PCR as a diagnostic tool for brucellosis

Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of human brucellosis or contamination of food products). In these cases, a genus-specific PCR assay is sufficient. Genus-specific assays tend to be simple, robust, and somewhat permissive of environmental influences. The main genetic targets utilized for these applications are the Brucella BCSP31 gene and the 16S–23S rRNA operon.

Other instances require identification of the Brucella species involved. For example, most government-sponsored brucellosis eradication programs include regulations that stipulate a species-specific response. For epidemiological trace back, strain-specific identification is helpful. Typically, differential PCR-based assays tend to be more complex and consequently more difficult to perform. Several strategies have been explored to differentiate among Brucella species and strains, including locus specific multiplexing (e.g. AMOS-PCR based on IS711), PCR-RFLP (e.g. the omp2 locus), arbitrary-primed PCR, and ERIC-PCR to name a few. This paper reviews some of the major advancements in molecular diagnostics for Brucella including the development of procedures designed for the direct analysis of a variety of clinical samples. While the progress to date is impressive, there is still room for improvement.     

Encephalitic listeriosis in ruminants: immunohistochemistry as a diagnostic tool

A retrospective analysis of 42 ruminants (sheep, goats and cattle) with suspected meningo-encephalitis was performed. The clinical findings and the post-mortem results of the animals have been specified. Bacteriological culture, Gram's stain and Listeria-specific immunohistochemistry were performed in order to confirm the diagnosis of these cases. The results of the different methods were evaluated for the detection of listerial antigens. Bacteriological culture was positive in 28.5% of the cases. In 47.6% of the cases, Gram-positive bacteria were found and 80.9% of the cases were immunohistochemically positive for the listerial antigen. The most important conclusion from this investigation is that the traditionally used Gram's stain and the bacteriological culture techniques are insufficient compared with immunohistochemistry for the confirmation of a Listeria monocytogenes infection.     

Use of high-resolution ultrasound as a diagnostic tool in veterinary ophthalmology

The recent development of a 20-MHz, high-frequency ultrasound probe has allowed tissue to be visualized at resolutions of 20 to 80 microm, which is similar to a low-power histologic view. This high degree of resolution, however, limits tissue penetration to 5 to 10 mm, which is ideal for examination of the anterior segment of the eye. The detail provided by high-resolution ultrasound readily permits the clinician to distinguish between various anterior segment entities that may appear similar but are treated quite differently, such as anterior uveal tumors, iridociliary cysts, and iris bombé. High-frequency ultrasound is also a valuable aid in creating a surgical plan for treatment of ocular disorders in which the cornea is opaque, such as feline corneal sequestrum and tumor invasion into the cornea. Other applications of this technology include elucidation of the pathogenesis of glaucoma in veterinary patients and evaluation of regions of the lens that are difficult to examine directly.     

Protein electrophoresis as a diagnostic and prognostic tool in raptor medicine.

Plasma proteins of 139 healthy adult birds of prey from 10 species were separated by electrophoresis to characterize and document normal reference ranges and species-specific electrophoretic patternsand to evaluate the value of this technique for health screening, disease diagnosis, and prognostic indication. Species studied included bald eagle (Haliaeetus leucocephalus), red-tailed hawk (Buteo jamaicensis), barn owl (Tyto alba), great horned owl (Bubo virginianus), turkey vulture (Cathartes aura), Harris' hawk (Parabuteo unicinctus), Stellar's sea eagle (Haliaeetus pelagicus), barred owl (Strix varia), screech owl (Otus asio), and black vulture (Coragyps atratus). Several clinical cases show the diagnostic/therapeutic value of protein electrophoresis in raptors. This study establishes species-specific reference ranges for several birds of prey and discusses the benefit of electrophoresis as a diagnostic technique in health screens, as a diagnostic aid in conjunction with other tests, and as a prognostic indicator in clinical evaluation of raptors.     

Ultrasound as a diagnostic tool in the investigation of a pony with intestinal lymphoma

A 21‐year‐old New Forest pony presented for evaluation of lethargy and colic. Transcutaneous abdominal ultrasonography revealed a cavitary, thick walled, mass‐like lesion that appeared continuous with the small intestine. The thick walls of the mass‐like lesion were of heterogeneous echogenicity with hyperechoic foci extending from the hyperechoic luminal surface to within the wall and there was loss of normal wall layering. These findings were confirmed grossly at exploratory celiotomy and histopathological examination of affected tissues confirmed lymphoma. The ultrasound findings facilitated a preoperative presumptive diagnosis that allowed informed decision‐making and aided case management.     

Testicular fine needle aspiration cytology as a diagnostic tool in dog infertility

The results of testicular aspirate cytology taken from clinical patients with a history of infertility were compared with the clinical and histological findings. Azoospermia was the most common and the most rewarding indication for the examination. Samples were also taken from cases with suspected testicular tumours, orchitis, epididymitis, severe oligo- and teratozoospermia, lack of libido and unilateral testicular atrophy. Histological and cytological findings were found to correlate well. Identification of cell types from normal germinal epithelium was relatively easy. No Immediate adverse effects of aspiration were noted. Five normospermic dogs were monitored for two to six months after aspiration, and there were no marked deleterious effects on testicular consistency, testicular histology or semen characteristics. Testicular cytology obtained by fine needle aspiration may, at least to some extent, be used to assist clinical diagnosis, especially In azoospermlc dogs and dogs with palpable changes of testicular tissue.     

Telomerase enzyme activity as a diagnostic tool to distinguish effusions of malignant and benign origin

Telomerase enzyme activity is high in populations of cells that are dividing, and is low or undetectable in quiescent cell populations. Activation of telomerase in tissues that normally lack the capacity for self-renewal is strongly correlated with neoplasia. Telomerase activity can be detected in samples containing very small numbers of cells and studies of human patients suggest that measurement of telomerase activity may be useful for the evaluation of samples that can be obtained in a minimally invasive manner. This study compares the presence or absence of telomerase activity with cytologic evaluation of body cavity effusions, to determine if neoplasia is the underlying cause for the effusion in dogs and cats. Detection of telomerase in effusions was no more sensitive than cytologic evaluation for the identification of underlying neoplasia, and was less specific (telomerase assay: sensitivity = 50%, specificity = 83%; cytology: sensitivity = 50%, specificity = 100%). We conclude that although the telomerase assay may constitute a useful adjunctive test for the diagnosis of neoplasia in some dogs and cats with body cavity effusions, the results of this assay are not sufficiently reliable to be used as a sole diagnostic test.     

Urinary alkaloid excretion as a diagnostic tool for fescue toxicosis in cattle.

Fescue toxicosis research studies have often included serum prolactin as a physiologic index of the disorder. Serum prolactin has not been used as a clinical measure of fescue toxicosis because of variation associated with sex and physiologic condition of the animal and climatic and seasonal factors. The primary excretory route of the alkaloids responsible for this toxicosis is the urine. Three pasture experiments were conducted to examine serum prolactin and urinary ergot alkaloid variability among steers continuously grazing endophyte-infected (E+) or endophyte-free (E-) tall fescue and among steers that were switched from one pasture form to the other. A fourth grazing experiment was used to examine how to best to manage the steers prior to sampling for urinary ergot alkaloid excretion. Coefficients of variability for urinary alkaloid excretion were consistently lower (46-65%) than serum prolactin (64-142%). Urinary alkaloid excretion patterns changed within 12 hours following switching steers from E+ to E- pasture or vice versa, but serum prolactin was recalcitrant to change. Because it is less variable and more dynamic than serum prolactin, urinary alkaloid excretion can be used for health assessment of steers grazing E+ and E- pastures. Regression analysis established a quadratic relationship between alkaloid excretion and average daily weight gain, with a regression coefficient of 0.86. Urinary alkaloid analysis was useful in determining whether cattle were consuming toxic tall fescue.     

Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool

In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus is no longer available. The sensitivity was improved by combining cycling optimization and visualization of PCR products in ethidium bromide and silver-stained acrylamide gels.     

Evaluation of nasotracheal aspiration as a diagnostic tool for Rhodococcus equi pneumonia in foals

The reliability of preparing bacteriological cultures from nasotracheal aspirates of foals routinely in order to diagnose R. equi pneumonia in foals was studied by isolating Rhodococcus equi from specimens obtained from 96 foals by nasotracheal aspiration with a silicon catheter. Results were compared with specimens obtained from 21 foals by transtracheal aspiration (percutaneous tracheal puncture). These 117 foals showed clinical signs of respiratory tract infection at sampling. R. equi was isolated from 14 of 21 (66.7%) specimens by transtracheal aspiration and from 59 of 96 (61.4%) specimens by nasotracheal aspiration, 649 of 655 isolates (99.1%) from the 73 positive specimens were virulent R. equi, and the culture-positive foals were diagnosed as having R. equi pneumonia. To assess the contamination of aspirates by organisms from the nasopharynx, the results of R. equi isolation from nasal swabs obtained from 56 of the 96 foals were compared to those obtained by nasotracheal aspiration from the same foals. R. equi was isolated from 2 of the 56 nasal swabs: one from a tracheal aspirate was positive, and the other was not. These results suggest that the nasotracheal aspiration technique, which is noninvasive and not associated with complications, could be used as an alternative to the transtracheal aspiration method, especially for the diagnosis of R. equi pneumonia in foals.     

Bacteriologic examination of equine fecal flora as a diagnostic tool for equine intestinal clostridiosis

The fecal flora of 56 clinically healthy and 23 sick horses were examined bacteriologically for counts of Clostridium perfringens, molds, coliforms, alpha- and beta-hemolytic streptococci, and microbes belonging to genus Bacillus, as well as for the presence of Salmonella spp. Of the healthy horses, 85.7% had a C perfringens count less than 10(1) colony-forming units/g of feces. Of the healthy horses, lowest counts were found in race-horses. Of the sick horses, equine intestinal clostridiosis was diagnosed in 2 horses with large C perfringens counts (10(4) to 10(7) colony-forming units/g) and with acute diarrhea. The 7 isolates of C perfringens were identified as serotype A. Salmonella spp were not detected from any of the horses. The study indicated that diagnosing equine intestinal clostridiosis based on the determination of the fecal C perfringens count was suitable.     

Impacts of intensification of pastoral agriculture on soils: Current and emerging challenges and implications for future land uses

Abstract

AIM: To find evidence for localisation in the uterus, and fetal infection, of Leptospira spp. in farmed deer in the lower North Island of New Zealand during and shortly after the breeding season.

METHODS: Between February and July 2008, 116 blood samples, 120 kidneys, 120 uteri and 27 fetuses were collected from 120 mixed-age hinds from lines from nine farms, at a deer slaughter premises. Serum samples were tested for antibodies against Leptospira borgpetersenii serovar Hardjo-bovis and Leptospira interrogans serovar Pomona, using the microscopic agglutination test (MAT). For both serovars, a titre of >1:48 was considered positive. Samples from kidneys, uteri and fetal tissue were subjected to bacterial culture, using Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and real-time PCR, using DNA gyrase subunit B gene primers.

RESULTS: Thirty-four of 116 (29.3%) serum samples were positive for serovar Hardjo-bovis, and 13 (11.2%) for serovar Pomona. Seven of 120 kidneys were positive for serovar Hardjo-bovis by culture, and five of these, but no others, were positive by real-time PCR. Of 120 uteri, none was culture- or PCR-positive. None of 27 fetal samples was culture-positive but one was positive by real-time PCR. The dam of the PCR-positive fetus was culture-negative from the kidney, but had an MAT titre of 1:192 for Hardjo-bovis.

CONCLUSIONS: Attempts to isolate Leptospira spp. from the genital tracts and early fetuses of farmed deer were unsuccessful. However, molecular evidence suggested fetal infection in one case. This finding justifies further study of the role of leptospires in the genital tract and fetus and its association with reproductive loss in farmed deer.     

Evaluation of colonoscopic allergen provocation as a diagnostic tool in dogs with proven food hypersensitivity reactions

OBJECTIVE: To evaluate the colonoscopic allergen provocation (COLAP) test as a new tool for the diagnosis of IgE-mediated food allergy. METHODS: Oral food challenges as well as COLAP testing were performed in a colony of nine research dogs with proven immediate-type food allergic reactions. In addition, COLAP was performed in five healthy dogs. RESULTS: When compared with the oral challenge test, COLAP accurately determined 18 of 23 (73 per cent) positive oral challenge reactions (73 per cent) in dogs with food allergies and was negative in the healthy dogs. CLINICAL SIGNIFICANCE: The accuracy of this new test may be higher than that for gastric sensitivity testing. Therefore, COLAP holds promise as a new test to confirm the diagnosis of suspect IgE-mediated food allergy in dogs.     

The use of electromyographic examination as a diagnostic tool and phenytoin sodium as treatment in a case of classic springhalt in a Dutch warmblood horse

This paper describes a case of unilateral stringhalt present for 18 months in the right hind limb of a 4-year-old Warmblood gelding. The only abnormalities detected by electromyography (EMG) were a prolonged insertion activity, fibrillation potentials, and positive waves at rest and enhanced EMG activity in the right lateral digital extensor muscle on muscle contraction. This was interpreted as denervation and hyperirritability of this muscle. Both similarities and differences with Australian stringhalt could be found. As described for horses suffering from Australian stringhalt, phenytoin sodium was administered orally in a dosage ranging from 15 to 9.3 mg/kg body weight in order to try to influence the hyperflexia. Therapeutic effects without side effects could be achieved at plasma concentrations between 5.1 and 9.9 mg/L at a dosage of 12 mg/kg body weight twice daily, which is consistent with data in the literature (5-10 mg/L). The EMG examination seems to help to clarify the aetiology of the classical form of stringhalt, since the only abnormality in this patient was an abnormal electrical activity in the lateral digital extensor muscle. As in Australian stringhalt, in this type of stringhalt phenytoin also relieved the hyperflexion of the tarsus.