Evaluation of efficacy of a biofilm-embedded bacteria-based vaccine against staphylococcal mastitis in sheep-A randomized, placebo-controlled field study.

Our objective was to evaluate the efficacy of a vaccine against staphylococcal mastitis in 5 dairy sheep farms, with 316 ewes in the vaccinated (V) group and 307 in the control (C) group studied throughout a lactation period. Two administrations of the vaccine were performed during the last stage of gestation of ewes. Starting 15 d after lambing and at monthly intervals thereafter, up to 9 milk samplings were performed for bacteriological and cytological examinations. Staphylococcal isolates recovered were examined for biofilm formation. Blood samples were collected for measurement of IgG poly-N-acetylglucosamine-specific antibodies. The most frequently isolated bacteria were staphylococci: 56.4 and 76.1%, respectively, of total isolates recovered from ewes of group V and C, respectively; staphylococci as causal agents of mastitis were isolated less frequently from V (5.3%) than in ewes in C (10.3%). Among mastitis-associated staphylococcal isolates recovered from V ewes, a smaller proportion was biofilm-forming than among ones from C: 53.2% versus 74.9% of isolates; biofilm-forming staphylococci as causal agents of mastitis were isolated less frequently from ewes in group V (2.3%) than in ewes in group C (6.0%). Anti-poly-N-acetylglucosamine-specific antibody values increased in V ewes and were higher than in C; a greater proportion of ewes with low antibody titers developed staphylococcal mastitis (41.4%) than of V ewes with high antibody titers (17.0%). Incidence risk of mastitis, staphylococcal mastitis, and biofilm-associated staphylococcal mastitis was smaller in V than in C: 36.7, 17.1, and 8.0% versus 44.3, 30.9, and 18.9%, respectively. The first case of staphylococcal mastitis occurred later in V than in C: third versus second sampling point. Overall, efficacy of the vaccine was 44.6% for staphylococcal mastitis, 57.7% for biofilm-associated staphylococcal mastitis, 33.1% for staphylococcal intramammary infection, and 51.5% for biofilm-associated staphylococcal intramammary infection. Nevertheless, vaccination should not be the only means for controlling mastitis; other udder health management measures should be included therein to improve control of the infection.

Study of potential environmental factors predisposing ewes to subclinical mastitis in Greece.

Objectives of the work presented herewith were to investigate association of prevalence of subclinical mastitis with environmental (climatic and topographic) factors and to identify factors potentially predisposing ewes to the disease. Milk samples were collected from 2198 sheep in 111 farms, in all 13 administrative regions of Greece, for bacteriological and cytological examination. Data on farm location were collected in the field using hand-held Global Positioning System Garmin units. The geo-references were resolved to specific farm level. Prevalence of subclinical mastitis was 0.260. Main aetiological agents were staphylococci (Staphylococcus aureus and coagulase-negative species), which accounted for 0.699 of all isolates recovered. In a multivariable mixed-effects analysis, the two environmental variables found to be associated with increased prevalence of subclinical mastitis were the minimum temperature of coldest month (coefficient: -0.084 ± 0.033, P = 0.014) and the mean temperature for 30 days prior to sampling date (coefficient: 0.031±0.014, P = 0.029).

Use of geographical information system and ecological niche modelling for predicting potential space distribution of subclinical mastitis in ewes.

Objective was to recognise areas potentially of high risk for increased frequency of subclinical mastitis in ewes. Milk samples were collected, for bacteriological and cytological examination, from 2198 clinically healthy ewes in 111 farms in all administrative regions of Greece. Data on farms were located in the field using hand-held Global Positioning System Garmin units. Collected data were analysed by an Ecological Niche Model under the framework of a geographic information system. Two separate analyses were performed: one for subclinical mastitis independently of causal agent (prevalence in population sampled: 0.260) and one for subclinical mastitis caused specifically by slime-producing staphylococci (prevalence in population sampled: 0.153). A model was constructed in which sheep farms were divided into two clusters, according to prevalence of subclinical mastitis: farms in the upper three quartiles of prevalence were used as occurrence points for the Ecological niche modelling procedure ('infected farms'); farms in the lower quartile of prevalence within each category were (pseudo)negative points. Significant differences in environmental parametres prevailing in locations of farms into the study, were identified for up to 13 parametres between locations of farms according to management system applied in farms. When farms in each management system were considered separately, differences became evident between farms in each management system, as well as between the two infections. The factor with the highest relative contribution in the analyses was the distance from other sheep farms; other factors also of importance in the predictive models were the altitude, the maximum temperature of warmest month and the total precipitation of driest month. Verification of the model revealed that ≥ 0.760 of infected farms' were located in areas predicted as high risk for prevalence of subclinical mastitis or slime staphylococcal subclinical mastitis. The paper describes for the first time potential association of mastitis with environmental factors and presents predictive models for mastitis in ewes taking into account environmental parametres.

Slime-producing staphylococci as causal agents of subclinical mastitis in sheep.

Hitherto, research work in slime production from staphylococcal strains of mastitis origin has focused in laboratory properties of these organisms. Objective of present work was to study subclinical mastitis in sheep, caused specifically by slime-producing staphylococci: to investigate its frequency and to identify potential factors playing a role therein. Slime production was evaluated in 708 staphylococcal isolates recovered from cases of subclinical mastitis in a field study in 2198 ewes performed in an extensive countrywide field investigation across Greece. Isolates were studied by means of microbiological and molecular methods. Of these strains, 262 were characterised as slime-producing, 227 as weak slime-producing and 219 as non slime-producing. Most frequently detected genes were eno and icaB; Staphylococcus aureus possessed more genes than coagulase-negative strains; greater number of genes was detected in slime-producing than in weak slime-producing or non-slime-producing strains. Subclinical mastitis caused specifically by slime-producing staphylococci was detected in 337 ewes: prevalence in population sampled was 0.153. A multivariable mixed-effects model revealed that milking mode (highest prevalence in hand-milked flocks) and flock management system (highest prevalence in semi-intensive flocks) were the two factors associated with increased prevalence of mastitis in flocks. The results confirmed the significance of slime producing staphylococcal strains of importance in the aetiology of subclinical mastitis of sheep. Hand-milking was identified as the most important factor predisposing to that infection.

Extensive countrywide field investigation of subclinical mastitis in sheep in Greece.

The objectives of this work were (1) to investigate prevalence of subclinical mastitis, (2) to identify etiological agents involved, and (3) to study factors potentially predisposing ewes to subclinical mastitis. Milk samples were collected from 2,198 ewes in 111 farms with a total population of 35,925 ewes, in all 13 administrative regions of Greece, for bacteriological and cytological examination. Prevalence of subclinical mastitis was 0.260. Main etiological agents were staphylococci (Staphylococcus aureus and coagulase-negative species), which accounted for 0.699 of all isolates recovered; prevalence of staphylococcal mastitis was 0.191. In a multivariable mixed-effects analysis, the primary factor found to be associated with increased prevalence of subclinical mastitis was the management system practiced in flocks (flocks under a semi-intensive system had the highest prevalence). Other factors that were included in the multivariable model were the stage of lactation period (ewes in the 2nd month postpartum showed the highest prevalence) and application of postmilking teat dipping. In contrast, measures taken at the end of a lactation period (e.g., intramammary administration of antimicrobial agents) were not found to have an effect on prevalence of subclinical mastitis. The results confirmed the significance of subclinical mastitis as a frequent problem of ewes, with staphylococci as the primary etiological agent. The findings confirm the multifactorial nature of subclinical mastitis and indicate that its control should rely on many approaches.

Gastrointestinal trichostrongylosis can predispose ewes to clinical mastitis after experimental mammary infection.

Objective was to study, in an experimental model, the possible role of gastrointestinal nematode infection in predisposing ewes to mastitis during the lactation period. Twenty-four ewes (A or B [n=12]), free from nematode and trematode helminths, were used. Group A animals received 5000 third-stage larvae of a trichostrongylid helminth cocktail and group B ewes were unparasitised controls. Animals in group A developed gastrointestinal trichostrongylosis confirmed by >500epg in faecal samples; mean epg of group B ewes were <20 (P<0.001). Ewes were challenged by deposition of Mannheimia haemolytica into the teat duct. In group A, 7 ewes developed clinical and 5 subclinical mastitis; no ewe in group B developed clinical mastitis, but only subclinical (12 ewes) (P=0.002). M. haemolytica was isolated from 132/132 and 121/132 udder samples from group A or B, respectively (P<0.015); increased leucocyte numbers were recorded in 66/66 and 61/66 samples, respectively (P=0.023). During post-mortem examination, mean number of helminths in gastrointestinal content was 2523 and 7.5 in group A or B, respectively (P<0.001); within group A, proportions of Teladorsagia and Haemonchus were significantly greater in ewes that developed clinical mastitis than in others which did not (0.709 and 0.162 versus 0.662 and 0.136, respectively; P<0.035). M. haemolytica was isolated from 36/36 and 19/36 udder tissue samples from group A or B, respectively (P<0.001). In ewes with subclinical mastitis (in group A or B), inducible-lymphoid-follicles were observed in the teat, which were not observed in ewes with clinical disease. Total pathology scores summed over all days were 127 and 73 for group A or B ewes, respectively (maximum possible 192; P<0.05). In general, there was positive correlation between intestinal helminth counts and pathology score (P<0.001) and between Teladorsagia counts and pathology score (P=0.002) in ewes that developed clinical mastitis. It is concluded that, in view of bacterial challenge, gastrointestinal trichostrongylosis and particularly Teladorsagia infection, might lead to clinical mastitis, through various pathogenetic pathways.

Trematode infections in pregnant ewes can predispose to mastitis during the subsequent lactation period.

Objective was to investigate if trematode infections predispose ewes to mastitis and/or metritis. We used 80 trematode-infected ewes: primigravidae in group P-A and multigravidae in M-A remained untreated, primigravidae in P-B and multigravidae in M-B were drenched with netobimin and multigravidae in M-C were given rafoxanide. We collected faecal samples for parasitological examination, blood samples for ß-hydroxybutyrate concentration measurement and uterine content, teat duct material and milk samples for bacteriological examination. We found significant differences in blood ß-hydroxybutyrate concentrations between M-A, M-B and M-C during pregnancy (P ⩽ 0.002). We did not observe significant differences between groups regarding development of metritis (P>0.83). We found that for M-A, M-B and M-C ewes, respectively, median time to first case of mastitis was 5.75, 21 and 6.75 days after lambing (P = 0.003) and incidence risk of mastitis was 0.308, 0.069 and 0.222 (P = 0.047). We postulate that trematode infections predispose ewes to mastitis; perhaps, increased ß-hydroxybutyrate blood concentrations adversely affect mammary cellular defences. This is the first report associating parasitic infections with mastitis in sheep.

Administration of a long-acting antiparasitic to pre-pubertal ewe-lambs in Greece results in earlier reproductive activity and improved reproductive performance.

We studied the reproductive effects of administration of a long-acting antiparasitic (moxidectin) given to pre-pubertal ewe-lambs in Greece at the beginning of the reproductive season. 45 animals, naturally infected with trichostrongylids, were allocated into treated (n=30, treatment on D0, 21 June) or control (n=15) group. Rams of confirmed fertility, were introduced from 15 August (D55) to 20 December (D182) into the ewe-lambs. Throughout the study (performed at latitude N 36°26', in a flock free from brucellosis, Chlamydophila infection and toxoplasmosis), epg counts were monitored and reproductive performance of ewes was assessed. Up to D112, arithmetic mean epg counts in treated animals were 0; thereafter and up to D350, they were 23-473. Respective figures for controls were 190-977 epg. Reproductive performance parameters for treated and control animals respectively, were as follows; median 'Interval to first mating after ram introduction': 36.5 d and 71.0 (P=0.04); median 'Age at first mating': 8.5m and 10.0m (P=0.045); 'Cycling rate': 20.0% and 6.7% (P=0.03); 'Mating rate': 86.7% and 66.7%; 'Return-to-oestrus rate': 26.7% and 26.7%; 'Abortion rate': 3.3% and 0%; 'Lambing rate': 83.3% and 66.7%; 'Total lambs born per ewe' and 'Liveborn lambs born per ewe': 1.5 and 1.1 (P=0.01); 'Stillbirth rate' 0% and 0% and 'Lamb bodyweight per ewe': 5.0 kg and 3.8 kg (P=0.005). Anthelmintic treatment of pre-pubertal ewes, in order to maximise reproductive performance may be employed as a management strategy according to targets set in individual flocks.

Transmission of Mannheimia haemolytica from the tonsils of lambs to the teat of ewes during sucking.

Objective of the work was to study whether Mannheimia haemolytica may be transmitted from the mouth of the lambs into the teat of the dam during sucking. We compared bacterial populations within the teat duct and milk of ewes immediately before and immediately after sucking by the lambs. Tonsils of lambs of the ewes were swabbed. M. haemolytica strain DAG21T recovered from a teat duct of a ewe was compared to strain DAG21R recovered from the tonsils of her lamb by using 16s rRNA sequencing. We used those two isolates and another one of known pathogenicity, for challenging ewes: (i) 2 mm deep into healthy teats, (ii) 2 mm deep into teats with chapping lesions or (iii) into the cistern of healthy mammary glands. Of samples collected before suckling, 20/792 were bacteriologically positive, and of those after, 50/792 were bacteriologically positive (P<0.001); in 37 cases, a negative sample became positive. One M. haemolytica (DAG21T) was recovered after suckling from a teat duct of a ewe. The organism was isolated from 57/90 tonsillar swabs from lambs. Risk of infection of ewe' teats was 0.004 throughout lactation, being greatest (0.021) during the 3rd week of lactation. The 16s rRNA sequences of strains DAG21T and DAG21R were identical over 1450 nucleotides. Phylogenetic analysis showed that the two isolates clustered together with isolates of M. haemolytica. Organism deposition into healthy teats caused subclinical mastitis; deposition into teats with lesions or directly into mammary gland caused clinical mastitis. When results of inoculation of the three strains were compared between them, statistical significance was always P>0.9. Results provide clear evidence that suckling by lambs can lead to transmission of M. haemolytica into the teats of the ewes; the bacteria have the potential to cause mastitis if circumstances are favourable.

Effects of lamb sucking on the bacterial flora of teat duct and mammary gland of ewes.

We aimed (i) to determine differences in bacterial flora of teat duct and mammary gland of ewes before and after suckling, (ii) to evaluate factors potentially affecting those. We collected samples of teat duct material and mammary secretion from 11 ewes immediately before and after sucking by lambs, as well as 120 min later. We processed samples bacteriologically and compared changes in infection by the Sign Test. We isolated bacteria from 3.5% duct and 1.5% secretion samples before suckling. Respective figures post-suckling were 10.6% and 2.0%, and 120 min later 6.8% and 1.5%. We recorded differences in infection of duct samples before and after suckling in 40 cases; bacteria were isolated before suckling from six samples, whereas after it from 34 (p < 0.001). Also, we recorded differences in samples collected after suckling and 120 min later in 12 cases; bacteria were isolated immediately post-suckling from eight samples, whereas 120 min later from four (p = 0.375). No significant changes were seen for secretion. We found neither difference between ewes with single or twin lambs, nor among stages of lactation. Mostly, we isolated staphylococci: 70% of isolates before suckling, 80% of isolates after it, 91% of isolates 120 min later. After suckling we also isolated two Mannheimia haemolytica strains. Suckling predisposes to entrance of bacteria into the teat; however, increased teat infections did not result in mammary infections. Isolation of M. haemolytica post-suckling indicates that lambs act as source of infection for this pathogen.

Subclinical mastitis changes the patterns of maternal-offspring behaviour in dairy sheep.

Subclinical mastitis was induced by inoculating one mammary gland in dairy ewes (n=8) with a Staphylococcus simulans isolate; control ewes (n=4) were included. The milk yield of inoculated glands decreased (P<0.001), the California Mastitis Test (CMT) score increased and the organism could be recovered from the inoculated glands. With time, there was significantly increased frequency of "hindering sucking" (P=0.016) and "head up posture" (P<0.001) in the control ewes. Infected ewes had a significantly increased frequency of "vocalisation" (P=0.013) compared to controls. There was a significant difference in the frequency of "sucking attempt" and "successful suck" (P<0.05) behaviours between lambs of the two groups. Lambs of the challenged ewes also showed significantly increased frequency and duration of these behaviours towards the uninoculated glands of their dams, rather than to the challenged glands (P<0.05); no such difference was evident for the lambs of control ewes. It was concluded that subclinical mastitis alters the sucking behaviour of both ewes and lambs.

Experimental staphylococcal mastitis in bitches: clinical, bacteriological, cytological, haematological and pathological features.

The objectives of the work were to study the features of experimentally induced canine mastitis and to present hypotheses regarding the pathogenesis of the disease. The right caudal abdominal mammary gland of six bitches was inoculated on day 8 after whelping with Staphylococcus intermedius to induce mastitis; adjacent mammary glands were used as controls. Clinical examination, bacteriological and cytological (whiteside test, Giemsa) examination of mammary secretion, as well as haematological tests were performed from 5 days before until 34 days after challenge. Mastectomy was sequentially performed 1, 2, 4, 18, 26 and 34 days after challenge in each of the bitches, in order to carry out a pathological examination of mammary glands. All animals developed clinical mastitis: challenged glands became painful, hot, enlarged and oedematous; secretion was brownish, purulent, with flakes or clots, subsequently becoming yellowish and thick. Staphylococci were isolated from all inoculated glands (up to 22 days). WST was positive in 41/46 samples from inoculated glands and 66/138 samples from control glands; neutrophils predominated during the acute stage. Blood leukocyte counts increased, whilst platelet counts decreased. Gross pathological findings initially included congestion, purulent discharge and subcutaneous oedema; then abscesses, brownish areas and size decrease were seen. Salient histopathological features were initially neutrophilic infiltration, haemorrhages, destruction of mammary epithelial cells and alveoli, and then infiltration by lymphocytes, shrunken alveoli, loss of glandular architecture and fibrous tissue proliferation. We conclude that in bitches, intrammamary inoculation of Staphylococcus intermedius can induce clinical mastitis, followed by subclinical disease. The disorder is characterized by bacterial isolation and leukocyte influx in challenged glands, by leukocyte presence in adjacent mammary glands, by increased blood leukocyte counts and by destruction of mammary parenchyma.