Comparison of isolation and expansion techniques for equine osteogenic progenitor cells from periosteal tissue

Stem cell therapy and cell-based therapies using other progenitor cells are becoming the treatment of choice for many equine orthopedic lesions. Important criteria for obtaining autogenous equine progenitor cells in vitro for use in clinical cell-based therapy include the ability to isolate and expand cells repeatedly to high numbers (millions) required for therapy, in a clinically relevant time frame. Cells must also maintain their ability to differentiate into the tissue type of choice. The objective of this study was to compare isolation and expansion techniques for preparation of periosteal-derived osteogenic progenitor cells for use in commercial autogenous cell-based therapy. Cells were allowed to migrate spontaneously from periosteal tissue or were enzymatically released. Isolated cells were expanded using enzymatic detachment of cells and subsequent monolayer or dynamic culture techniques. Viable osteogenic progenitor cells from each group were counted at 2 weeks, and osteogenic potential determined. Cells isolated or expanded using the explant or bioreactor technique yielded cells at a much lower number per gram of tissue compared with that of enzyme digestion and monolayer expansion, but all cells were able to differentiate into the ostoblast phenotype. Osteogenic progenitor cells isolated by enzymatic release and expanded using monolayer culture reached the highest number of viable cells per gram of donor periosteal tissue while maintaining the ability to differentiate into bone forming cells in vitro. This technique would be an easy, consistent method of preparation of equine osteogenic cells for clinical cell based therapy for orthopedic conditions.     

Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum

Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was amplified and sequenced, and the resultant sequences were aligned with published sequences for Pythium aphanidermatum, P acanthicum, and P myriotylum. A pair of P insidiosum-specific primers (PI-1 and PI-2) were designed from variable regions within the ITSI region. A nested PCR assay was developed in which the 1st round amplified the ITSI region by use of universal fungal primers. Second-round amplification utilized the internal P insidiosum-specific primers PI-1 and PI-2. Specificity of the assay was tested with DNA extracted from cultures of the following: 10 clinical isolates of P insidiosum and 1 isolate each of P graminicola, P irregulare, P arrhenomanes, P myriotylum, P deliense, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a canine-pathogenic Lagenidium species. Nested PCR produced a single 105-base pair amplicon for each of the P insidiosum isolates, but did not produce amplicons for any of the other isolates. Results of this study suggest that PCR is a useful tool for the identification of P insidiosum.     

Comparison of three culture techniques for the isolation of Brucella abortus from bovine supramammary lymph nodes

Three different culturing techniques were compared and evaluated to determine the most effective method for isolating Brucella abortus from bovine supramammary lymph nodes (SM's). In method I, the SM was sliced in half, and the inner surface was minced finely with a sterile scalpel. The minced surface was spread onto the agar surface of 4 selective media. In method II, the SM was cut into small pieces and placed in a bag with a volume of phosphate-buffered saline equal to the volume of the lymph node. The bag was placed in a laboratory blender and the SM was macerated for 5 min. The tissue suspension was spread with a sterile cotton swab onto the agar surface of 4 selective media. In method III, the SM was processed in the laboratory blender. One milliliter of the suspension was pipetted into a flask of biphasic medium, and 2 ml of the suspension was pipetted into another flask of biphasic medium. A total of 626 SM's from 285 cows were cultured. Brucella abortus was isolated from 149 (52.3%) cows by 1 or more methods. Brucella abortus was isolated from 136 cows by method I. 137 cows by method II, and 86 cows by method III. Nine (3.2%) cows were positive by method I only, 11 (3.9%) cows by method II only, and 2 (0.7%) cows by method III only. The isolation rate for method III was significantly lower than for method I or II. There was no significant difference between methods I and II.     

Laparoscopic techniques for investigating the equine oviduct

Reasons for performing study: The diagnostic and therapeutic options for oviduct disorders in the mare are limited. The current best techniques require exploratory surgery under general anaesthesia or flank laparotomy. Hypothesis: The orthograde flushing of the oviduct for diagnostic or therapeutic options is possible using laparoscopic techniques in the standing sedated mare. Methods: Development of a laparoscopic technique for catheterisation of the infundibulum and flushing of the oviduct (sterile methylene blue solution) in the standing sedated mare was examined in 2 experiments. The first involved a transvaginal laparoscopic approach, the second a laparoscopic flank approach. Passage of fluid into the uterus was assessed by post operative hysteroscopy. Results: In Experiment I, visualisation of the infundibulum was possible (left side 7/8 cases, right side in 6/8 cases). The beginning of the oviductal ampulla could be seen in 3 of 8 cases on the left side. An adequate opening of the infundibulum and visualisation or catheterisation of the abdominal ostium were not possible. In Experiment II, catheterisation of the ampulla was successful in 7 of 11 cases, and in 5 of these 7 cases the injected fluid could be identified in the uterus by post operative hysteroscopy. Conclusion: A transvaginal laparoscopic approach to the oviduct is not appropriate for oviductal flushing in the mare. However, a laparoscopic flank‐approach permits investigation and flushing of the oviduct. Potential relevance: Laparoscopic flushing could become a practical method for diagnosis and therapy of oviduct disorders and a minimally invasive technique for collection of young embryos or the transfer of gametes (GIFT).     

Evaluation of the effects of intra-articular injection of dimethylsulfoxide on normal equine articular tissues

To evaluate the effects of intra-articular injection of dimethylsulfoxide (DMSO) on normal equine articular structures, 7 adult horses with clinically normal carpi were allotted to 2 treatment groups (group A, n = 4; group B, n = 3). In each horse after collection of synovial fluid samples, the right antebrachial carpal and middle carpal joints were aseptically injected with 2 ml of a 40% solution of 90% medical grade DMSO in lactated Ringer solution, and the corresponding joints of the left forelimb (controls) were injected with 2 ml of lactated Ringer solution. In group-A horses, 2 ml of synovial fluid was obtained prior to injections of 40% DMSO at 24 hours and 72 hours, for a total of 3 injections. At necropsy, synovial fluid, synovial membrane, and articular cartilage specimens were obtained. Group-B horses were injected with 40% DMSO in the same sequence; however, the series was repeated following a 1-week interval. Clinical evaluation of these horses revealed no evidence of carpal inflammation associated with any injection in any group. Synovial fluid analysis of DMSO-injected and control joints revealed insignificant differences in leukocyte counts and total protein content. There was no evidence of cartilage degradation on gross, histologic, or histochemical evaluation of any of the joints. Intercellular matrix staining of the articular cartilage failed to reveal any observable difference in glycosaminoglycan content between injection with DMSO or lactated Ringer solution.     

Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses

In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-Tcl3, can be used for isolation and propagation of equine herpesviruses.     

Fracture repair techniques for the equine mandible and maxilla

The purpose of this article is to review the principles of fracture repair for the rostral mandible and maxilla and to demonstrate a variety of repair techniques. Because each fracture is unique, knowledge of the different repair techniques will allow the practitioner to choose a repair method best suited to the fracture configuration, equipment available, and the skill level and preferences of the surgeon.     

Improved culture methods for isolation of Salmonella organisms from swine feces

OBJECTIVE: To compare 3 alternative culture techniques for the detection of Salmonella organisms in swine feces with a modification of the International Standard Organization (ISO) 6579 standard protocol. SAMPLE POPULATION: Fecal samples from swine herds suspected of having Salmonella infections. PROCEDURE: 4 experiments were performed to evaluate the following: 1) diagnostic sensitivity of the selective preenrichment and rapid isolation novel technology (SPRINT) protocol, compared with that of the modified ISO protocol; 2) detection limit of the SPRINT protocol for Salmonella organisms; 3) use of tetrathionate-novobiocin (TTN) broth, compared with selenite cysteine (SC) broth for selective enrichment; and 4) use of universal preenrichment (UPE) broth, compared with buffered peptone water (BPW) for preenrichment of samples prior to the use of modified semisolid Rappaport-Vassiliadis (MSRV) plates. RESULTS: Comparing the Salmonella culture results of 183 swine fecal samples, the diagnostic sensitivity of the SPRINT protocol (0.86) was not significantly different than the diagnostic sensitivity of the modified ISO protocol (0.80), although it was 24 hours faster. The SPRINT protocol could detect 5 of the 6 investigated Salmonella serotypes at inoculation concentrations of < 10 colony-forming units (CFU)/25 g of uncontaminated feces. The TTN broth performed significantly better than the SC broth for selective enrichment of Salmonella organisms. There was no significant difference in results of preenrichment of samples between the use of UPE broth or BPW. CONCLUSIONS AND CLINICAL RELEVANCE: The SPRINT protocol may provide a faster alternative for isolation of Salmonella organisms from swine fecal samples. Furthermore, the use of TTN broth instead of SC broth may increase the sensitivity of the modified ISO 6579 protocol.     

Studies of bovine Mycoplasma mastitis. 2. Testing of various culture media and culture methods for the isolation of Mycoplasma from milk samples]

The recipes Medium-I broth, Medium-B broth, and Weissenseemycoplasma broth as well as Medium-I agar, Medium-B agar, and MRL agar were tested for their applicability to culturing mycoplasma from milk samples, using the direct and indirect techniques. No dependable information on the occurrence of mycoplasma was obtainable from tested material unless several nutritive media and techniques were combined. Medium I, for which almost no imported substances were needed, was in no way inferior to common international nutritive substrates. Its use in conjunction with the indirect culturing technique is recommended for routine diagnosis.     

Occurrence of equine coital exanthema in pastured draft horses and isolation of equine herpesvirus 3 from progenital lesions

During the period from 2001 to the following year, progenital diseases had been epidemic among the draft stallions and mares pastured together in Iwate Prefecture, the northeastern district of Japan. A stallion and 8 of 31 mares were affected in 2001, and 1 of 2 stallions and 10 of 36 mares in 2002. The clinical symptoms consisted of the formation of papules, pustules, ulcers and scabs on the progenital skin and mucosa in stallions and mares. In 2002, Equine herpesvirus 3 (EHV3) was isolated from 2 mares and the glycoprotein G gene of the virus detected from a stallion and 4 mares by polymerase chain reaction. Serum neutralizing tests showed that 12 of 38 horses, 10 clinically and 2 subclinically affected, changed to be positive for the EHV3 antibody. The results suggest that the horses were affected with equine coital exanthema (ECE) through coitus. Five mares with the antibody at the pre-pastured period may have been the possible origins of EHV3 infection in 2002, although the exact origin in 2001 remains unknown. The artificial insemination was performed for the prevention of ECE spreading through coitus on the pasture in 2003. There was no epidemic of the disease in 31 mares, although 3 mares with the antibody at the pre-pastured period showed the significant increase in the titers during the pastured period.     

Local anaesthetic techniques for the equine head,towards guided techniques and new applications

Perineural nerve blocks are often used in equine practice, especially since the use of diagnostic and surgical procedures in the standing sedated horse have expanded over recent decades. The purpose of this review is to discuss the different perineural nerve blocks for the equine head. The review starts with the currently most used blind approaches as described in textbooks and scientific studies. In human medicine, the role of guided techniques, such as ultrasound guidance, advanced imaging guidance and nerve stimulator guided techniques, is very extensively described. These techniques are promising to use in equine medicine as well. The first studies that describe these techniques in equine cases are also discussed in this review, as well as the possibilities for neuromodulation in equine pain syndromes like equine trigeminus-mediated headshaking and the role of perineural nerve blocks in diagnosing this syndrome.     

Optimization of a culture technique for the isolation of Listeria monocytogenes from faecal samples

A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.     

An evaluation of a biphasic medium for the isolation of Brucella abortus from bovine tissues

The isolation of virulent Brucella abortus from specimens taken from cattle was studied using culture in biphasic medium in which solid and liquid media were contained in the same flask and on solid medium in Petri plates. A total of 8638 specimens from 441 cattle, reactors to one or more of several serological tests or to an allergic test, were used. The specimens consisted of lymph nodes, udder, spleen, uterine caruncles, cotyledons, foetal tissues, foetal stomach contents and mammary secretions. Following maceration each specimen was inoculated into 2 flasks of biphasic medium one with 1 ml (Flask A) and the other with 2 ml (Flask B), and on to solid medium in one Petri plate. The biphasic medium and solid medium were shown to have equal ability to support the growth of B. abortus. The increased rate of detection in the biphasic medium was due to the increased volume of the inoculum used. Brucella were isolated from 1151 specimens. By direct plating on the solid medium 685 specimens were positive whereas Flask A positive for 961 specimens and Flask B for 1031 specimens. The success of the biphasic medium can be judged by the increase in the number of infected animals detected. Of the 150 infected cattle, both techniques identified 126 animals in common, 4 were identified only by the plate technique and 20 only by the biphasic technique. Culture on both solid medium and in biphasic medium is recommended for optimum results.     

A comparison of techniques for the quantitative analysis of hyaluronic acid in equine synovial fluid.

A comparison of methods of preparing the hyaluronic acid of equine synovial fluid for quantitative spectrophotographic analysis is presented. A new method is proposed which appears superior to the previous methods.