Detection of Antinuclear Antibodies in Canine Serum by Immunoperoxidase in MDCK and Indirect Immunofluorescence in HEp2 Cells

Antinuclear antibodies are found in animals suffering from Systemic Lupus Erythematosus (SLE) and some other diseases, their presence in the blood is determined by antinuclear antibody (ANA) test using indirect immunofluorescence (IF) with HEp2 cells as a substrate. In this work, an immunoperoxidase (IP) assay was developed to evaluate the ANAs in canine sera, using canine kidney cell lines (MDCK) and compared with a commercial immunofluorescence test on Hep2 cells for this system, a fluoresceinated anti-canine Ig antibody was standardized. The study was performed on 50 sera from dogs submitted to the laboratory with different clinical diagnoses of autoimmune-associated diseases. The procedures on both cells were unified to perform comparisons of the reactions, direct sera or at different dilutions were added to a monolayer of permeabilized MDCK cells, followed by a peroxidized anti-canine IgG conjugate, and a substrate for the IP reaction. The same sera were tested on the commercial IF assay on Hep2 cell system. In 22/50 cases, the presence of LE cells in peripheral blood was determined. A high correlation was found in the detection of antinuclear antibodies between both cell lines and techniques, however there were differences in the reaction patterns in the nucleus and cytoplasm between cell lines. The diffuse nuclear pattern observed in MDCK cells was more related to the presence of high percentages of LE cells in peripheral blood. The differences found in the results were possibly associated with the presence of homologous antigens between the MDCK cells and the dog. In addition, the methodology and standardization for the use and interpretation of a reference serum was developed to unify the interpretation criteria in the laboratory.

Stimulation of Phagocytosis and Production of Antibodies against Canine Herpesvirus Type 1 by Pidotimod (Adimod^TM)

Neutrophils are the most important circulating phagocytes. Circulating mono-cytes and precursors of tissue macrophages also have the ability to phagocytize. Pidotimod (ADIMODTM) exerts immunostimulatory and immunoregulatory effects through the stimulation and regulation of cellular immune responses by lymphocytes Canine herpesvirus (CHV) mainly affect puppies between the first and second weeks of age, causing high morbidity in the litter. To date, there is only one commercial vaccine in Europe to prevent disease. In this work, inactivated CHV cultures were inoculated in rabbits, adsorbed and not adsorbed to chitosan nanoparticles. Phagocytosis in the presence or absence of specific antibodies was measured. Response of virus neutralizing antibodies was also evaluated. AdimodTM enhanced the nonspecific and specific phagocytotic response. The association of the virus to the nanoparticles increased the phagocytic ability of blood cells;however, AdimodTM alone had a greater effect on phagocytic activity and generated a stronger immune response that corresponded to the increased phagocytosis (p TM was used.

Canine Herpesvirus Seroprevalence and Associated Factors in Dogs of Mexico

Canine herpesvirus (CHV-1) causes disease associated with high mortality in infect-ed puppies, which represents large financial losses for dog breeders. Since CHV-1 at the time of the study he had not been reported in Mexico, the main objective of this study was to determine the prevalence of antibodies against CHV-1 in canine kennels in the metropolitan area of Mexico City. A commercial enzyme-linked immuno-sorbent assay (ELISA) was used, and the results were compared to those of a viral neutralization test. The ELISA kit uses the complete viral particle as the antigen. The plaque reduction neutralization test was combined with the immunoperoxidase technique because of the low cytopathic effect of CHV-1. Neutralizing antibodies were also detected in 20 randomly selected samples. The prevalence of CHV-1 with ELISA was 87%. The concordance between ELISA and serum neutralization (SN) was 0.1129, the sensitivity of the ELISA against SN was 1.0 (100%), the positive predic-tive value was 0.39 (39%), and the negative predictive value was 1 (100%). These results show that ELISA is useful for monitoring the dog population for CHV-1;a positive test result requires confirmation with an SN test, and a negative ELISA result indicates a high probability of being SN-negative. The only variables that were sta-tistically associated with CHV-1 prevalence were breed and kennel. A statistically significant relationship between the degree of ELISA and SN titer was obtained, with a confidence level of 95%. None of the clinical presentation factors was statistically significant. These results suggest that most of the canine population studied in Mex-ico is in a herpesvirus latency state.

Pathology Isolation and Identification of Canine Herpesvirus (CHV-1) in Mexico

This work presents the pathology description, isolation and identification of canine herpesvirus (CHV-1) in Mexico, a virus that causes a generalized hemorrhagic infection in puppies from the canidae family. Methods: Isolates were obtained from puppies that died within the first four weeks of life and had lesions consistent with canine herpesvirus. Results: The main gross lesions were petechial and ecchymotic hemorrhages in kidneys, liver and lungs;proliferative interstitial nephritis;multifocal necrosis in liver and kidneys;and encephalitis with intranuclear inclusion bodies. Herpesvirus was confirmed through direct immunofluorescence, electron microscopy and polymerase chain reaction for DNA polymerase and glycoprotein B genes. Discussion: Eight strains were isolated and identified as canine herpesvirus corresponding to three of the working cases with gross and microscopic lesions very similar to those described in the literature;then, isolates were confirmed by PCR gene amplification, positive reactions on immunofluorescence and observations from electron microscopy. This work represents the first report of this disease, including gross and histological lesions, and confirmation by isolation and identification of the canine herpesvirus in Mexico.