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Improving the serodiagnosis of canine Leishmania infantum infection in geographical areas of Brazil with different disease prevalence.

ABSTRACT: Serodiagnosis of Leishmania infantum infection in dogs relies on the detection of antibodies against leishmanial crude extracts or parasitic defined antigens. The expansion of canine leishmaniasis from geographical areas of Brazil in which the infection is endemic to regions in which the disease is emerging is occurring. This fact makes necessary the analysis of the serodiagnostic capabilities of different leishmanial preparations in distinct geographical locations. In this article sera from dogs infected with Leishmania and showing the clinical form of the disease, were collected in three distinct Brazilian States and were tested against soluble leishmanial antigens or seven parasite individual antigens produced as recombinant proteins. We show that the recognition of soluble leishmanial antigens by sera from these animals was influenced by the geographical location of the infected dogs. Efficacy of the diagnosis based on this crude parasite preparation was higher in newly endemic regions when compared with areas of high disease endemicity. We also show that the use of three of the recombinant proteins, namely parasite surface kinetoplastid membrane protein of 11 kDa (KMP-11), and two members of the P protein family (P2a and P0), can improve the degree of sensitivity without adversely affecting the specificity of the diagnostic assays for canine leishmaniasis, independently of the geographical area of residence. In addition, sera from dogs clinically healthy but infected were also assayed with some of the antigen preparations. We demonstrate that the use of these proteins can help to the serodiagnosis of Leishmania infected animals with subclinical infections. Finally, we propose a diagnostic protocol using a combination of KMP-11, P2a y P0, together with total leishmanial extracts.

SUBMITTER: Ramirez L

PROVIDER: S-EPMC6890974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania protozoan and has an important biological role in host-parasite interactions both in the midgut epithelium of the sand fly vector and in the vertebrate macrophages. Canine leishmaniasis (CanL) is a chronic infectious disease predominantly caused by Leishmania infantum. An early and accurate immunodiagnosis of the disease is crucial for veterinary clinical practice and for disease control. In this work, we evaluated L. infantum LPG as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for CanL immunodiagnosis (LPG-ELISA) by testing serum samples from 97 naturally infected dogs with diverse clinical presentations ranging from subclinical infection to severe disease, as evaluated by veterinarian infectologists. Serum samples from healthy dogs from non-endemic areas (n = 68) and from dogs with other infectious diseases (n = 64) were used as controls for assay validation. The performance of the LPG-ELISA was compared with that of an ELISA using the soluble fraction of L. infantum total lysate antigen (TLA). LPG-ELISA presented a superior performance in comparison to TLA-ELISA, with 91.5% sensitivity, 98.5% specificity and 99.7% accuracy. A distinguishing feature of the LPG-ELISA compared to the TLA-ELISA was its higher ability to identify subclinical infection in clinically healthy dogs, in addition to the absence of cross-reactivity with other canine infectious diseases. Finally, LPG-ELISA was compared to TR DPP visceral canine leishmaniasis test, the immunochromatographic test recommended by the Brazilian Ministry of Agriculture. LPG-ELISA exhibited higher values of specificity (98.5% versus 93.1%) and sensitivity (91.5% versus 90.6%) compared to TR DPP. In conclusion, L. infantum-derived LPG was recognized by antibodies elicited during CanL in different infection stages and was shown to be a suitable antigen for specific clinical settings of veterinary diagnosis and for public health usage.

Project description:BACKGROUND:Visceral leishmaniasis (VL) is an infectious disease with a variety of clinical signs. The main form of parasite transmission to humans and other mammalian hosts is through the bite of infected arthropod females with Lutzomyia longipalpis serving as the main vector in the Americas. Dogs are the main urban domestic reservoirs of the parasite and the main source of vector infection due to their high prevalence in endemic areas and the large number of parasites in the skin of infected animals. Although miltefosine has been used in Europe since 2002 for treatment of VL infected dogs, in the Americas the treatment of dogs has not been recommended. Therefore, this study aimed to evaluate efficacy of miltefosine observing a reduction of clinical signs in infected dogs and the infectiveness to the vector by Leishmania (L.) infantum. METHODS:To our knowledge, this is the first controlled study using qPCR and xenodiagnosis to evaluate the efficacy of miltefosine (Milteforan®, Virbac) as a single treatment in Brazil. Thirty-five adult dogs with canine visceral leishmaniasis (CVL), confirmed by clinical and laboratory tests, were included in this study. They received miltefosine at a dose of 2 mg/kg every 24 h for 28 days. The dogs were observed over a three-month period, during which clinical evaluations based on a scoring system were conducted at pre-established times. Parasite load was assessed by cytology and real-time polymerase chain reaction (qPCR). Transmissibility to the vector was evaluated by xenodiagnosis. RESULTS:At the end of the period, the following were observed: (i) the remission of clinical signs with a reduction in clinical scores for 94.2% of the animals; (ii) a statistically significant reduction (98.7%) in parasitic load by qPCR; and (iii) a reduction in infectivity to sand flies. After treatment, 74.2% of the animals remained or had become non-infectious. CONCLUSIONS:Our study indicates that the use of miltefosine administered orally for 4 weeks contributes to a clinical improvement and reduction in infectivity of dogs to L. infantum.

Project description:BackgroundCanine leishmaniasis (CanL) is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China), which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp) fragment of the conserved region in the minicircle kinetoplast DNA (kDNA) and the results of phylogenetic analyses based on the sequence of the amplified fragment.ResultsThe proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease.ConclusionsMore than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of maintaining dogs, must be taken against these infected dogs due to their role in the transmission of the infection to vectors. The phylogenetic tree based on the sequences of conserved region in kDNA of Leishmania can effectively distinguish species of Leishmania.

Project description:BackgroundThe sand fly Nyssomyia neivai is one of the most abundant species in Southern Brazil. It is frequently found in areas that are foci of visceral leishmaniasis in the state of Santa Catarina, caused by Leishmania infantum. In this region, the main vector of L. infantum, Lutzomyia longipalpis, has not been detected. In the absence of L. longipalpis, this study aimed to identify the sand fly fauna and diagnose any potential Leishmania spp. infection in sand flies and in dogs in a region of Southern Brazil that experienced a recent canine visceral leishmaniasis outbreak.MethodsThis report includes a survey of the sand fly fauna at the Zoonosis Control Center of the Municipality of Tubarão (Santa Catarina, Brazil). Molecular tests were conducted to investigate Leishmania spp. natural infection in sand flies using polymerase chain reaction (PCR). In positive females, in addition to morphological identification, molecular analysis through DNA barcoding was performed to determine the sand fly species. Additionally, the dogs were tested for the presence of Leishmania spp. using a non-invasive technique for the collection of biological material, to be assessed by PCR.ResultsA total of 3419 sand flies, belonging to five genera, were collected. Nyssomyia neivai was the most abundant species (85.8%), followed by Migonemyia migonei (13.3%), Pintomyia fischeri (0.8%), Evandromyia edwardsi (< 0.1%), and species of the genus Brumptomyia. (0.1%). Out of the 509 non-engorged females analyzed by PCR, two (0.4%) carried L. infantum DNA. The naturally infected females were identified as Ny. neivai, in both morphological and molecular analysis. In addition, two out of 47 conjunctival swabs from dogs tested positive for L. infantum, yielding an infection rate of 4.2%.ConclusionsThese results confirm the presence of Ny. neivai naturally infected with L. infantum in an area where dogs were also infected by the parasite, suggesting its potential role as a vector in Southern Brazil.

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Improving the serodiagnosis of canine Leishmania infantum infection in geographical areas of Brazil with different disease prevalence.

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