The upper respiratory tract is a natural reservoir of haemolytic Mannheimia species associated with ovine mastitis

Lamb suckling has been suggested to be an important way of infecting a ewe's udder with different bacteria, including Mannheimia haemolytica. To test the potential role of lambs in transferring Mannheimia species to the ewe’s udder, the restriction endonuclease cleavage patterns of isolates obtained from nasopharyngeal swabs were compared with those obtained from cases of mastitis. Sterile cotton swabs were used to collect nasopharyngeal samples from 50 ewes and 36 lambs from three flocks. M. haemolytica and Mannheimia glucosida as well as haemolytic Mannheimia ruminalis-like organisms were detected in the upper respiratory tract of lambs and ewes. Comparison of the restriction endonuclease cleavage patterns of the isolates suggested that the M. haemolytica isolates obtained from different milk samples from ewes with mastitis were more clonal than those obtained from the nasal swabs. However, some nasal isolates within both Mannheimia species had restriction endonuclease cleavage patterns identical to those obtained from milk samples from ewes with mastitis, indicating that lambs may have a role in transferring these organisms to the udder. More clonality was observed between the M. glucosida isolates than between M. haemolytica isolates.

Mastites ovinas: Epidemiologia, factores de virulência e antigénios imunorrelevantes de agentes etiológicos

O presente trabalho teve como objectivo o estudo da prevalência de mastites ovinas em explorações do Alentejo e a identificação dos agentes etiológicos, seus factores de virulência e epitopos imunorrelevantes. A prevalência de mastite clínica e subclínica foi 1,7% e 32,2%, respectivamente. O agente etiológico mais prevalente foi Staphylococcus epidermidis (N=115), tendo sido também identificados Staphylococcus aureus (N=27) e Streptococcus agalactiae (N=17). A pesquisa de factores de virulência permitiu identificar os padrões de susceptibilidade (N=404) e as Concentrações Inibitórias Mínimas de princípios activos (N=130). De 109 isolados de Staphylococcus epidermidis; oito revelaram capacidade para produzir biofilme in vitro. Os isolados estudados aderiam e eram internalizados por células epiteliais mamárias (N=12). A pesquisa de cinco superantigénios resultou negativa (N=27). Foram estudados os perfis proteicos de Staphylococcus epidermidis, tendo sido identificados os epitopos imunorrelevantes, reconhecidos por imunoglobulinas séricas e mamárias. Verificou-se uma resposta imunológica local específica nos animais infectados./SUMMARY - OVINE MASTITIS: EPIDEMIOLOGY, VIRULENCE FACTORS AND IMMUNORELEVANT ANTIGENES OF AETIOLOGICAL MICRORGANISMS The present work aimed at investigating the prevaleance of ovine mastitis in farms from Aletenjo and the identification of causative microrganisms, their virulence factors and immunorelevant epitopes. The preva lence of clinical and subclinical mastitis was 1.7% and 32.2%,respect ively. The most preva lent aet iologica l agent was Staphylococcus epidermidis (N=115); Staphylococcus aureus (N=27) and Streptococcus agalactiae (N=17) were also identified. The investigation of virulence factors allowed the identification of susceptibility patterns (N=404) and drug Minimal Inhibitory Concentrations (N=130). From 109 Staphylococcus epidermidis isolates; eight showed the ability to produce biofilm in vitro. The isolates studied adhered and were internalised by mammary epithelial cells (N=12). None of the five superantigens studied was detected (N=27). The protein profile of Staphylococcus epidermidis was determined, and the immunorelevant epitopes, recognised by blood and milk immunoglobulins, were identified. It was possible to detect a specific local immune response in infected animals.

Aspectos citológicos e microbiológicos na mastite em ovelhas da raça Bergamácia em lactação

Pós-graduação em Medicina Veterinária - FMVZ

Ocorrência de mastite subclínica ovina durante duas lactações consecutivas em rebanho da raça Santa Inês

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoramento microbiológico da mastite ovina na região de Bauru - SP

Mastitis is an acute or a chronic inflammation of the udder, represented for physical, chemical and bacteriological milk alterations. It is notable that ovine milk represents high economic importance, and it can be used in the production of cheeses and other derivates, contributing for the farmer’s recipe enlargement. However, the mastitis in animals can determine the reduction of milk quantity and quality in infected animals. This may determine lambs’ weight loss and increase animal’s mortality. By the other hand, considering Public Health issues, mastitis can cause milk nutrients losses and promote the microorganisms’ multiplication that may lead to diarrheas and to other zoonosis in humans. This study aimed to evaluate 159 sheep mastitis status in 20 small farms located in Bauru (SP) region. Of the 309 colected samples, in 97 (31.4%) were isolated microorganisms of the genus Staphylococcus sp., which them 27.5% being coagulase negative Staphylococcus and 3.9% coagulase positive Staphylococcus. Of 177 isolates, 97 (54.8%) were represented by the genus Staphylococcus sp., which them 57 (58.8%) from pure culture and 40 (41.2%) in association. Most agents isolated were sensitive to antibiotics used in the test. Thus, we could verify the isolation of infectious microorganisms such as coagulase positive and negative Staphylococcus and, as well in dairy cattle, preventive measures to obtain hygienic milk are extremely important in order to get a final product with quality and safe in food and microbiological point of view.

Streptococcus equi subsp ruminatorum subsp nov., isolated from mastitis in small ruminants

Six isolates of an unknown Gram-positive, catalase-negative, chain-forming, coccus-shaped organism isolated from ovine and caprine mastitis were characterized by phenotypic and molecular taxonomic methods. On the basis of cellular morphology and the results of biochemical tests, the organism was tentatively identified as a streptococcal species. Comparative 16S rRNA gene sequencing studies confirmed that the organism is a member of the genus Streptococcus, with Streptococcus equi as its closest phylogenetic relative (98(.)8% similarity). DNA-DNA pairing studies showed that the unidentified organism displayed more than 70% relatedness to the type strains of S. equi subsp. equi and subsp. zooepidemicus. Despite the relatively high DNA-DNA reassociation values, biotyping and ribotyping allowed clear differentiation of the unknown bacterium from the two recognized subspecies of S. equi. On the basis of phenotypic and molecular genetic evidence, it is proposed that the unknown Streptococcus isolates from ovine and caprine mastitis be classified as a novel subspecies, Streptococcus equi subsp. ruminatorum subsp. nov. The type strain is CECT 5772(T) (=CCUG 47520(T) = Mt 167(T)).

Ovine leptospirosis in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Microbiological evaluation of milk samples positive to California Mastitis Test in dairy buffalo cows (Buballus bubalis)

In order to observe the microbiological status of CMT positive samples, 734 apparently health mammary quarters from buffalo cows were submitted to physical evaluation, strip cup test and CMT. After milk samples inoculation in 10% ovine blood agar base media and in MacConkey agar and incubation under aerobic condition for 72 hours at 37 degrees C, identification was proceeded. According to CMT, 227 quarters (30,93%) were positive, among them 73 (32,16%) presented 1+ reaction, 53 (23,35%) were 2+ and 101 (44,49%) were 3+. Microbiological exams of such samples were positive in 147 (64,76%) out of 227 CMT positive samples and among the remaining 72 (31,72%) were negative and 8 (3,52) were contaminated. In the 147 microbiological positive samples 204 bacteria were found in pure or associated growth and the most frequent agents were: Corynebacterium sp (59,25%); Staphylococcus sp (17,65%) among which 86,11% were coagulase negative and 13,89% were coagulase positive; and Micrococcus sp (6,37%). The results revealed that, excluding the eight contaminated samples, 147 (67,12%) quarters out of 219 CMT positive could be considered as bacteria-carrier and that even in a smaller percentage false-positive results can cause problems in a sanitary program for mastitis control in dairy buffalo cows.

Distribution of contagious and environmental mastitis agents isolated from milk samples collected from clinically health buffalo cows between brazilian dry and rainy seasons of the year

The present study was performed to evaluate the microbiological characteristics of clinically health quarters submitted to milking and also to observe the distribution of contagious and environmental agents between brazilian dry and rainy seasons of the year. During nine months 734 quarters from 37 buffalo cows were submitted monthly to udder inspection, palpation and strip cup test before milking. 734 asseptic milk samples were inoculated in 10% ovine blood agar and in MacConkey agar media, then incubated for 72 hours at 37 C. Among the 580 isolated microrganisms, 182 (31,38%) were recovered from samples collected during the rainy season and 398 (68,62%) from the dry season. In the rainy period the most prevalent agents were: bacteria from the genus Corynebacterium sp (53,30%), Staphylococcus sp (19,78%) and Rhodococcus equi (13,74%). In the dry period, the commonest ones were: Corynebacterium sp (44,97%), Staphylococcus sp (18,84%) and Micrococcus sp (9,55%). The results demonstrated that the methods used to select health quarters in brazilian dairy buffalo farms allow the transmission of contagious bacteria during both seasons of the year, maintaining agents known to cause mainly subclinical inflammatory reactions that compromise cronically the physiology and production of the mammary gland.

Validation of 3D models of the outer and inner surfaces of an ovine femur

The validation of Computed Tomography (CT) based 3D models takes an integral part in studies involving 3D models of bones. This is of particular importance when such models are used for Finite Element studies. The validation of 3D models typically involves the generation of a reference model representing the bones outer surface. Several different devices have been utilised for digitising a bone’s outer surface such as mechanical 3D digitising arms, mechanical 3D contact scanners, electro-magnetic tracking devices and 3D laser scanners. However, none of these devices is capable of digitising a bone’s internal surfaces, such as the medullary canal of a long bone. Therefore, this study investigated the use of a 3D contact scanner, in conjunction with a microCT scanner, for generating a reference standard for validating the internal and external surfaces of a CT based 3D model of an ovine femur. One fresh ovine limb was scanned using a clinical CT scanner (Phillips, Brilliance 64) with a pixel size of 0.4 mm2 and slice spacing of 0.5 mm. Then the limb was dissected to obtain the soft tissue free bone while care was taken to protect the bone’s surface. A desktop mechanical 3D contact scanner (Roland DG Corporation, MDX 20, Japan) was used to digitise the surface of the denuded bone. The scanner was used with the resolution of 0.3 × 0.3 × 0.025 mm. The digitised surfaces were reconstructed into a 3D model using reverse engineering techniques in Rapidform (Inus Technology, Korea). After digitisation, the distal and proximal parts of the bone were removed such that the shaft could be scanned with a microCT (µCT40, Scanco Medical, Switzerland) scanner. The shaft, with the bone marrow removed, was immersed in water and scanned with a voxel size of 0.03 mm3. The bone contours were extracted from the image data utilising the Canny edge filter in Matlab (The Mathswork).. The extracted bone contours were reconstructed into 3D models using Amira 5.1 (Visage Imaging, Germany). The 3D models of the bone’s outer surface reconstructed from CT and microCT data were compared against the 3D model generated using the contact scanner. The 3D model of the inner canal reconstructed from the microCT data was compared against the 3D models reconstructed from the clinical CT scanner data. The disparity between the surface geometries of two models was calculated in Rapidform and recorded as average distance with standard deviation. The comparison of the 3D model of the whole bone generated from the clinical CT data with the reference model generated a mean error of 0.19±0.16 mm while the shaft was more accurate(0.08±0.06 mm) than the proximal (0.26±0.18 mm) and distal (0.22±0.16 mm) parts. The comparison between the outer 3D model generated from the microCT data and the contact scanner model generated a mean error of 0.10±0.03 mm indicating that the microCT generated models are sufficiently accurate for validation of 3D models generated from other methods. The comparison of the inner models generated from microCT data with that of clinical CT data generated an error of 0.09±0.07 mm Utilising a mechanical contact scanner in conjunction with a microCT scanner enabled to validate the outer surface of a CT based 3D model of an ovine femur as well as the surface of the model’s medullary canal.

Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence commercialization, is referred to as the ‘Valley of Death’ and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes (GMP) and scalable designs and to apply these in pre-clinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopaedic bone engineering from bench to bedside byestablishing a pre-clinical ovine critical-sized tibial segmental bone defect model and discuss our preliminary data relating to this decisive step.

The mechanical response of the ovine lumbar anulus fibrosus to uniaxial, biaxial and shear loads

Analytical and computational models of the intervertebral disc (IVD) are commonly employed to enhance understanding of the biomechanics of the human spine and spinal motion segments. The accuracy of these models in predicting physiological behaviour of the spine is intrinsically reliant on the accuracy of the material constitutive representations employed to represent the spinal tissues. There is a paucity of detailed mechanical data describing the material response of the reinforced­ground matrix in the anulus fibrosus of the IVD. In the present study, the ‘reinforced­ground matrix’ was defined as the matrix with the collagen fibres embedded but not actively bearing axial load, thus incorporating the contribution of the fibre-fibre and fibre-matrix interactions. To determine mechanical parameters for the anulus ground matrix, mechanical tests were carried out on specimens of ovine anulus, under unconfined uniaxial compression, simple shear and biaxial compression. Test specimens of ovine anulus fibrosus were obtained with an adjacent layer of vertebral bone/cartilage on the superior and inferior specimen surface. Specimen geometry was such that there were no continuous collagen fibres coupling the two endplates. Samples were subdivided according to disc region - anterior, lateral and posterior - to determine the regional inhomogeneity in the anulus mechanical response. Specimens were loaded at a strain rate sufficient to avoid fluid outflow from the tissue and typical stress-strain responses under the initial load application and under repeated loading were determined for each of the three loading types. The response of the anulus tissue to the initial and repeated load cycles was significantly different for all load types, except biaxial compression in the anterior anulus. Since the maximum applied strain exceeded the damage strain for the tissue, experimental results for repeated loading reflected the mechanical ability of the tissue to carry load, subsequent to the initiation of damage. To our knowledge, this is the first study to provide experimental data describing the response of the ‘reinforced­ground matrix’ to biaxial compression. Additionally, it is novel in defining a study objective to determine the regionally inhomogeneous response of the ‘reinforced­ground matrix’ under an extensive range of loading conditions suitable for mechanical characterisation of the tissue. The results presented facilitate the development of more detailed and comprehensive constitutive descriptions for the large strain nonlinear elastic or hyperelastic response of the anulus ground matrix.

Creation of a validated 3D finite element model of an ovine tibia

Introduction Ovine models are widely used in orthopaedic research. To better understand the impact of orthopaedic procedures computer simulations are necessary. 3D finite element (FE) models of bones allow implant designs to be investigated mechanically, thereby reducing mechanical testing. Hypothesis We present the development and validation of an ovine tibia FE model for use in the analysis of tibia fracture fixation plates. Material & Methods Mechanical testing of the tibia consisted of an offset 3-pt bend test with three repetitions of loading to 350N and return to 50N. Tri-axial stacked strain gauges were applied to the anterior and posterior surfaces of the bone and two rigid bodies – consisting of eight infrared active markers, were attached to the ends of the tibia. Positional measurements were taken with a FARO arm 3D digitiser. The FE model was constructed with both geometry and material properties derived from CT images of the bone. The elasticity-density relationship used for material property determination was validated separately using mechanical testing. This model was then transformed to the same coordinate system as the in vitro mechanical test and loads applied. Results Comparison between the mechanical testing and the FE model showed good correlation in surface strains (difference: anterior 2.3%, posterior 3.2%). Discussion & Conclusion This method of model creation provides a simple method for generating subject specific FE models from CT scans. The use of the CT data set for both the geometry and the material properties ensures a more accurate representation of the specific bone. This is reflected in the similarity of the surface strain results.

Ovine bone and marrow derived progenitor cells : isolation, characterization, and osteogenic potential

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) for their potential clinical use in bone engineering. Prior to clinical application, MPC-based treatment concepts need to be evaluated in preclinical, immunocompetent, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, ovine MPC and their osteogenic potential remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than osteoblasts (OB) derived from tibial compact bone as assessed using standard 2D culture. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14-/CD31-/CD45- /CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed >90%. Cells displayed a spindle-like morphology and formed an interconnected network. Implanted subcutaneously into NOD/SCID mice on type I collagen coated polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds, MPC presented a higher developmental potential than osteoblasts. In summary, this study provides a detailed in vitro characterisation of ovine MPC from a bone engineering perspective and suggests that MPC provide promising means for future bone disease related treatment applications.