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.     

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P₄) (5 ng/ml or 1 μg/ml) or estradiol 17β (E₂β) (50 pg/ml or 1 μg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E₂β (1 μg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.     

Bacterial and fungal pathogens isolated from corneal ulcerations in domesticated elephants (Elephas maximus maximus) in Sri Lanka

Infective corneal diseases are common among the domesticated elephants in Sri Lanka, causing morbidity including blindness leading to loss of revenue to their owners. From 140 elephants of different ages and both sexes, 36 animals (25.7%) had evidence of keratitis, corneal ulcers, corneal opacities, and some had foreign bodies in their eyes. Nine elephants (6.4%) had lesions in both eyes (6.41%). Cultures for both bacteria and fungi were obtained from 26 corneal ulcers, including the nine elephants with bilateral lesions. The other 10 animals could not be restrained for sample collection. Swabs from the normal corneas of an additional 20 elephants without signs of any ophthalmic diseases were also collected. Twenty-three of the 35 (65.71%) samples from affected corneas yielded bacterial pathogens, and 14 (40%) also had fungal isolates. None of them yielded a fungal isolate alone. The predominant bacteria isolated were Staphylococcus aureus , beta hemolytic streptococci and coliforms. Fusarium , Cladosporium , Curvularia and Aspergillus species were the primary fungal isolates. No bacteria or filamentous fungi were isolated from the eyes with the normal corneas. Microbial identification including fungal isolations is suggested in the management of infective corneal diseases in elephants.     

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.     

Subcutaneous administration of paclitaxel in dogs with cancer: A preliminary study

Intravenous paclitaxel has been underused in dogs due to severe and acute hypersensitivity reactions. Subcutaneous (SC) administration of paclitaxel and its safety are unknown. In this preliminary study, SC administration of paclitaxel was evaluated for hypersensitivity reactions and toxicity in 21 dogs with advanced cancer. Dogs received 1 to 5 paclitaxel doses, ranging from 85 to 170 mg/m², SC every 14 or 21 days. A total of 40 paclitaxel doses were administered and none of the 21 dogs developed systemic or acute local hypersensitivity reactions. Severe skin lesions at the injection site developed in 2 dogs after the 4th injection at the same location. Grade 4 neutropenia was observed in 50% of the dogs 5 days after the first treatment at 115 mg/m² (n = 14). Two animals developed Grade 5 diarrhea and died likely due to hemodynamic failure or sepsis. Paclitaxel can be administered SC in dogs with no hypersensitivity reaction.     

Bovine Oocyte Quality in Relation to Ultrastructural Characteristics of Zona Pellucida,Polyspermic Penetration and Developmental Competence

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus–oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP’s pores were evaluated in squares of 6.4‐μm width. In group B, oocytes were fertilized in vitro. After 48 h, non‐cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 ± 0.07 μm, which was smaller than the observed on oocytes of quality 2 (0.83 ± 0.10 μm), quality 3 (1.02 ± 0.22 μm) and quality 4 (1.38 ± 0.59 μm) (p ≤ 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p ≤ 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore’s diameter.     

Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil

Dogs naturally infected with Leishmania Infantum (=L. chagasi) were treated with miltefosine using different therapeutic regimens. The animals were evaluated for clinical evolution, biochemical parameters, parasite load (by real-time PCR), cytokine levels and humoral response. After treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive. In general, all animals presented a significant increase in parasite load 6 months after treatment. The IFN-γ levels in all the groups tended to increase during follow-up period, regardless of the miltefosine dose administered. The IL-4 and IL-10 levels of the animals tended to decrease during follow-up, except after 300 days when only IL-10 increased. The serum antibodies identified antigens that ranged from 116 kDa to less than 29 kDa in the Western blot assay. Furthermore, 300 days after treatment, qualitative and quantitative differences in the antigen profiles were observed. Antigens of 97 and 46 kDa were the most intensely recognized. Higher levels of antigen-specific Leishmania IgG were detected before and 300 days after treatment in all groups. Taking together, the improvement in the clinical symptoms was not followed by parasitological clearance, suggesting that treatment with miltefosine is not recommended, especially in endemic areas like Brazil, where children are the major victims and dogs are involved in the maintenance of the parasite cycle.     

Using BCG,MPT-51 and Ag85 as antigens in an indirect ELISA for the diagnosis of bovine tuberculosis

This study evaluated the Mycobacterium tuberculosis protein antigen MPT-51, the trimeric antigen 85 (Ag85) complex, and Bacillus Calmette-Guérin (BCG) in an indirect ELISA to diagnose bovine tuberculosis (TB) from serum samples. Serum was collected from 208 intra-dermal tuberculin test (ITT)-positive and 54 ITT-negative animals from a region where bovine TB is endemic. Using the Ag85 and BCG antigens, the indirect ELISA was able to discriminate ITT-positive from ITT-negative animals. This level of discrimination was not achieved when using the MPT-51 antigen. The highest sensitivity (Se) and specificity (Sp) of the test was found when BCG was used (Se, 82%; Sp, 91%). Further work in different epidemiological settings and with larger numbers of animals will be required to validate these findings.     

Cone beam computed tomography and intraoral radiography for diagnosis of dental abnormalities in dogs and cats

The development of veterinary dentistry has substantially improved the ability to diagnose canine and feline dental abnormalities. Consequently, examinations previously performed only on humans are now available for small animals, thus improving the diagnostic quality. This has increased the need for technical qualification of veterinary professionals and increased technological investments. This study evaluated the use of cone beam computed tomography and intraoral radiography as complementary exams for diagnosing dental abnormalities in dogs and cats. Cone beam computed tomography was provided faster image acquisition with high image quality, was associated with low ionizing radiation levels, enabled image editing, and reduced the exam duration. Our results showed that radiography was an effective method for dental radiographic examination with low cost and fast execution times, and can be performed during surgical procedures.