A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle

SUMMARY This paper reviews the laboratory diagnosis of Leptospira hardjo infection in cattle. Two genotypes of L hardjo, Hardjoprajitno and Hardjobovis, have been identified in cattle, but only Hardjobovis has been isolated in Australia. There are problems with diagnosis and control of bovine leptospirosis. Infection is usually subclinical and the serological titres vary greatly in peak and duration. Leptospires may be excreted in urine for up to 18 months. Low microscopic agglutination test titres may be significant in unvaccinated herds as indicators of endemic infection. Vaccines differ in their composition, and their efficacy is difficult to evaluate. The serological response after vaccination is difficult to differentiate from the response after infection. Pregnant cows that become infected may abort, but this is usually after the serological response has peaked. Therefore, paired serum samples are of little use in diagnosing abortion caused by L hardjo. Fluorescent antibody techniques are more sensitive than dark field microscopy for detection of leptospires in urine and tissue samples. Techniques for culture have improved but are still difficult to perform and take 3 months or longer for results to be known. DNA probes and polymerase chain reaction tests are very sensitive and specific, quick to perform, and can be used on fluid and tissue samples.     

Clinicopathological Features of an Equine Ovarian Teratoma

The present study describes the clinicopathological features of a mature cystic ovarian teratoma occurred in a 4 year old, cyclic, nulliparous Andalusian mare. The mass was discovered in the left ovary during a breeding soundness examination, and was hard in consistency and variously echogenic. Laparoscopy was performed to confirm the neoplastic change, followed by a unilateral laparotomic ovariectomy in the standing animal. On the cut surface the teratomatous ovary showed a large cyst filled with hair and sebaceous material and three small cysts containing mucous fluid, surrounded by solid areas where bone and cartilage could be recognized. Exceptionally, functional remnants of the ovary, including small follicles and a diestral corpus luteum were found near the tubal extremity. Histology revealed mature tissues foreign to the ovary including stratified squamous and respiratory epithelia, mucous, sebaceous and sweat glands, muscle fibres, fat and nervous tissue. The appearance of tracheal and lung architecture was occasionally observed.     

Cytochrome-P450 enzymes and autoimmunity: expansion of the relationship and introduction of free radicals as the link

The Cytochrome-P-450 enzymes (CYP) are among the most important xenobiotic-metabolizing enzymes, which produce reactive oxygen species (ROS) as the result of metabolizing xenobiotics.ROS are believed to play important roles in the pathophysiology of autoimmune diseases. ROS can alter the structure of cellular antigens to produce a "neo-antigen" which could mount an autoimmune response against the original antigen through molecular mimicry. ROS are involved in apoptosis, activation of antigen presenting cells and initiation or amplification of diverse immunologic reactions.Taking all these facts together, it could be speculated that CYP may be involved in the initiation and/or amplification of autoimmune phenomena.     

Expression of Prostate Glycoconjugates in the Stallion and Castrated Horse

This work was undertaken to determine the glycoconjugates secreted by the epithelium of the prostate in the intact stallion and castrated horse using lectin histochemical procedures in conjunction with enzymatic digestion and deglycosylation treatments. Additionally, anti‐5 and 13‐16‐cytokeratin antibodies were used to localize epithelial basal cells. In the stallion, lectin histochemistry showed the following sugar residues in the Golgi zone of the glandular cells: α‐Glu/Man, α‐Fuc and β‐Gal included in both O‐ and N‐linked oligosaccharides as well as β‐GalNAc, GlcNAc and α‐Gal, which belonged to O‐glycoproteins. β‐Gal and β‐GalNAc moieties were also noted subterminal to sialyl residues. Sialic acid specific lectins identified Neu‐5Ac(α2,3‐6)‐β‐Gal or Neu5Ac(α2,6)‐β‐GalNAc sequences in both N‐ and O‐bound glycoproteins. The prostatic glandular cells of the castrated horse expressed some of the same sugar moieties found in the stallions, such as α‐Glu/Man, α‐Gal and GlcNAc, but significant differences were also noted. In particular, β‐D‐GalNAc was only detected subterminal to sialic acid, β‐D‐Gal‐(1‐3)‐D‐GalNAc was found in N‐linked glycans, whereas β‐D‐Gal‐(1‐4)‐D‐GlcNAc and Neu5Acα2,6Gal/GalNAc were noted only in O‐glycoproteins. These results indicate that the lectin binding patterns in glandular cells may be modified by sex hormones. No specific lectin labelling of basal cells was found in either the stallion or the castrated horse even though they were immunostained with specific anti‐cytokeratin antibodies. These cells stained more strongly in the castrated horse than in the intact stallion suggesting that they are androgen responsive. The glycomolecules detected in the equine prostate secretions may contribute to the remodelling of the sperm surface, which occurs during sperm transit through the male genital tract and also after ejaculation in the seminal plasma. These changes may be important in the understanding of the stallion fertility.     

Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Synchronizing Cell Cycle of Goat Fibroblasts by Serum Starvation Causes Apoptosis

Cell cycle stage and synchronization of donor cells are important factors influencing the success of somatic cell nuclear transfer. This study examined whether serum starvation has any effect on specific cell death. We also studied the effects of serum starvation, culture to confluence, and full confluency (confluent + 72 h) on cell cycle characteristics and apoptosis of goat dermal fibroblast cells. The cells were obtained from the ear of a 1.5‐year‐old female goat. The following experimental groups were analysed for fibroblast cells: (i) normally growing, (ii) confluent, (iii) full confluency, (iv) cells starved for 48 h and (v) cells starved for 72 h. Analysis of cell cycle distribution by flow cytometry showed that 4.56 and 51.88% of normal cycling cells were at the G0 and G1 phases respectively. In the confluent group, 80% of the cells were arrested in the G0/G1 phase. Serum starvation for 48 and 72 h arrested 84.78% and 90.1% cells at the G0/G1 phase respectively which showed a significant difference when compared with the control group (p < 0.05). Double staining by PI and FITC distinguishes G0 phase from G1 phase. In the full confluency group, 91.53% of cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Under normal culture conditions, 6.39% of cells underwent early apoptosis. In the confluent group 8.93% of cells showed early apoptosis. Serum starvation for 48 and 72 h caused early apoptosis in 8.91 and 39.83% of the cells respectively. Full confluency treatment did not increase the number of apoptotic cells significantly (8.67%). After 72 h, serum starvation significantly increased early apoptosis (p < 0.05). In conclusion, the use of full confluency is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase and also induces less apoptosis in comparison with the serum starvation group.