Production and characterization of a panel of monoclonal antibodies for the identification of antigens of Babesia bovis

A panel of monoclonal antibodies was produced and characterized by an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and Western blotting with the aim of identifying antigens of Babesia bovis. After fusion, the resultant hybrids were selected by the IFAT, cloned, maintained in culture in vitro, and cryopreserved in liquid nitrogen. Ten clones producing monoclonal antibodies were found to react against the entire merozoites, three reacted on the surface of the merozoites, and one clone reacted against the polar region of the merozoites. All monoclonal antibodies reacted in ELISA, with the optical density varying from 0.368 to 0.502 (cut off = 0.022). The bands recognized by the monoclonal antibodies in Western blotting had molecular weights ranging from 162 to 19 kDa. Four clones recognized a single band of 73 kDa, and another four did not react in Western blotting.     

A pre-synchronization program at early postpartum might increase the chances of Bos indicus cows cycling prior to 50 days regardless of the length of calf separation

The aim of this study was to establish if pre-synchronization would enhance the number of animals cycling prior to conventional breeding at 45 days irrespective of the length of calf separation. Multiparous Bos indicus cows were allotted in four groups (n = 10). Control group (C) dams remained with their calves; groups G24, G48 and G72, which were partially weaned for 24, 48 and 72 h, respectively, were estrus synchronized using a controlled internal drug. These procedures were performed at 25 days and again at 45 days postpartum. The number of follicles, presence of a corpus luteum and back fat thickness (BFT) were determined by ultrasound. The proportion of cows with estrus and ovulation at day 25 postpartum was statistically different between the control and treated groups, with the values being 20, 60, 50 and 70 for the control, G24, G48 and G72 groups respectively (P < 0.05). At days 45 postpartum, the proportion of cows with estrus and ovulation was different in group G48 compared with the other groups (P <0.05). The average BFT and body condition score for the four experimental groups in the two periods were similar (P >0.05). Animals with a higher proportion of follicles from 17 to 21 mm, BFT values above 3.5 mm and a regular body condition were significantly different regardless of whether the dams remained with their calves or were separated, regardless of the length of this event. It can be concluded that (1) a pre-synchronization program at day 25 could trigger the onset of ovarian activity and facilitate a breeding program at day 50 and (2) temporary weaning enhances the effect of a pre-synchronization program.     

Microtomographic characterization of calcifications in canine mammary tumours

The present work describes the microtomographic characterization of macro‐ and microcalcifications present in excised canine mammary glands. In human breast cancer, microcalcifications are highly relevant for diagnosis and prognosis, often being the sole element determining biopsy. Canine mammary tumours are considered a model for human breast cancer, but the morphological features of calcifications had still to be studied in this species. The objective of this research is to contribute to the characterization of the mineralization features of the canine mammary gland. In the present study, the excised mammary glands of 33 bitches underwent fluoroscopic examination. In 30 of the samples, the presence of calcification was suspected, and multiple biopsies were taken of these areas. Biopsy fragments underwent microtomographic scanning. Microcalcifications were found in non‐neoplastic glandular tissue, benign and malign lesions, as it is known to happen in humans. Qualitative evaluation regarding morphology of the imaged calcifications showed similarities to breast cancer findings, based on the BI‐RADS 2013 classification, such as pleomorphism and shape. No differences in the quantitative morphological parameters of volume, surface, surface/volume, SMI and structure thickness were found when macrocalcifications were considered. However, although significant differences existed in these parameters between microcalcifications from malignant canine mammary tumours and the two other groups, none were found between non‐neoplastic and benign tumours. Findings further support the use of this spontaneous animal model for the study of human breast cancer, considering how clinically relevant microcalcifications are in humans.     

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass (Dicentrarchus labrax) during the course of an experimental challenge

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain (cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.     

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.     

Assessment of regional systolic and diastolic myocardial function using tissue Doppler and strain imaging in dogs with dilated cardiomyopathy

BACKGROUND: Tissue Doppler Imaging (TDI) or strain (St) imaging could provide sensitive indices for early detection and treatment follow-up of canine dilated cardiomyopathy (DCM). Analysis of TDI and St features in dogs with overt DCM is a prerequisite before using these new criteria in prospective screenings of predisposed families or in clinical trials. HYPOTHESIS: Radial and longitudinal right and left myocardial motion, assessed by TDI and St variables, is altered in dogs with DCM. ANIMALS: Case records for 26 dogs; 14 with DCM and 12 healthy controls of comparable age and weight were reviewed. METHODS: A retrospective analysis was conducted of conventional echocardiography, 2-dimensional color TDI, and St imaging data. RESULTS: The DCM group was characterized by decreases in radial and longitudinal systolic velocity gradients of the left ventricular free wall (LVFW), radial and longitudinal absolute values of peak systolic St of the LVFW, and longitudinal systolic right ventricular (RV) velocities (all P < .001 versus control) associated with longitudinal postsystolic contraction waves in 7/14 dogs. Early diastolic LVFW velocities also were decreased for longitudinal (P < .01) and radial (P < .05) motions. All radial LVFW, longitudinal basal LVFW, and RV systolic velocities were negatively correlated with heart rate (P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: LV contractility along both the short and long axes is impaired in dogs with spontaneous DCM, as is systolic RV and diastolic LVFW function. These myocardial alterations are associated with an inverse force-frequency relationship. Studies now are needed to determine the comparative sensitivity of TDI and St variables for the early detection of canine DCM.     

Risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil

From September 1995 to February 2001, blood samples were collected from 865 neotropical felids belonging to 8 different species. These animals were housed in 86 institutions located in 78 cities of 20 Brazilian states. Our goal was to identify the risk factors associated with sero-positivity to Toxoplasma gondii in captive neotropical felids from Brazil. All serum samples were tested by the modified agglutination test (MAT), using formalin-fixed whole tachyzoites and mercaptoethanol. For each animal an individual questionnaire was filled with questions about tattoo number, felid species, age, sex, origin, number of animals in the group, introduction of new animals in the group, time in the institution, eating meat previously frozen for a period <7 days in the last 6 months, eating meat of run-over or euthanized animals in the last 6 months, predation of rodents or birds in the last 6 months and presence of domestic cats near the enclosures in the last 6 months. The total sero-prevalence was 55% (95% CI: 52%, 57%). We estimated a prevalence of 46% (95% CI: 40%, 54%) for jaguarundi (Puma yagouaroundi); 58% (95% CI: 53%, 63%) for ocelot (Leopardus pardalis); 50% (95% CI: 45%, 56%) for oncilla (L. tigrinus); 54% (95% CI: 46%, 62%) for margay (L. wiedii); 12% (95% CI: 4%, 31%) for Pampas-cat (L. colocolo); 83% (95% CI: 65%, 93%) for Geoffroy's-cat (L. geoffroyi); 64% (95% CI: 50%, 68%) for jaguar (Panthera onca) and 48% (95% CI: 42%, 54%) for puma (Puma concolor). Multiple logistic regression was used to evaluate the association between the variables in the questionnaire and sero-positivity to T. gondii. We concluded that the independent risk factors for toxoplasmosis were: age >3 years (OR = 4.75 [2.75; 8.2]), eating meat previously frozen for a period <7 days (OR = 2.23 [1.24; 4.01]), and consumption of animals that were run-over or euthanized (OR = 1.64; [1.14; 2.37]).