The Use of Stromal Vascular Fraction in Long Bone Defect Healing in Sheep.

The objectives of the present study were to evaluate (a) the feasibility of using stromal vascular fraction (SVF) and nanocrystalline hydroxyapatite (nHA) paste in combination for the treatment of segmental bone defect, (b) the quality of the callus produced, (c) the potential improvement of the autograft technique, and (d) the direct comparison of the biomaterial to the use of autogenous cancellous bone. Unilateral, segmental mid-diaphyseal bone defect was created on the right metatarsus of skeletally mature sheep animals (n = 24) under anesthesia (D0). Residual segments were stabilized by stainless-steel plates and appropriate screws. Defects were managed as follows: group A: use of nHA paste to filling, group B: use of autogenous bone graft mixed with nHA bone paste, placed in defect, group C: use of SVF mixed with nHA bone paste injected into defect, group D: use of bone graft and SVF with nHA paste before apposition in bone defect. SVF had been previously isolated from adipose tissue of the animals intra-operatively after digestion with collagenase solution and neutralization. Animals were evaluated clinically and by X-raying and ultrasonographic examination of the defect, at regular intervals, until D90. Ultrasonographic assessment performed along the length of the defect included calculation of the length of the bone defect and assessment of vascularization. SVF was successfully isolated from group C and D animals, with the average yield being 1.77 × 106 cells. The comparison of clinical scores (based on the 'Kaler scale') on each post-operative day indicated significant differences between the four groups on D1 to D30 (p < 0.01); the median clinical score within group A was 2.5 for D1-D30 and 1 for the entire period; respective scores for other groups were 1.5 (p = 0.07) and 0 (p = 0.033). Differences in radiographic assessment scores were significant for scores obtained on D60 (p = 0.049) and D90 (p = 0.006). There was a significant difference between the four groups in the length of the bone defect, as assessed ultrasonographically, for the entire length of the study; median values were 8, 8.5, 6, and 8 mm for groups A, B, C, and D, respectively (p = 0.008). There was a significance in the differences between median scores obtained during the histopathological examination: 2, 11, 13.5, and 12 for group A, B, C, and D (p = 0.022). There was an inverse correlation between the overall scores of histopathological evaluations and the length of the bone defect (observed on D90) (p < 0.0001) and a correlation between the overall scores and the radiographic assessment scores (obtained on D90) (p < 0.0001). This is the first study in which the efficacy of fresh autologous Stromal Vascular Fraction (SVF) from adipose tissue in enhancing bone healing in a long, weight-bearing, diaphyseal bone was evaluated. It is concluded that the lumbosacral region was an attractive site for harvesting adipose tissue, the use of SVF contributed to faster rehabilitation post-operatively, and SVF significantly enhanced bone formation; in general, the results indicated an osteogenic potential of SVF comparable to the gold standard autologous bone graft.

Recovery of Staphylococci from Teatcups in Milking Parlours in Goat Herds in Greece: Prevalence, Identification, Biofilm Formation, Patterns of Antibiotic Susceptibility, Predictors for Isolation.

The objectives of this work are (a) to describe staphylococci on the teatcups of milking parlours in goat farms and identify predictors for the presence of staphylococcal isolates on the teatcups, (b) to evaluate relationships with total bacterial counts and somatic cell counts in bulk-tank milk, and (c) to establish patterns of susceptibility to antibiotics for the staphylococcal isolates and identify predictors for the recovery of resistant isolates. In a cross-sectional study of 66 goat farms across Greece, swab samples were collected from 303 teatcups (upper and lower part) for staphylococcal recovery, identification, and assessment of biofilm formation. Details regarding health management on the farms (including conditions in the milking parlour) and the socio-demographic characteristics of farmers were collected by means of a structured questionnaire. A total of 87 contaminated teatcups (28.7%) were found on 35 goat farms (53.0%). Staphylococci were more frequently recovered from the upper than the lower part of teatcups: 73 versus 43 teatcups, respectively. After identification, 67 staphylococcal isolates (i.e., excluding similar isolates) were recovered from the teatcups; Staphylococcus aureus, Staphylococcus capitis, and Staphylococcus equorum predominated. Of these isolates, 82.1% were biofilm-forming. In multivariable analysis, the annual incidence of clinical mastitis in the herd emerged as the only significant factor associated with the isolation of staphylococci from the teatcups. Of the 67 isolates, 23 (34.3%) were resistant to at least one antibiotic, and 14 (22.4%) were multi-resistant. Resistance was found most commonly against penicillin and ampicillin (22.4% of isolates), fosfomycin (17.9%), clindamycin (14.9%), erythromycin, and tetracycline (13.4%). In multivariable analysis, the annual incidence of clinical mastitis in the herd and the use of detergent for parlour cleaning emerged as significant factors associated with the isolation of staphylococci resistant to antibiotics.

Natural Co-Exposure to Borrelia burgdorferi s.l. and Anaplasma phagocytophilum: Unraveling the Hematological Profile in Sheep.

The occurrence of co-infected hosts and questing ticks with more than one tick-borne pathogen-as in the case of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato-is expected in endemic regions. Their synergy-in terms of pathogenesis and disease severity-has been suggested previously in humans. Limited data exist on the clinicopathological alterations in co-infected sheep. In this study, we investigated the impact of A. phagocytophilum and B. burgdorferi s.l. seropositivity, alone and in combination, on the hematological parameters of naturally infected sheep. A complete blood count was performed, and indirect immunofluorescence assays were used to detect IgG antibodies against A. phagocytophilum and IgG and IgM antibodies against B. burgdorferi s.l. Single natural exposure to B. burgdorferi s.l. was characterized by low Packed Cell Volume (PCV) values and platelet (PLT) counts, while single exposure to A. phagocytophilum was characterized by low PCV values, low white blood cell (WBC) counts, and an increased risk for leukopenia and neutropenia. Co-exposure resulted in the most severe blood abnormalities; all the blood parameters decreased, and the sheep presented an increased risk for anemia. Our study showed that natural co-exposure to A. phagocytophilum and B. burgdorferi s.l. in sheep leads to more severe blood abnormalities and enhances the pathogenic processes. More studies are needed to clarify the possible background mechanisms.

High Milk Somatic Cell Counts and Increased Teladorsagia Burdens Overshadow Non-Infection-Related Factors as Predictors of Fat and Protein Content of Bulk-Tank Raw Milk in Sheep and Goat Farms.

This paper presents the results of an extensive countrywide investigation performed in 325 dairy sheep flocks and 119 goat herds throughout Greece. The objectives of the study were (a) to investigate fat and protein content in the bulk-tank raw milk of small ruminant farms in Greece and (b) to identify factors potentially influencing that content and factors that can contribute to increased content. The mean fat/protein contents in bulk-tank raw milk of sheep and goats were 6.16 ± 0.05%/4.43 ± 0.01% and 4.77 ± 0.44%/3.23 ± 0.30%, respectively. Significant differences were seen in protein content between farms in the various parts of the country. For sheep, multivariable analyses revealed breed and age of lamb removal from dams as significant for fat content, and somatic cell counts, management system in the farm, administration of anthelmintic treatment during the last stage of pregnancy, and farmer education as significant for protein content. For goats, significant factors were month into lactation period, age of kid removal from dams, and breed for fat content, and somatic cell counts, month into lactation, grazing duration, and % Teladorsagia larvae in faecal samples for protein content. For concurrently high fat and protein content, in multivariable analyses, the following emerged as significant factors: somatic cell counts in milk, numbers of parasite eggs in faeces, and veterinary collaboration (sheep), and month into lactation and somatic cell counts in milk (goats). The results indicate that high somatic cell counts in milk (reflecting the presence of mastitis) and gastrointestinal parasitic infections (mainly Teladorsagia infection) appear to exert a more significant influence on fat and protein content of milk, in comparison to non-infection-related factors.

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 (>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.

Antibiotic Resistance of Staphylococci from Bulk-Tank Milk of Sheep Flocks: Prevalence, Patterns, Association with Biofilm Formation, Effects on Milk Quality, and Risk Factors.

The objectives of this work were to study prevalence and characteristics of resistance to antibiotics of staphylococcal isolates from the bulk-tank milk of sheep flocks across Greece, to assess possible associations of the presence of antibiotic resistance with the quality of milk in these flocks and to evaluate flock-related factors potentially associated with antibiotic resistance among these isolates. A cross-sectional study was performed in 325 sheep flocks in Greece. Bulk-tank milk samples were collected for bacteriological examination; staphylococcal isolates were evaluated for resistance to 20 antibiotics. Oxacillin-resistant staphylococcal isolates, isolates resistant to any antibiotic, and multi-resistant isolates were recovered from 8.0%, 30.5%, and 12.0% of flocks, respectively. Of 232 isolates, 11.6% were resistant to oxacillin, 46.1% were resistant to at least one antibiotic, and 16.4% were multi-resistant. Resistance was seen more frequently among coagulase-negative (50.6%) than among Staphylococcus aureus (31.5%) isolates. Resistance was more frequent against penicillin and ampicillin (34.1% of isolates), clindamycin (17.7%), and fosfomycin (14.2%). An association was found between biofilm formation by staphylococci and resistance to fosfomycin. For recovery of oxacillin-resistant isolates, the lack of experience by farmers emerged as a significant factor; respective factors for the isolation of staphylococci resistant to any antibiotic or multi-resistant isolates were the early stage of the lactation period (0th-1st month) and the intensive management system applied in the flocks, respectively.

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.

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.

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.

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).

The bacterial flora in the teat duct of ewes can protect against and can cause mastitis.

We studied the possible effects of bacterial populations within the teat duct, in the pathogenesis of ovine mastitis. In experiment I, 32 ewes were allocated into group A (ewes from which we isolated (+++ growth) coagulase-negative staphylococci), B (ewes from whose duct we isolated (+ growth) coagulase-negative staphylococci) or C (ewes from which we isolated Bacillus spp.) and subdivided into A1, B1, C1 (n=4; challenged by deposition of 1.250 cfu of Mannheimia haemolytica into the teat duct) or A2, B2, C2 (n=4; used as uninoculated controls); group D (n=8) contained ewes with no bacteria in their teat ducts and were challenged as above. There were less bacteriological isolations of flora (P = 0.018) and challenge (P<0.05) organisms from A1 than from A2 and D ewes; the severity of pathological findings in A1 (summed up score: 27) ewes was smaller than in D (summed up score: 36) ewes (P = 0.038). No such findings were evident with B1 or C1 ewes (P>0.4). In experiment II, ewes (groups E and F, n=6) from whose duct we isolated coagulase-negative staphylococci (+ growth) were used; in group G (n=6) ewes with no bacteria in their teat ducts were included. Teat chapping was applied in E and G ewes. All E ewes developed acute clinical mastitis within 24 h after teat chapping, although we had carried out no challenge; there were more bacteriological isolations of flora organisms from E than from F and G ewes (P < 0.001); the severity of pathological findings in E (score: 28) was greater than in F (score: 3) or G (score: 14) ewes. In experiment III, eight ewes with no bacteria in their teat ducts were allocated into group H or I (n=4) and challenged into the teat (group H) or into the gland (group I) with 10(6) cfu of a Staphylococcus simulans recovered from the teat duct of a group E ewe. Group H ewes developed transiently clinical followed by subclinical mastitis (based on bacteriological and cytological evidence), whilst group I ewes developed severe clinical disease. We conclude that staphylococcal flora present in high numbers within the teat duct of ewes can afford some protection against invading microorganisms. However with impeded defence mechanisms of the teat, the same flora may invade the mammary parenchyma and cause clinical mastitis.