Detection of Leishmania infantum in Dogs by PCR with Lymph Node Aspirates and Blood

Abstract

The PCR technique was applied to the diagnosis of leishmaniasis in dogs, both serologically negative and positive. DNA was taken from lymph node aspirates and blood. The primers 13a and 13b, derived fromLeishmania amazonies and Leishmania braziliensis kinetoplast DNA (kDNA), also amplifiedLeishmania infantum IPT1 constant region of minicircle kDNA. The amplified fragment is 116 bp long. It was cloned and the sequence was determined. A 70-bp inner fragment was designed and used as a probe in dot blot hybridization. A group of 124 dogs was examined, 37 of which showed typical symptoms of disease. PCR was performed on 124 blood samples and 52 lymph node aspirates. Using microscopic examination as the “gold standard,” we calculated sensitivity, specificity, and positive and negative predictive values of 100% using lymph node aspirates and values of 85, 80, 95, and 57%, respectively, using blood samples. We found that 40% of the animals without lesions and 38% of the animals with clinical signs gave false-negative results by indirect immunofluorescence antibody testing. These animals could contribute to the spreading of infection among dogs, and represent a potential risk for human health as well.

Utility of Diagnostic Tests Used in Diagnosis of Infection in Dogs Experimentally Inoculated with a North American Isolate of Leishmania infantum infantum

“…False-negative results of up to 38% have been reported in chronically infected dogs and in numbers of dogs unable to mount an immune response to infection. 22 In the United States, a broad range of Leishmania antibody activity in Foxhounds and other dogs has been reported. 3,4 Serological examinations of dogs experimentally infected with LIVT-1 also have yielded erratic results when tested by IFAT.…”

Atypical presentation of Old‐World cutaneous leishmaniasis, diagnosis and species identification by PCR

“…Specific PCR for direct diagnosis was performed by using the forward and reverse primers 13 A (GTG GGG GAG GGG CGT TCT) and 13B (ATT TTA CAC CAA CCC CCA GTT) to amplify the 120-bp conserved region of the Leishmania kinetoplast minicircle. 13 Species-specific primers, LINR4 (forward, GGG GTT GGT GTA AAA TAG GG) and LIN17 (reverse, TTT GAA CGG GAT TTC TG), were carried out as described by Aransay et al . to amplify the variable region of the Leishmania KDNA, the length of which varies in different Leishmania species.…”

Prevalence of Leishmania infantum and co-infections in stray cats in northern Italy

“…The rPCR assay targeted a 123bp fragment within the constant region of the mini-circle kinetoplast DNA (kDNA) (NCBI accession no. AF291093) and was carried out as previously described [21]. The following primers were used: QLK2-U 5 -GGCGTTCTGCGAAAACCG-3 and QLK2-D5 -AAAATGGCATTTTCGGGCC-3 .…”