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      Detection of Leishmania infantum DNA in the non-parasitized lung of dogs with visceral leishmaniasis

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          Abstract

          Background

          Despite the very low or absent parasitism in the lungs, the interstitial pneumonitis is a common lesion found in humans and dogs with visceral leishmaniasis. The lung is a neglected organ in the study of dogs and humans with visceral leishmaniasis, but interstitial pneumonitis represents an important lesion characterized by thickening of the alveolar septum due to fibrosis and inflammatory exudate, and its pathogenesis is still uncertain. In this study, the polymerase chain reaction (PCR) was used to detect Leishmania infantum in paraffin-embedded lung biopsies from naturally infected dogs from an endemic area in Minas Gerais State, Brazil; PCR was compared to histological and immunohistochemical techniques for detecting Leishmania.

          Results

          Eighteen dogs in which leishmaniasis had been diagnosed by serological tests - indirect immunofluorescence assay (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation tests (CFT) - were classified as asymptomatic, oligosymptomatic or symptomatic. Nine of the 18 dogs studied had a positive PCR (50%) but parasites were not detected by histopathological and immunocytochemistry methods.

          Conclusions

          These data indicate that PCR on DNA extracted from paraffin-embedded tissue is a valuable method for detecting Leishmania infantum parasites in lungs of naturally infected dogs, despite the apparent absence of parasites from standard HE (hematoxylin and eosin) stained slides and of labeled parasites from immunocytochemical preparations.

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          Most cited references18

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          Purification of DNA from formaldehyde fixed and paraffin embedded human tissue.

          The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.
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            An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues.

            Canine visceral leishmaniasis (CVL) is a zoonosis and a chronic systemic disease of the dog caused by a protozoan by the species Leishmania infantum in the Old World and Leishmania chagasi in the New World. Several methods are currently employed for the diagnosis of CVL including microscopic detection of the parasite in bone marrow and lymph node aspirates, demonstration of specific antibodies anti-Leishmania in sera from infected animals, and isolation of the parasite by in vitro culture or by inoculation of laboratory animals. However, a definitive diagnosis is based on the actual detection of the parasite, which is conventionally achieved by examining Giemsa-stained smears or histopathological sections stained with hematoxylin and eosin. These methods have a low sensitivity, and therefore, they are often inconclusive. This is particularly true in canine organs that have a low level of parasitism such as kidneys, lungs, central nervous system, and testis, or, in some cases, the skin. The technique for immunohistochemical detection of leishmanial amastigotes in canine tissues has been reported previously and has proved to be undoubtedly efficient for the diagnosis. In this paper, we describe a straightforward and inexpensive immunohistochemical approach for Leishmania detection in formalin-fixed paraffin-embedded canine tissues. Amastigote forms of Leishmania were easily observed within macrophages in several organs from naturally infected dogs using the streptavidin-biotin immunohistochemical method with canine hyperimmune serum as the primary antibody. In addition, the secondary antibody used was not specific to canine immunoglobulin, characterizing a cross-immune reaction. Our results indicate that this technique could be a useful tool for epidemiological, clinical, and histopathological studies.
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              Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides.

              The "polymerase chain reaction" (PCR) procedure for amplifying specific gene sequences has recently been combined with sequence-specific oligonucleotide (SSO) probe hybridization to develop a highly sensitive, rapid, and simple method for analyzing allelic variations in genomic DNA. In the present study we have used PCR/SSO to analyze partially purified DNA extracted from formalin-fixed, paraffin-embedded tissue specimens. We report that this DNA, including samples that were partially degraded, proved to be suitable for analysis by the PCR/SSO procedure.
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                Author and article information

                Contributors
                + 55 31 3499-3037 , ricardogoncalves2007@gmail.com
                silvaso@icb.ufmg.br
                vet.greg@gmail.com
                carolinacarvalhos@gmail.com
                wagnertafuri@gmail.com
                melo@icb.ufmg.br
                Journal
                BMC Vet Res
                BMC Vet. Res
                BMC Veterinary Research
                BioMed Central (London )
                1746-6148
                17 December 2018
                17 December 2018
                2018
                : 14
                : 403
                Affiliations
                [1 ]ISNI 0000 0001 2181 4888, GRID grid.8430.f, Departamento de Patologia Geral, Instituto de Ciências Biológicas, , Universidade Federal de Minas Gerais, ; Av Antonio Carlos 6627, Belo Horizonte, MG CEP31270-901 Brazil
                [2 ]ISNI 0000 0001 2181 4888, GRID grid.8430.f, Departamento de Parasitologia, Instituto de Ciências Biológicas, , Universidade Federal de Minas Gerais, ; Av Antonio Carlos 6627, Belo Horizonte, MG CEP31270-901 Brazil
                Article
                1730
                10.1186/s12917-018-1730-7
                6296075
                0eaba9e1-8164-48b7-8ca8-2041cd4d276b
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 24 May 2018
                : 3 December 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100004901, Fundação de Amparo à Pesquisa do Estado de Minas Gerais;
                Funded by: FundRef http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Funded by: FundRef http://dx.doi.org/10.13039/501100002322, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior;
                Funded by: FundRef http://dx.doi.org/10.13039/501100007375, Pró-Reitoria de Pesquisa, Universidade Federal de Minas Gerais;
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2018

                Veterinary medicine
                leishmania infantum,leishmania (leishmania) chagasi,pcr,dog,histopathology,immunocytochemistry

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