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.

Association of Staphylococcal Populations on Teatcups of Milking Parlours with Vaccination against Staphylococcal Mastitis in Sheep and Goat Farms.

There is a paucity of information regarding staphylococcal populations on teatcups of milking parlours in sheep and goat farms. The objectives were to describe the populations of staphylococci on teatcups in milking parlours in sheep or goat farms in two field investigations throughout Greece and to potentially associate the findings with the use of anti-staphylococcal mastitis vaccinations in the farms visited during the two investigations. In a cross-sectional (255 sheep and 66 goat farms across Greece) and a longitudinal (12 sheep farms, four samplings, throughout lactation) study, swab samples were collected from 1418 teatcups (upper and lower part) for staphylococcal recovery, identification and assessment of biofilm-formation. A total of 328 contaminated teatcups (23.1%) were found in 105 sheep (41.2%) and 35 goat (53.0%) farms. Staphylococci were more frequently recovered from the upper than the lower part of teatcups: 269 versus 139 teatcups, respectively. After identification, 253 staphylococcal isolates were found: Staphylococcus aureus, Staphylococcus equorum, Staphylococcus lentus, and Staphylococcus capitis predominated. Of these isolates, 87.4% were biofilm-forming. The proportion of contaminated teatcups was smaller in farms where vaccination against anti-staphylococcal mastitis in general or vaccination specifically against mastitis caused specifically by biofilm-forming staphylococcal strains was applied, 19.7% or 10.9%, respectively, versus 25.5% in farms without vaccination. In the longitudinal study, contaminated teatcups were identified in 28 (58.3%) sampling occasions, with staphylococci being recovered more frequently from their upper part. The same species as in the cross-sectional study predominated. Of these isolates, 61.9% were biofilm-forming. In farms where vaccination against mastitis caused specifically by biofilm-forming staphylococcal strains was applied, the proportion of contaminated teatcups was smaller: 20.4% versus 48.3% in farms without vaccination. There were no differences in proportions of contaminated teatcups between sampling occasions. In conclusion, the great majority of staphylococci recovered from teatcups of milking parlours in sheep and goat farms included biofilm-forming isolates. Reduced staphylococcal isolation was noted in farms where anti-staphylococcal vaccination was performed; this was possibly the effect of reduced excretion of staphylococci in the milk of vaccinated animals.

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.

Extensive Countrywide Field Investigation of Somatic Cell Counts and Total Bacterial Counts in Bulk-Tank Raw Milk in Sheep Flocks in Greece.

Objectives were to investigate somatic cell counts (SCC) and total bacterial counts (TBC) in the raw bulk-tank milk of sheep flocks in Greece, to study factors potentially influencing increased SCC and TBC in the bulk-tank milk of sheep and to evaluate possible associations of SCC and TBC with milk content. Throughout Greece, 325 dairy sheep flocks were visited for collection of milk sampling for somatic cell counting, microbiological examination and composition measurement. Geometric mean SCC were 0.488 × 106 cells mL-1; geometric mean TBC were 398 × 103 cfu mL-1; 228 staphylococcal isolates were recovered form 206 flocks (63.4%). Multivariable analyses revealed annual incidence risk of clinical mastitis, age of the farmer and month into lactation period (among 53 variables) to be significant for SCC > 1.0 × 106 cells mL-1 and month into lactation period at sampling and availability of mechanical ventilators (among 58 variables) to be significant for TBC > 1500 × 103 cfu mL-1. Negative correlation of SCC with fat, total protein and lactose and positive correlation of SCC with added water were found. With SCC > 1.0 × 106 cells mL-1, significant reduction of protein content (2%) was observed, whilst in flocks with SCC > 1.5 × 106 cells mL-1, significantly lower annual milk production per ewe (42.9%) was recorded.

Experimental Study of the Potential Role of Salmonella enterica subsp. diarizonae 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.

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.

A Detailed Questionnaire for the Evaluation of Health Management in Dairy Sheep and Goats.

The objective of this work was to develop, use and present a detailed questionnaire for the evaluation of health management in dairy small ruminants; it includes 442 questions organised in seven sections: general, infrastructure, animals, production characteristics, health management, nutrition, human resources. Consistency of replies was evaluated in 27 farmers, interviewed twice. Inconsistent replies were given by all farmers to 30 different questions (Cronbach's coefficient alpha: 0.987). Then, interviews were performed in 444 farms around Greece. Mean duration of an interview was 63.6 min. Clarifications were requested by 273 farmers to 22 different questions (maximum per farmer: 8). The experience of the investigator, the primary language of farmers and asking clarifications by the farmers affected the duration of the interview. The questionnaire can be used for research work in the field, to record details in the farms under study. In accord with the needs of a particular study, it can be modified, by adding more specific questions or omitting others deemed of less importance. Moreover, it can also be used for routine monitoring purposes, as a useful means to record and maintain details of farms during clinical work. To the best of our knowledge, the questionnaire is the most extensive and detailed one available internationally for dairy small ruminants.

Detailed data from experimentally-induced mastitis in ewes, with the aim to evaluate cathelicidin-1 in milk.

Bacteriological, cytological and proteomics data have been obtained from ewes in two experiments, after intramammary challenge with Mannheimia haemolytica or Staphylococcus chromogenes. Animals were sampled before and sequentially after challenge. Conventional techniques were employed for bacterial isolation and somatic cell counting in milk samples; milk whey samples were subjected to proteomics evaluation by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. There was a correlation between leucocyte content and cathelicidin-1 spot densities in milk samples, although the protein was detected in milk earlier than the increase in leucocyte content. There was also a significant association between presence of mastitis in a mammary gland and detection of cathelicidin-1 in the respective milk sample; the degree of association was greater during the first 24 h post-inoculation. The data are further discussed in the research article "Detection of cathelicidin-1 in the milk as an early indicator of mastitis in ewes" [1].

Clinical, Ultrasonographic, Bacteriological, Cytological and Histopathological Findings of Uterine Involution in Ewes with Uterine Infection.

The objectives of the study were (a) to study the characteristics of uterine involution in ewes that had developed subclinical uterine infection in the immediately post-partum period and (b) to evaluate effects of the infection in the subsequent reproductive performance of ewes. Uterine infection was induced in ewes (I, n = 10) by intrauterine inoculation of Escherichia coli; uninoculated controls were included (C, n = 12). Animals were examined at regular intervals before and post-inoculation. Clinical and ultrasonographic examinations were performed. Vaginal swab samples and biopsy uterine tissue samples were collected for bacteriological, cytological and histological examination. Finally, ewes were put to rams and reproductive performance was monitored. After challenge, it was ultrasonographically found that caruncular dimensions, myometrial thickness and diameter of uterine lumen were greater in I ewes. In these ewes, particular reduction of dimensions occurred during the second week post-partum, whilst in C ewes during the first week. The uterine artery diameter and the blood flow into the uterus were also greater in I than in C ewes. E. coli infection was more frequent and of longer duration in I than in C ewes: in 68.1% and 50.0% of ewes and 19.5 and 14 days, respectively. There was lower proportion of neutrophils and higher of lymphocytes in group I than in C. In inoculated ewes, there was histological evidence of uterine epithelial destruction, increased cellular infiltration, hyperaemia and extracasation, which persisted up to 42 days post-partum. During the subsequent reproductive season, all ewes in group I lambed normally and produced healthy and viable lambs. No significant difference in reproductive performance parameters were seen in I comparison to C ewes. It is concluded that the innate immunity of the uterus sufficed to counteract the bacterial infection, although the process of involution took longer than in healthy animals; moreover, the ultrasonographic examination is a useful means for assessment of the genital tract of ewes post-partum; finally, no adverse effects were noted in the subsequent reproductive performance of ewes.

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.

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).