Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations

Three serological methods, indirect fluorescent immunoassay (IFI), enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT) that are commonly employed in the diagnosis of canine visceral leishmaniasis (CVL), have been assessed. A total of 234 domestic dogs, drawn from an area in the municipality of Belo Horizonte, Minas Gerais, Brazil, endemic for visceral leishmaniasis, were submitted to clinical and parasitological examinations and serological assay. Sera collected from confirmed non-infected dogs (n=20), and from dogs with other parasitic diseases including Trypanosoma cruzi (n=7), Leishmania braziliensis (n=5), Toxoplasma gondii (n=5) and Ehrlichia canis (n=3), were also included in the study. IFI presented a lower sensitivity (72%) than ELISA (95%), although the specificities of these assays were low (52 and 64%, respectively) and both exhibited cross-reactivity with sera from dogs infected with T. cruzi, L. braziliensis and E. canis. In contrast, DAT exhibited a high sensitivity (93%) and a high specificity (95%) and cross-reacted with only one serum sample derived from an E. canis-infected dog. The reproducibilities of the ELISA and DAT assays were excellent, whilst that of IFI was considered to be acceptable. The results produced by ELISA and DAT were in complete agreement, those between ELISA and IFI were at an acceptable level of agreement, whilst the concurrence between the IFI and DAT results were either acceptable or poor depending on the clinical conditions of the group of dogs examined. Since there is no readily accessible method for the diagnosis of CVL that offers 100% specificity and sensitivity, the choice of technique employed must depend on the aim of the investigation.     

A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.     

Evaluation of agreement between numerical rating scales, visual analogue scoring scales, and force plate gait analysis in dogs

Objective— To evaluate the accuracy of numerical rating (NRS) and visual analogue (VAS) scoring scales compared with force plate gait analysis and agreement between observers for each scoring scale.
Study Design— Experimental study.
Animals— Mixed breed dogs (n=21) with a right limb tibial osteotomy repaired with an external fixator.
Methods— Three small-animal veterinarians with orthopedic training scored lameness using NRS and VAS before surgery, and at 4 and 8 weeks after surgery. Peak force and impulse were determined at the same time points using a force plate. Agreement between observers and with force plate data was assessed. Significance was set at P ≤.05.
Results— Agreement was generally low among observers for both NRS and VAS scores. When evaluated at each time point, an acceptable level of agreement was present only at 4 weeks after surgery. Only impulse had a significant relationship with some of the observers' subjective scores. No significant relationships between any observer's scores and force plate data existed if very lame dogs were omitted.
Conclusions— Subjective scoring scales do not replace force plate gait analysis. Agreement is low unless lameness is severe, and each observer uses an individually unique scale. Subjective scoring scales most accurately reflect force plate gait analysis when lameness is severe.
Clinical Relevance— Subjective lameness scoring scales may not accurately reflect lameness and do not replace force plate gait analysis. Observers must stay the same during the duration of a study for accurate analyses.     

Cytoarchitecture, morphology, and lumbosacral spinal cord projections of the red nucleus in cattle

OBJECTIVE: To analyze the morphology, cytoarchitecture, and lumbosacral spinal cord projections of the red nucleus (RN) in cattle. ANIMALS: 8 healthy Friesian male calves. PROCEDURES: Anesthetized calves underwent a dorsal laminectomy at L5. Eight bilateral injections (lateral to the midline) of the neuronal retrograde fluorescent tracer fast blue (FB) were administered into the exposed lumbosacral portion of the spinal cord. A postsurgical calf survival time of 38 to 55 days was used. Following euthanasia, the midbrain and the L5-S2 spinal cord segments were removed. Nissl's method of staining was applied on paraffin-embedded and frozen sections of the midbrain. RESULTS: The mean length of the RN from the caudal to cranial end ranged from 6,680 to 8,640 microm. The magnocellular and parvicellular components of the RN were intermixed throughout the nucleus, but the former predominate at the caudal portion of the nucleus and the latter at the cranial portion with a gradual transitional zone. The FB-labeled neurons were found along the entire craniocaudal extension of the nucleus, mainly in its ventrolateral part. The number of FB-labeled neurons was determined in 4 calves, ranging from 191 to 1,469 (mean, 465). The mean cross-sectional area of the FB-labeled neurons was approximately 1,680 microm2. CONCLUSIONS AND CLINICAL RELEVANCE: In cattle, small, medium, and large RN neurons, located along the entire craniocaudal extension of the RN, contribute to the rubrospinal tract reaching the L6-S1 spinal cord segments. Thus, in cattle, as has been shown in cats, the RN parvicellular population also projects to the spinal cord.     

Determination of tumor necrosis factor-alpha responsiveness in piglets around weaning using an ex vivo whole blood stimulation assay

Ex vivo whole blood stimulation with endotoxin has proved to be a useful method for quantitative evaluations of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) response ability in humans. In the present study, a dose- and time-response study was carried out in order to develop an ex vivo whole blood stimulation assay for the quantification of TNF-alpha production in pigs. The TNF-alpha response was enhanced with increasing endotoxin stimulation dose. The maximum TNF-alpha response occurred after 2-8 h of stimulation. Subsequently, the assay was used to evaluate the TNF-alpha response in pigs (n=32) in relation to weaning. The TNF-alpha response was 332 pg ml(-1) (+/-59 pg) plasma 2 days before weaning and 127 pg ml(-1) (+/-23 pg) plasma 2 days after weaning, which was a significant reduction (p<0.001). Total and differential counts of leucocytes were the same before and after weaning. Thus the lower TNF-alpha response may be due to reduced monocyte responsiveness to the endotoxin, rather than caused by a reduction in monocyte numbers. The reduced TNF-alpha response in piglets after weaning may be a factor of importance to the increased disease susceptibility seen in piglets in this period.     

Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium-intracellulare complex (MAC)

Pathogens that are transmitted between the environment, wildlife, livestock and humans represent major challenges for the protection of human and domestic animal health, the economic sustainability of agriculture, and the conservation of wildlife. Among such pathogens, the genus Mycobacterium is well represented by M. bovis, the etiological agent of bovine tuberculosis, M. avium ssp. paratuberculosis (Map) the etiological agent of Johne disease, M. avium ssp. avium (Maa) and in a few common cases by other emergent environmental mycobacteria. Epidemiologic surveys performed in Europe, North America and New Zealand have demonstrated the existence and importance of environmental and wildlife reservoirs of mycobacterial infections that limit the attempts of disease control programmes. The aim of this review is to examine the zoonotic aspects of mycobacteria transmitted from the environment and wildlife. This work is focused on the species of two main groups of mycobacteria classified as important pathogens for humans and animals: first, M. bovis, the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, captive wildlife, domestic livestock, non-human primates and humans; the second group examined, is the M. avium-intracellulare complex (MAC) which includes M. avium ssp. avium causing major health problems in AIDS patients and M. avium ssp. paratuberculosis the etiological agent of Johne disease in cattle and identified in patients with Crohn disease. MAC agents, in addition to a broad host range, are environmental mycobacteria found in numerous biotopes including the soil, water, aerosols, protozoa, deep litter and fresh tropical vegetation. This review examines the possible reservoirs of these pathogens in the environment and in wildlife, their role as sources of infection in humans and animals and their health impact on humans. The possibilities of control and management programmes for these mycobacterial infections are examined with regards to the importance of their natural reservoirs.     

Cloning and expression of pigeon IFN-γ gene

This is the first paper describing the cloning of pigeon IFN-γ gene (PiIFN-γ) and the analysis of the in vitro expressed recombinant protein. The PiIFN-γ gene was identified by RT-PCR as a 498 bp, fragment coding for a precursor protein of 165 amino acids instead of 164 amino acids, as observed in the other avian species. The recombinant protein was expressed in vitro by an eukaryotic system and the biological properties of the cytokine were tested using a chicken macrophage cell line. The high degree of amino acid and nucleotide identity, shared with the ChIFN-γ, and the fact that the pigeon protein was functional on chicken cells, indicates a cross-reactivity between pigeon and chicken IFN-γ. The detection of the PiIFN-γ could represent an useful instrument in understanding the role played by this cytokine in immune response related to vaccinations and infectious diseases in the pigeon.     

NECROTIZING ENCEPHALITIS IN A YORKSHIRE TERRIER: CLINICAL, IMAGING, AND PATHOLOGIC FINDINGS

A 5-year-old, male Yorkshire Terrier had chronic and progressive neurologic signs compatible with lesions in the right brain stem and right forebrain. In magnetic resonance images of the brain there were multifocal lesions at different stages of evolution, consistent with an inflammatory disease. The lesions were located in the white matter of the cerebrum, in the diencephalon and mesencephalon. These lesions were hypointense in T1-weighted images and hyperintense in T2-weighted images with no mass effect and minimal enhancement with gadolinium. Necrotizing encephalitis was confirmed post mortem.     

Histopathological findings in the early diagnosis of granulosa cell tumour in bitches

Granulosa cell tumour (GCT) is a majorly observed ovarian tumour in female dogs. It is essential to diagnose GCT in its initial phase before any symptoms occur, as histological and physiological differences may be observed based on the evolution of this neoplasia. This study aimed to analyse the anatomic histopathology of GCT in its initial stage, with findings of ovaries not yet with the suspicion of neoplasms in the Canis familiaris. A sample including 55 ovaries presented GCT in 40 female dogs. The histopathological analysis was performed considering the intensity of pleomorphism, vascularization and inflammatory infiltrate. Furthermore, we evaluated the mitoses count in 10 fields using 40× magnification. Out of the 40 animals evaluated, 62.5% (25/40) presented the tumour in only one ovary. The Call‐Exner corpuscle was present in 65% (26/40) of the cases. The follicular histological pattern was present in 52.5% (21/40) of the animals. The presence of the Call‐Exner bodies and the degree of tumour cell pleomorphism (p = 0.033) were associated. Moreover, the degree of vascularization and the intensity of the inflammatory infiltrate were also related (p = 0.001). In addition, there was a positive relationship between the increase in pleomorphism and the mean age of the animals (p = 0.044). This study confirmed that the appearance of this tumour may precede any clinical symptomatology. In this study, the most frequent histopathological pattern was the follicular. The characteristics of the granulosa cell tumour diagnosed early were poorly pleomorphic cells, low mitotic index and presence of Call‐Exner body.