Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

The development of a species‐specific test to detect Hymenoschyphus pseudoalbidus in ash tissues

Ash dieback, caused by the pathogen Hymenoscyphus pseudoalbidus, is an emerging lethal disease of Fraxinus excelsior in large parts of Europe. To develop a method for the early detection of H. pseudoalbidus, we designed primers for 46 microsatellites (simple sequence repeats, SSRs) of the pathogen. Seven pairs of primers (SSR38, SSR58, SSR114, SSR198, SSR206, SSR211 and SSR212) were found to bind only to the genome of H. pseudoalbidus, but not to the genome of H. albidus or to 52 different fungal endophytes isolated from F. excelsior and F. angustifolia. Using these seven primer pairs, H. pseudoalbidus was identified in fruiting bodies and different types of ash tissues including dead leaves, dead petioles and discoloured or non‐discoloured wood. Along one twig, H. pseudoalbidus was detected at different levels of intensity, which depended on the distance from symptomatic tissue. The detection limit was 0.9–1.8 pg of genomic DNA per PCR. Of 50 analysed commercially available seedlings, six were infected with H. pseudoalbidus. Two SSR loci (SSR198 and SSR211) showed fragment length polymorphism. Our results showed that the new primers not only provide an easy and inexpensive means of detecting H. pseudoalbidus in ash tissues, but can also provide information on the genetic heterogeneity of the species.     

Multiple congenital urogenital abnormalities in a Tennessee Walking Horse colt

A 9-month-old, Tennessee Walking Horse colt was examined for urinary incontinence. Cystoscopy revealed a single identifiable ureter that appeared abnormal, and sabulous urolithiasis. Only the left kidney, which appeared lobulated and hydronephrotic, could be located using ultrasound. Results of serum chemistries were consistent with renal failure. Necropsy revealed unilateral agenesis of the right kidney and ureter with severe left ureterolithiasis, bilateral cryptorchidism and segmental aplasia of the ductus deferens. Histopathological examination revealed cystitis and diffuse interstitial fibrosis of the left kidney. Congenital malformations should be included as differential diagnoses for urinary incontinence and urogenital disease. Ultrasonography and cystoscopy can be useful to diagnose suspected agenesis of the urinary system in adult horses.     

Faecal shedding, alimentary clearance and intestinal spread of prions in hamsters fed with scrapie

Shedding of prions via faeces may be involved in the transmission of contagious prion diseases. Here, we fed hamsters 10mg of 263K scrapie brain homogenate and examined the faecal excretion of disease-associated prion protein (PrP(TSE)) during the course of infection. The intestinal fate of ingested PrP(TSE) was further investigated by monitoring the deposition of the protein in components of the gut wall using immunohistochemistry and paraffin-embedded tissue (PET) blotting. Western blotting of faecal extracts showed shedding of PrP(TSE) in the excrement at 24-72 h post infection (hpi), but not at 0-24 hpi or at later preclinical or clinical time points. About 5% of the ingested PrP(TSE) were excreted via the faeces. However, the bulk of PrP(TSE) was cleared from the alimentary canal, most probably by degradation, while an indiscernible proportion of the inoculum triggered intestinal infection. Components of the gut-associated lymphoid tissue (GALT) and the enteric nervous system (ENS) showed progressing accumulation of PrP(TSE) from 30 days post infection (dpi) and 60 dpi, respectively. At the clinical stage of disease, substantial deposits of PrP(TSE) were found in the GALT in close vicinity to the intestinal lumen. Despite an apparent possibility of shedding from Peyer's patches that may involve the follicle-associated epithelium (FAE), only small amounts of PrP(TSE) were detected in faeces from clinically infected animals by serial protein misfolding cyclic amplification (sPMCA). Although excrement may thus provide a vehicle for the release of endogenously formed PrP(TSE), intestinal clearance mechanisms seem to partially counteract such a mode of prion dissemination.     

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene‐Expressing Embryos

The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.     

Disseminated Rhodococcus equi in an Anglo‐Nubian goat

Disseminated Rhodococcus equi infection was diagnosed in an Anglo‐Nubian goat presenting for non‐weight bearing lameness of the right pelvic limb. Radiographs showed a moth‐eaten osteolytic lesion in the proximal tibia suggestive of an aggressive bone lesion. Two pulmonary nodules were also present on thoracic radiographs. Initial antemortem cytology of the tibial lesion was suggestive of Rhodococcosis and the goat was sent to necropsy. Necropsy and bacterial culture confirmed the diagnosis of disseminated R. equi infection in the right tibia, lungs, and liver.     

DNA fragmentation kinetics and postthaw motility of flow cytometric-sorted white-tailed deer sperm

This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials).     

Detection of Mycoplasma conjunctivae in sheep affected with conjunctivitis and infectious keratoconjunctivitis

AIM: To determine the aetiolog y of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep.

METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp.

RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected.

CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks.