Development of Immunological Assays Based on Leishmania donovani Antigen for Diagnosis of Canine Visceral Leishmaniasis and Their Multicenter Evaluation in Brazil and Italy.

Canine visceral leishmaniasis (CVL) due to Leishmania infantum infection is a zoonotic disease prevalent in the areas of South America and the Mediterranean. Infected dogs as reservoirs can contribute to disease transmission and can be a scourge to public health. Therefore, early diagnosis of infected dogs may play a pivotal role in circumscribing disease progression. Invasive tissue aspiration and insufficient serological methods impair a single assay for prompt CVL diagnosis. In the present study, we aimed to evaluate the potential of Leishmania donovani isolated membrane protein, LAg, for the diagnosis of CVL through immunological assays. Initially, enzyme-linked immunosorbent assay was done with Brazilian dog sera to evaluate the performance of LAg in diagnosing CVL and found sensitivity and specificity of 92.50% and 95%, respectively. The study further confirmed the diagnostic efficacy of LAg in a dipstick format. The dipstick test of canine sera from three centers in Brazil and one center in Italy collectively showed sensitivity values in the range of 53.33% to 100% in recognizing symptomatic dogs and specificity values between 75% and 100% to rule out healthy dogs. Moreover, a rapid immunochromatographic test was developed and optimized using LAg. This test was able to identify 94.73% of CVL of Brazilian origin with specificity of 97.29%. The current results highlight the reactive potential of the L. donovani antigen, LAg, for L. infantum CVL diagnosis and support our previous findings, which suggest the utility of LAg for the diagnosis of both L. donovani and L. infantum human VL in a variety of endemic regions. LAg as a diagnostic candidate may be employed to identify comprehensive CVL cases in epidemiological areas.

Microbiological, molecular, and histopathological findings in goats experimentally infected with Actinobacillus seminis.

The objective of this study was to experimentally evaluate the pathogenicity of an Actinobacillus seminis isolate named SAAS01 in goats. Animals were challenged with 2 mL of a suspension containing 1,5 × 108 CFU/mL of A. seminis (SAAS01 isolate) through the intrapreputial, epididymis tail, and conjunctival routes. Epididymis and testicular fragments were submitted to histopathological exam, and semen samples underwent microbiological and molecular diagnoses. Clinically, a unilateral increase in firm consistency was observed in the epididymis and testicles of two animals inoculated in epididymis tail and in one animal inoculated through conjunctival sac; this firmness continued until the day of euthanasia. Two goats inoculated through epididymis tail and conjunctival sac routes presented histopathological findings with macroscopically and microscopically significant changes. A. seminis was isolated from semen samples collected from goats inoculated through the epididymis tail and conjunctival sac routes. A. seminis DNA was amplified from six semen samples of three goats inoculated through the epididymis tail, two in conjunctival sac and one through intrapreputial route. The experimental infection model using goats confirmed the pathogenicity of the A. seminis isolate, demonstrating the predilection of the agent for the epididymis, with clinical signs, histopathological lesions, bacterial isolation, and a positive molecular diagnosis.

Survey of Ehrlichia canis, Babesia spp. and Hepatozoon spp. in dogs from a semiarid region of Brazil.

This study assessed the occurrence of Ehrlichia spp., Babesia spp. and Hepatozoon spp. infections in 100 tick-harboring dogs from a semiarid region of the State of Paraíba, Northeastern Brazil. Blood samples and ticks were collected from the animals, and a questionnaire was submitted to dog owners to obtain general data. Blood samples were used to perform hemogram, direct blood smear and immunological and molecular hemoparasite detection. The 1,151 ticks collected were identified as Rhipicephalus sanguineus; direct smears revealed E. canis-like morulae in the monocytes of 4% (4/100) of the non-vaccinated female dogs, and 34% and 25% of the dogs tested positive for Ehrlichia canis by indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR), respectively. Blood smear examination revealed Babesia-suggestive merozoites in the erythrocytes of 2% (2/100) of the animals. Babesia vogeli was detected by PCR in ten animals (10%) and was correlated with young age (p = 0.007) and thrombocytopenia (p = 0.01). None of the animals showed Hepatozoon spp. positivity. These results indicate that E. canis is the main tick-borne canine pathogen in the study area and provide the first report of B. vogeli infection in dogs from Paraiba State.

An assessment of whole blood and fractions by nested PCR as a DNA source for diagnosing canine ehrlichiosis and anaplasmosis.

Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB) and blood fractions potential in nested PCR (nPCR) to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G), mononuclear cells (M), and buffy coat (BC) samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C). There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.