Development of a PCR test for rapid diagnosis of contagious equine metritis

In order to establish a rapid diagnostic method for contagious equine metritis (CEM), we developed and evaluated a polymerase chain reaction (PCR) test. Species-specific PCR primer sets were derived from the DNA sequence of a cloned DNA fragment of Taylorella equigenitalis that did not hybridize with the genome of a taxomonically related species, Oligella urethralis. Single step PCR with primer set P1-N2 and two-step semi-nested PCR with primer sets P1-N2 and P2-N2 detected as low as 100 and 10 CFU of the bacteria, respectively. Single-step PCR detected T. equigenitalis from genital swabs of experimentally infected mares with sensitivity comparable to that of bacterial isolation. Furthermore, two-step PCR was more sensitive than the culture method. Upon examination of field samples, 12 out of 3,123 samples were positive by single-step PCR while only 2 were positive by bacterial culture. The 12 PCR-positive samples originated from 5 mares, of which 3 animals were considered to be carriers based on previous bacteriologic and serologic diagnoses for CEM. The PCR test described in this study would provide a specific and highly sensitive tool for the rapid diagnosis of CEM.     

Is AZT/3TC therapy effective against FIV infection or immunopathogenesis?

In vitro and in vivo prophylactic and therapeutic efficacy of AZT/3TC treatment was evaluated against feline immunodeficiency virus (FIV) infection. In vitro studies utilized FIV-infected peripheral blood mononuclear cells (PBMCs) or FIV-infected T-cell lines treated with AZT (azidothymidine) alone, 3TC alone, or AZT/3TC combination and tested for anti-FIV activity and drug toxicity. AZT/3TC combination had additive to synergistic anti-FIV activities in primary PBMC but not in chronically infected cell lines. In vivo studies consisted of four treatment groups (n=15) of SPF cats receiving AZT/3TC combination (5-75 mg/kg/drug PO BID for 8 or 11 weeks) and one control group (n=9) receiving oral placebo. Group I (n=6, 150 mg/kg/drug/day) was treated starting 3 days pre-FIV inoculation, whereas Group II (n=3, 150 mg/kg/drug/day) and Group III (n=3, 100 mg/kg/drug/day) treatments were simultaneous with FIV inoculation. Group IV treatment (n=3, 100 mg/kg/drug/day) was initiated 2 weeks post-FIV inoculation. All cats were monitored for drug toxicity and FIV infection. Eighty-three percent of cats in Group I and 33% of cats in Groups II and III were completely protected from FIV infection. A significant delay in infection and antibody seroconversion was observed in all unprotected cats from Groups I, II and III. Group IV cats had only a slight delay in FIV antibody seroconversion. Adverse drug reactions (anemia and neutropenia) were observed at high doses (100-150 mg/kg/drug/day) were reversible upon lowering the dose (20 mg/kg/drug/day). In contrast, AZT/3TC treatment had no anti-FIV activity in chronically infected cats. Furthermore, severe clinical symptoms caused by adverse drug reactions were observed in some of these cats. Overall, AZT/3TC treatment is effective for prophylaxis but not for therapeutic use in chronically FIV-infected cats.     

Muscular architecture and VIP-like immunoreactive nerves in the gastroduodenal junction of the chicken

The muscular architecture and the distribution of nerves with vasoactive intestinal polypeptide-like (VIP-like) immunoreactivity were examined in the gastroduodenal junction of the chicken. Both the outer and the inner muscle layers (tunica muscularis) and the muscularis mucosae were examined. The inner muscle layer was divided into two parts, namely its inner and outer parts. The outer muscle layer was not present in the pyloric part but was found in the proventriculus and the duodenum. The muscle strands of the outer muscle layer ran longitudinally but were absent at the isthmus and the pylorus. Most of the outer part of the inner muscle layer was located in or close to the pyloric part. The muscle strands in the outer part connected the pyloric part with other regions, namely the craniodorsal muscle, the tendinous centres and the isthmus. The inner part of the inner muscle layer was widely distributed in the stomach. In the pyloric part, the muscle strands of this layer followed a circular or spiral course. The muscularis mucosae was not present in the caudodorsal and cranioventral muscles of the gizzard. In addition, VIP-like immunoreactive nerve fibres were predominantly distributed in the inner part of inner muscle layer of the pyloric part. These results suggest that the ventriculopyloric orifice and the pyloric part itself constrict with a sphincter-like action.Abbreviations VIP vasoactive intestinal polypeptide     

Effects of a high-protein diet versus dietary supplementation with ammonium chloride on struvite crystal formation in urine of clinically normal cats

OBJECTIVE: To evaluate the effects of a high-protein diet versus dietary supplementation with ammonium chloride (NH4Cl) on struvite crystal formation in the urine of clinically normal cats by measuring the urine concentration of hydrochloric acid (HCl)-insoluble sediment, urine pH, struvite activity product (SAP), number of struvite crystals in urine, and urine volume. ANIMALS: 23 healthy adult cats. PROCEDURE: Urine was fractionated by centrifugation with subsequent extraction of the sediment with 1 N HCl (study 1). Diets containing either 29% crude protein or 55% crude protein were fed to cats in a crossover trial of 3 weeks/period (study 2). Diets supplemented with either sodium chloride (NaCl) or NH4Cl were fed, by use of a 3 x 3 Latin-square design with 3 wk/period (study 3). In studies 2 and 3, urine samples were collected for the last 7 days of each period. RESULTS: The HCl-insoluble sediment contained Tamm-Horsfall glycoprotein (THP; study 1). The high-protein diet (study 2) and dietary supplementation with NH4Cl (study 3) resulted in a decrease in urine pH, SAP, and the number of struvite crystals in urine. However, the high-protein diet decreased urine concentrations of HCl-insoluble sediment containing THP (study 2), in contrast to the NH4Cl supplementation that increased urine volume without a significant effect on the urine concentration of the HCl-insoluble sediment (study 3). CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that compared with dietary supplementation with NH4Cl, the high-protein diet is preferable as a urine acidifier for the prevention of struvite crystal formation in clinically normal cats.     

Morphology of the intermandibular gland of the Lesser mouse deer, Tragulus javanicus

The morphology of the intermandibular gland of the Lesser mouse deer (Tragulus javanicus), which plays an important function in marking area and territory and in the reproductive behaviour of the animal, was examined using immunohistochemistry, lectin histochemistry and scanning electron microscopy. The gland was composed of sebaceous and apocrine glandular material. Sebaceous glands occupied a greater area of the total gland and consisted of many large lobules with polyhedral cells having a pale cytoplasm. The sebaceous gland, being holocrine, possessed no special secretory ducts. The apocrine gland was lined by cuboid cells and the secretory products were often seen in the apical portions of the cells. Myoepithelial cells contained actin filaments lining the basal membranes of the apocrine gland and were surrounded by nerve fibres which immunostained with protein gene product 9.5. The secretion of the gland appears to be a mixture of larger amounts of lipid material from sebaceous glands, and glycoconjugates secreted by both sebaceous and apocrine glands. Lectin histochemistry detected these as galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-mannose and D-glucose. The male gland was larger in size and contained more N-acetyl galactosamine and N-acetyl glucosamine in its secretion than the gland of the female. This implied the presence of sexual differences in secretions in the intermandibular gland of the Lesser mouse deer.     

Mycoplasma meleagridis of turkeys: probability of eliminating egg-borne infection

An equation was developed that describes the probability of eliminating egg-borne Mycoplasma meleagridis (MM) of turkeys by egg treatment. Probability (P) of MM elimination was described in terms of four parameters: pretreatment prevalence of MM, alpha; treatment failure rate, beta; number of eggs treated, n; and hatching rate, h. The eradication equation, based on the Poisson probability distribution, was P(0) = e-n alpha beta h. Probabilities derived from 12 hypothetical MM-eradication situations showed that increasing the size of any or all of the parameters decreased the likelihood of eradication. Use of the predictive equation as a planning tool in a successful MM-eradication program is demonstrated.     

Ultrastructure of the thyroid gland of the one-humped camel (Camelus dromedarius)

Follicular epithelium of the thyroid gland of the one-humped camel was examined by light and electron microscopy. It consisted of a single type of epithelial cell which varied from flattened to columnar in shape. Follicular epithelial cells were characterized by the presence of markedly dilated cisternae of rough-surfaced endoplasmic reticulum, well developed Golgi apparatus, abundant small vesicles (150 nm to 200 nm in diameter) in the apical cytoplasm, and electron-dense colloid droplets measuring from 250 nm to 1600 nm. Follicular epithelial cells frequently showed apocrine secretion into colloidal lumens. Apocrine protrusions with smooth surface were dome-like or balloon-like structures and contained a fine granular matrix. These findings indicate that the morphological features of the follicular epithelial cells of the thyroid gland of the camel are essentially similar to those of mammals except for the presence of apocrine secretion, which is unique to the camel.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of Mycoplasma pulmonis in clinical isolates

To develop a diagnostic tool to identify Mycoplasma pulmonis (M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases.