC, and nalD genes. Whole genome sequencing analysis revealed the presence of ß-lactamase encoding genes, with blaPAO present in all isolates. Additionally, seven different genes of the oxacillinase family (blaOXA-35, blaOXA-50, blaOXA-395, blaOXA-396, blaOXA-486, blaOXA-488, blaOXA-494) were identified, with each strain harboring one to three of these. Regarding the OprD, five strains had truncated structures, at Loop 2, Loop 3, Loop 4, and Loop 9, while the remaining strains carried previously reported amino acid changes. Further, an additional strain had a truncated MexR; whereas, two other strains had totally modified NalC sequences. The active form of MexT, responsible for the downregulation of OprD production, as the intact sequence of the NalD protein, was found in all the strains studied. It is concluded that the truncated OprD, MexR, and NalC proteins, detected in eight strains, probably led to inactive proteins, contributing to carbapenem resistance. However, four strains carried known modifications in OprD, MexR, and NalC, as previously reported in both susceptible and resistant strains, a finding that indicates the complexity of carbapenem resistance in P. aeruginosa.

Ultrasonography of Testicular Maturation and Correlation with Body Growth and Semen Evaluation in Beagle Dog Model.

This prospective study investigated the ultrasonographic appearance of the canine testis from birth to adulthood. Eight purpose-bred laboratory Beagle-breed dogs were monitored from 4 to 40 weeks of life. The following parameters were evaluated every two weeks: bodyweight and height, scrotal and testicular volume, ultrasonographically measured testicular volume, echogenicity, heterogeneity, blood-flow score, ratio of the grayscale intensity value of the testis to the capsule, ejaculate volume, motility, viability, and number of spermatozoa. A correlation analysis was carried out between the various measurements obtained. Fertility was achieved in the 36th week of life. The echogenicity of the testicular parenchyma increased with age, and subsequently to the 30th week of life remained constant. The heterogeneity of the testicular parenchyma, as was evaluated by the standard deviation of the values of grayscale intensity of the parenchyma, also increased with age and was >19 at the onset of fertility. The ratio of grayscale intensity of testicular parenchyma had values < 200 at maturity. A colour Doppler evaluation first detected blood flow in the testis in the 22nd week. After the 32nd week, distinct signals were visible. In the 36th week, >80% of the testes imaged had visible vessels. A significant correlation was found between all the evaluation methods. The findings of the study may help clinicians detect the onset of fertility in dogs, especially when semen evaluation is not feasible; however, their applicability in all breeds or individuals might possibly vary due to genetic, physiological, and developmental differences. In summary, the study ultrasonographically explores the testicular maturity in dogs, with the aim to improve clinical assessments and health management in these animals.

Extensive Countrywide Molecular Identification and High Genetic Diversity of Haemonchus spp. in Domestic Ruminants in Greece.

The gastrointestinal nematode parasite Haemonchus spp. is one of the most pathogenic parasites of ruminants, due to its blood-sucking activity, which causes large economic losses in the ruminant industry. The latest epizootiological data recorded an increase in the infection, not only in Greece but also in other countries, mainly attributed to climatic changes. The study of the population structure and the investigation of the phylogenetic relationships of Haemonchus spp. are essential for the understanding of its biology and epizootiology to implement appropriate control and prevention strategies. In addition, the molecular approach allows the determination of evolutionary relationships between different species of this parasite, the diverse hosts they infect, as well as the different geographic compartments from which they originate. Therefore, the aim of the present study was to identify the species of the sympatric populations of the genus Haemonchus, a nematode parasite infecting ruminants (sheep, goats, cattle, and buffaloes) from different regions of Greece (continental and insular) using molecular methods. At the same time, an attempt was made to identify the possible subpopulations of Haemonchus spp. in Greece, to investigate their phylogenetic relationships, as well as to determine the genetic diversity of each population. A total of 288 worms of the genus Haemonchus were processed using molecular methods; of these, 96 were collected from sheep, 96 from goats, 48 from cattle, and finally, 48 from buffaloes. A fragment of 321 base pairs of the second internal transcribed spacer (ITS2) sequence of nuclear DNA was amplified for species identification, and, after basic local alignment search tool (Blast) analysis, it was revealed that they belonged to H. contortus. A fragment of 820 base pairs of subunit 4 of the nicotinamide dehydrogenase (ND4) gene of mitochondrial DNA was amplified for genetic diversity analysis. The Greek mitochondrial ND4 sequences of H. contortus were classified into 140 haplotypes, and the values of the average nucleotide and haplotype diversity were lower compared to the respective values derived from Italy, Malaysia, the USA, and China. The phylogenetic analysis of the ND4 gene revealed a clear grouping of the Greek haplotypes when compared with Asian ones, and, at the same time, there was no profound grouping of the same haplotypes with regard to their different hosts and geographical origin within different regions of Greece. The aforementioned findings confirmed that H. contortus prevails in our country and can infect all species of ruminants, without geographical boundaries, when the right conditions (i.e., common grazing) are created.

Lameness in Adult Sheep and Goats in Greece: Prevalence, Predictors, Treatment, Importance for Farmers.

The present study refers to an extensive investigation of lameness performed countrywide in Greece, on 325 sheep and 119 goat farms. The specific objectives of this work were to present data on the occurrence of lameness on sheep and goat farms and to identify variables (including variables related to climatic factors) associated with the disorder on the farms. Farms were visited and animals on the farm were assessed for the presence of lameness; further, an interview was carried out with the farmer to obtain information regarding practices applied on the farm. Climatic variables at the location of each farm were derived from NASA research. The within farm prevalence rate varied from 0.0% to 25.0% in sheep flocks and from 0.0% to 30.0% in goat herds. The mean ± standard error (median (interquartile range)) within farm prevalence rate among sheep farms was 1.9% ± 0.2 (0.0% (0.0%)); among goat farms, it was 2.6% ± 0.5% (0.0% (0.0%)). Multivariable analysis for within farm prevalence of lameness revealed three significant predictors in sheep farms: application of vaccination against foot-rot, increased precipitation at the farm location and longer annual grazing period for sheep, and one in goat farms: increased precipitation at the farm location. Treatment of lameness involved mostly administration of antibiotics (on 104 farms); the antibiotics administered most often were lincomycin (on 69 farms) and oxytetracycline (on 33 farms). There was a tendency for higher median within farm prevalence of lameness among farms where no antibiotic administration was practiced. Finally, 6.2% of sheep farmers and 4.2% of goat farmers considered lameness as an important health problem for the animals, specifically the third and fifth most important problem on the respective farms.

Scientometrics Evaluation of Published Scientific Papers on the Use of Proteomics Technologies in Mastitis Research in Ruminants.

The objective of this study was the presentation of quantitative characteristics regarding the scientific content and bibliometric details of the relevant publications. In total, 156 papers were considered. Most papers presented original studies (n = 135), and fewer were reviews (n = 21). Most original articles (n = 101) referred to work involving cattle. Most original articles described work related to the diagnosis (n = 72) or pathogenesis (n = 62) of mastitis. Most original articles included field work (n = 75), whilst fewer included experimental (n = 31) or laboratory (n = 30) work. The tissue assessed most frequently in the studies was milk (n = 59). Milk was assessed more frequently in studies on the diagnosis (61.1% of relevant studies) or pathogenesis (30.6%) of the infection, but mammary tissue was assessed more frequently in studies on the treatment (31.0%). In total, 47 pathogens were included in the studies described; most were Gram-positive bacteria (n = 34). The three bacteria most frequently included in the studies were Staphylococcus aureus (n = 55 articles), Escherichia coli (n = 31) and Streptococcus uberis (n = 19). The proteomics technology employed more often in the respective studies was liquid chromatography-tandem mass spectrometry (LC-MS/MS), either on its own (n = 56) or in combination with other technologies (n = 40). The median year of publication of articles involving bioinformatics or LC-MS/MS and bioinformatics was the most recent: 2022. The 156 papers were published in 78 different journals, most frequently in the Journal of Proteomics (n = 16 papers) and the Journal of Dairy Science (n = 12). The median number of cited references in the papers was 48. In the papers, there were 1143 co-authors (mean: 7.3 ± 0.3 co-authors per paper, median: 7, min.-max.: 1-19) and 742 individual authors. Among them, 15 authors had published at least seven papers (max.: 10). Further, there were 218 individual authors who were the first or last authors in the papers. Most papers were submitted for open access (n = 79). The median number of citations received by the 156 papers was 12 (min.-max.: 0-339), and the median yearly number of citations was 2.0 (min.-max.: 0.0-29.5). The h-index of the papers was 33, and the m-index was 2. The increased number of cited references in papers and international collaboration in the respective study were the variables associated with most citations to published papers. This is the first ever scientometrics evaluation of proteomics studies, the results of which highlighted the characteristics of published papers on mastitis and proteomics. The use of proteomics in mastitis research has focused on the elucidation of pathogenesis and diagnosis of the infection; LC-MS/MS has been established as the most frequently used proteomics technology, although the use of bioinformatics has also emerged recently as a useful tool.

The Use of Machine Learning to Predict Prevalence of Subclinical Mastitis in Dairy Sheep Farms.

The objective of the study was to develop a computational model with which predictions regarding the level of prevalence of mastitis in dairy sheep farms could be performed. Data for the construction of the model were obtained from a large Greece-wide field study with 111 farms. Unsupervised learning methodology was applied for clustering data into two clusters based on 18 variables (17 independent variables related to health management practices applied in farms, climatological data at the locations of the farms, and the level of prevalence of subclinical mastitis as the target value). The K-means tool showed the highest significance for the classification of farms into two clusters for the construction of the computational model: median (interquartile range) prevalence of subclinical mastitis among farms was 20.0% (interquartile range: 15.8%) and 30.0% (16.0%) (p = 0.002). Supervised learning tools were subsequently used to predict the level of prevalence of the infection: decision trees, k-NN, neural networks, and Support vector machines. For each of these, combinations of hyperparameters were employed; 83 models were produced, and 4150 assessments were made in total. A computational model obtained by means of Support vector machines (kernel: 'linear', regularization parameter C = 3) was selected. Thereafter, the model was assessed through the results of the prevalence of subclinical mastitis in 373 records from sheep flocks unrelated to the ones employed for the selection of the model; the model was used for evaluation of the correct classification of the data in each of 373 sets, each of which included a test (prediction) subset with one record that referred to the farm under assessment. The median prevalence of the infection in farms classified by the model in each of the two categories was 10.4% (5.5%) and 36.3% (9.7%) (p < 0.0001). The overall accuracy of the model for the results presented by the K-means tool was 94.1%; for the estimation of the level of prevalence (<25.0%/≥25.0%) in the farms, it was 96.3%. The findings of this study indicate that machine learning algorithms can be usefully employed in predicting the level of subclinical mastitis in dairy sheep farms. This can facilitate setting up appropriate health management measures for interventions in the farms.

Detection of Gastrointestinal Pathogens with Zoonotic Potential in Horses Used in Free-Riding Activities during a Countrywide Study in Greece.

The objectives of this study were (a) to detect zoonotic gastrointestinal pathogens in faecal samples of horses using the FilmArray® GI Panel and (b) to identify variables potentially associated with their presence. Faecal samples collected from 224 horses obtained during a countrywide study in Greece were tested by means of the BioFire® FilmArray® Gastrointestinal (GI) Panel, which uses multiplex-PCR technology for the detection of 22 pathogens. Gastrointestinal pathogens were detected in the faecal samples obtained from 97 horses (43.3%). Zoonotic pathogens were detected more frequently in samples from horses in courtyard housing (56.0%) than in samples from horses in other housing types (39.7%) (p = 0.040). The most frequently detected zoonotic pathogens were enteropathogenic Escherichia coli (19.2% of horses) and Shiga-like toxin-producing E. coli stx1/stx2 (13.8%). During multivariable analysis, two variables emerged as significant predictors for the outcome 'detection of at least one zoonotic pathogen in the faecal sample from an animal': (a) the decreasing age of horses (p = 0.0001) and (b) the presence of livestock at the same premises as the horses (p = 0.013). As a significant predictor for the outcome 'detection of two zoonotic pathogens concurrently in the faecal sample from an animal', only the season of sampling of animals (autumn) emerged as significant in the multivariable analysis (p = 0.049). The results indicated a diversity of gastrointestinal pathogens with zoonotic potential in horses and provided evidence for predictors for the infections; also, they can serve to inform horse owners and handlers regarding the possible risk of transmission of pathogens with zoonotic potential. In addition, our findings highlight the importance of continuous surveillance for zoonotic pathogens in domestic animals.