Molecular investigation of Ehrlichia spp. and Anaplasma platys in domestic cats: clinical signs, hematological and biochemical alterations

Ehrlichia sp. e Anaplasma platys são micro-organismos Gram negativos, parasitos intracelulares obrigatórios, residindo em vacúolos citoplasmáticos de leucócitos e plaquetas, encontrados no sangue periférico ou em tecidos. Poucos relatos têm sido feitos sobre erliquiose e anaplasmose em gatos no Brasil, os quais são baseados na presença de mórulas em leucócitos e plaquetas, ou pela detecção de anticorpos. O objetivo deste trabalho foi investigar a infecção natural por Ehrlichia sp. e A.platys em gatos no Município de Campos dos Goytacazes-RJ, através da hematoscopia e pela detecção do DNA desses agentes. Foram utilizadas amostras de sangue total e de soro de 91 gatos, independente de raça, sexo e idade. Realizaram-se hemograma, bioquímica sérica e PCR, utilizando oligonucleotídes para Ehrlichia sp. e A.platys. Os dados de hematoscopia mostraram que 9,89% dos gatos apresentaram mórulas em macroplaquetas. O DNA de A.platys foi detectado em 13,18% dos 91 animais e em 44,44% das amostras positivas à hematoscopia. O DNA de Ehrlichia sp. não foi detectado em nenhuma amostra. Nenhuma alteração foi observada nos sinais clínicos nem nos resultados laboratoriais nos animais estudados. Os dados sugerem que os felinos domésticos podem atuar como potenciais reservatórios para A. platys, como forma não sintomática das enfermidades relacionadasEhrlichia sp. and Anaplasma platys are Gram-negative micro-organisms, obligate intracellular parasites, residing in cytoplasmic vacuoles of leukocytes and platelets, found in peripheral blood or tissue. Few reports have been made about ehrlichiosis and anaplasmosis in cats in Brazil, which are based on the presence of morulae in leukocytes and platelets, or by detecting antibodies. The objective of this study was to investigate the natural infection with Ehrlichia sp. and A. platys in cats in Campos dos Goytacazes-RJ, by hematoscopia and DNA detection of these agents. Samples of whole blood and serum from 91 cats, regardless of race, gender and age. Blood count, serum biochemistry and PCR using primers for Ehrlichia sp. and A. platys were perfomed. Data from hematoscopia showed 9.89% of morulae only in platelets. The DNA of A. platys was detected in 13.18% of the 91 samples and 44.44% of the positive at hematoscopia. The DNA of Ehrlichia sp. was not detected in any sample. All animals studied did not show clinical signs neither positive laboratory results. The data suggest that domestic cats can serve as potential reservoirs for A. platys as asymptomatic form of related disease

miR-20a is upregulated in serum from domestic feline with PKD1 mutation.

Polycystic kidney disease (PKD), also known as autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous condition characterized by cysts in renal parenchyma. It is the most prevalent inherited disease of domestic cats. MicroRNAs (miRNAs or ncRNA) are short, noncoding, single-stranded RNAs that may induce PKD cytogenesis by affecting numerous targets genes as well as by directly regulating PKD gene expression. We compared the relative expression profile of miR-20a, -192, -365, -15b-5p, and -16-5p from plasma and serum samples of nine domestic cats with PKD1 mutation, detected by polymerase chain reaction (PCR), and a control group (n = 10). Blood samples from cats with PKD1 mutation provide similar concentrations of microRNAs either from plasma or serum. Serum miR-20a is upregulated in PKD group with p < 0.005; Roc curve analysis showed an AUC of 90,1% with a cut-off value sensitivity of 77.8% and specificity of 100%. This data provides important information regarding renal miRNA expression in peripheral blood sampling

Fold change of serum microRNAs (miR-20a, miR-192, miR-365, miR-16-5p, miR-15b-5p) from domestic cat.

RT-rtPCR assays. Normalized with cel-miR-39-3p. miR-20a is upregulated in PKD group *(p p values were p > 0.05.</p

Extraction protocol for miRNA.

(PDF)</p

Cycle threshold values (mean ± SEM) for microRNA derived from feline plasma and serum samples of the PKD group.

Cycle threshold values (mean ± SEM) for microRNA derived from feline plasma and serum samples of the PKD group.</p

microRNAs, stem-loop RT primers, mature microRNA sequences, and physiologic or pathologic correlation [8].

microRNAs, stem-loop RT primers, mature microRNA sequences, and physiologic or pathologic correlation [8].</p

Clinical parameters (blood count, biochemical and urinalysis) of individual samples.

Hematocrit (HCT), Red Blood Cell (RBC), Hemoglobin (HGB), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration (MCHC), White blood cell (WBC), Blood Urea Nitrogen (BUN), Phosphorus (P), Potassium (K), Symmetric dimethilarginine (SDMA), Urine Protein/Creatinine Ratio (UPCR), Gamma-Glutamyl Transpeptidase (GGT). (PDF)</p

Reverse transcription protocol for miRNA.

(PDF)</p

Mean, standard deviation (SD) and standard error of the mean (SE) from clinical parameters (blood count, biochemical and urinalysis) of PKD group.

Where Hematocrit (HCT), Red Blood Cell (RBC), Hemoglobin (HGB), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration (MCHC), White blood cell (WBC), Blood Urea Nitrogen (BUN), Phosphorus (P), Potassium (K), Symmetric dimethilarginine (SDMA), Urine Protein/Creatinine Ratio (UPCR), Gamma-Glutamyl Transpeptidase (GGT). (PDF)</p

Real time PCR protocol for miRNA.

(PDF)</p