Diagnosis Of Canine Leishmaniasis Research Articles

Refinement of the rKLi8.3-Based Serodiagnostic ELISA Allows Detection of Canine Leishmaniosis in Dogs with Low Antibody Titers.

The diagnosis of canine leishmaniasis (CanL) still represents a challenge due to the variable clinical manifestations and the large number of asymptomatic dogs. Serological tests are most commonly used to detect infected animals, revealing anti-Leishmania antibodies, mainly of the IgG isotype. Recently, a new diagnostic antigen, rKLi8.3, containing 8.3 kinesin tandem repeats (TR) from a Leishmania infantum strain from Sudan, has been shown to provide excellent specificity and sensitivity for the detection of Leishmania-infected humans and dogs. However, asymptomatic animals with very low antibody titers are often difficult to detect by serodiagnosis. Thus, we wondered whether the addition of an anti-IgG-enhancing step in the protein A/G-based rKLi8.3-ELISA will improve the diagnostic performance without decreasing the specificity. For this, parasitologically confirmed CanL cases with low or high clinical scores, uninfected healthy controls and dogs with other infections were tested by rKLi8.3-ELISA as well as two different immunochromatographic rapid tests, rKLi8.3-lateral flow test (LFT) and Dual Path Platform (DPP®) based on the rK28 antigen. Our results show that the diagnostic accuracies of the rKLi8.3-ELISA and LFT were similar to that of DPP, missing several asymptomatic animals. However, the addition of a secondary, amplifying anti-dog IgG antibody in the protein A/G-based rKLi8.3-ELISA enabled the detection of nearly all asymptomatic dogs without compromising its specificity.

Canine Leishmania spp. infection in two distinct foci of visceral and cutaneous leishmaniasis in Tunisia.

Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) infantum and L. major, respectively, are endemic in Tunisia. The aim of the study was to assess canine Leishmania spp. infection prevalence as well as to identify the Leishmania species involved in two well-documented and geographically distinct VL and ZCL foci. One hundred seventy-six dogs were randomly recruited in the VL focus of Sbikha-Zaghouan (n = 100) and the ZCL focus of Echrarda-Nasrallah (n = 76). Physical examination and blood collection were systemically performed. Needle aspiration was done in case of lymph node (LN) enlargement. All sera were tested by ELISA. kDNA RT-PCR was performed on DNA extracts from (i) buffy coats of seropositive dogs and (ii) LN aspirates. Leishmania species identification was done by ITS1 PCR-sequencing. Thirty-three dogs (18.8%) were infected by Leishmania; 30 having anti-Leishmania antibodies and 3 were seronegative dogs with Leishmania DNA in LN aspirates. Prevalence of infection was significantly higher in VL foci than in ZCL foci (27% versus 7.9%, p = 0.002). Leishmania species was identified in 11 dogs and corresponded to L. infantum. Combination of serology and qPCR on LN aspirates seems to be the best option for canine leishmaniasis diagnosis. Infection is more frequent in VL foci and L. infantum is the only identified species.

Canine Leishmaniasis diagnosis by direct examination: Case report

Leishmaniasis is a zoonosis caused by a protozoan of the kingdom Protista of the genus Leishmania, it is a public health problem with an unfavorable prognosis for infected animals. The city of Unaí - MG in northwest Minas Gerais, is considered an endemic area of the disease, with great casuistry animals and humans. A dog with a complaint of rectal prolapse was examined and, after clinical investigation, the presence of seborrhea, lesions on the tip of the ear, skin peeling, exaggerated nail growth, perilabial ulcer was noted, and direct examination of the ulcers was performed, which demonstrated the presence of amastigote forms of Leishmania sp. and the animal was euthanized.

Serodiagnosis of leishmaniasis in asymptomatic and symptomatic dogs by use of the recombinant dynamin-1-like protein from Leishmania infantum: A preliminary study

Visceral leishmaniasis (VL) is a fatal manifestation of an infection caused by intracellular protozoa of the Leishmania genus. In New World countries, VL is classified as a zoonotic disease with domestic dogs acting as its main reservoir. Asymptomatic dogs are as competent to transmit Leishmania to the vectors as symptomatic dogs, however current diagnostic tests are limited and present low sensitivity for this important group. The development of accurate tests is fundamental to the early diagnosis, treatment, and control of canine leishmaniasis. In this study, we investigated the use of a recombinant protein (dynamin-1-like protein, Dyn-1) from L. infantum, as a potential target antigen for leishmaniasis serodiagnosis in both symptomatic and asymptomatic dogs. The antigenic performance of the protein was evaluated by means of ELISA assays using sera from symptomatic (n = 25), asymptomatic (n = 34) and non-infected dogs (n = 36) using ELISA. In addition, sera from dogs experimentally infected with Trypanosoma cruzi (n = 49) and naturally infected with Babesia sp. (n = 8) were tested to evaluate possible cross-reactivity. A crude soluble antigen (CSA) of Leishmania was used as an antigen control and K39 and K26 were used as reference antigens because they are already widely used in commercial tests. rDyn-1-based assay showed the highest sensitivity (97%) compared to the antigens K39 (88%), K26 (86%) and crude extract (95%). The highest specificity among the tests was also obtained with the protein rDyn-1 (94%), compared with the other antigens K39 (81%), K26 (87%), and crude extract (77%). This study showed that the rDyn-1 ELISA assay was able to identify 100% of asymptomatic dogs, establishing its potential as a target for the diagnosis of canine leishmaniasis.

Diagnosis and prevalence of canine leishmaniasis in the Atlas shepherd dog.

The aim of this study was to determine the prevalence of canine leishmaniasis in the Atlas shepherd dogs from the Tiaret region of Algeria. A total of 161 dogs were included in this study and four diagnostic techniques were used, namely lymph node cytology, PCR, IFAT and ELISA. 110 out of 161 dogs were positive by at least one diagnostic technique, a percentage of 68.32% of the total number, which represent very high prevalence of canine leishmaniasis in this canine breed. 152 dogs underwent the two serological tests namely IFAT and ELISA among which 137 dogs underwent in addition the molecular examination by PCR. IFAT was positive in 9.86% of the dogs, ELISA was positive in 12.5% while 68% of the dogs proved positive by PCR. 47 dogs with lymph node enlargement underwent lymph node cytology, of which 26 dogs showed forms of amastigotes in their smears after microscopic examination, confirming the usefulness of this approach for the diagnosis of canine leishmaniasis. The clinical signs of the disease in this breed are similar to those of other canine breeds The dominant clinical signs in these dogs were onychogryphosis, poor general condition and lymph node hypertrophy. The autopsy was carried out on 7 dogs that died of canine leishmaniasis, all of which showed splenomegaly, while 5 dogs also showed hepatomegaly. Renal damage was found in 1 dog with frank jaundice. The splenic cytology was positive in all 7 dogs with the presence of amastigote forms in the smears after microscopic examination.

Pre-test probability and likelihood ratios for clinical findings in canine leishmaniasis.

Canine leishmaniasis is a parasitic zoonosis mainly caused by L. infantum; an obligate intracellular protozoan transmitted by haematophagous insects of the genus Phlebotomus, which affects dogs and wild canids. The clinical implications of this disease are highly variable, since infected animals may remain asymptomatic (absence of observable clinical signs) or present a wide spectrum of clinical alterations and degrees of severity, including the death of the animal. Symptoms such as lymphadenomegaly, alopecia, weight loss, keratoconjunctivitis and onychogryphosis are usually the first diagnostic reference available. The objectives of this study are to evaluate the validity (sensitivity, specificity and likelihood ratios) and diagnostic utility (pre-test probability) of the clinical signs commonly associated with canine leishmaniasis based on the prevalence in the area and to explore the combination of symptoms that best predicts the diagnosis of canine leishmaniasis. It is a matched case-control study in the canine population of southern Spain based on the comparison of the findings collected in the clinical history and the results of the LeisSCAN quantitative ELISA. A total of 39 cases and 78 controls were analysed. Approximately 80% of the infected animals showed signs compatible with the disease. The most frequent alterations were cutaneous (64.1%), systemic (51.3%) and oculo-nasal (30.7%). The most useful signs to support this diagnosis were alopecia and epistaxis (LR+ 6.69 and 6.0, respectively) (pre-test leishmaniasis probability is ≥70% for prevalence ≥28% when alopecia or epistaxis is present), followed by lameness (LR+ 5.0). The combinations of signs that showed greater validity were alopecia with hyperkeratosis of the snout and alopecia with onychogryphosis (LR+ >10). None of the observed signs or their combinations resulted useful to rule out the diagnosis (LR- 0.55 to 1.15). The results found show notable differences in the diagnostic value of the clinical signs, individually and in combination, so we believe that medical decisions should be based on their diagnostic validity (LR+) and the estimation of the pre-test and post-test probability.

Canine Leishmaniasis: Update on Epidemiology, Diagnosis, Treatment, and Prevention

Simple SummaryDogs are the main host of Leishmania infantum, a parasite that causes an incurable disease called canine leishmaniasis. This parasite is transmitted through the bite of a sandfly (a small insect related to mosquitoes and flies) in tropical and subtropical countries, but direct transmission between dogs, and from pregnant dogs to their puppies, exists. We reviewed the advances in tools and techniques for the surveillance of the disease, its diagnosis, treatment, and prevention. Canine leishmaniasis is expanding to the Northern Hemisphere, where it is barely known, due to climate change and the importation of dogs. Surveillance is therefore necessary in order to determine the extent of the disease in these areas and to monitor the appearance of the sandflies. Molecular techniques and rapid serological tests are now widespread for diagnosis and epidemiological studies. Several vaccines have been developed in the last decade, and even though their efficacy is limited, these advances will pave the way for the development of better vaccines against Leishmania and other parasites. Although new pharmacological tools are available, we are still waiting for the ideal drug that can eliminate the parasite from target organs and limit transmission to sandflies, without the side effects of current antileishmanials.Dog are the main reservoir of Leishmania infantum, causing canine leishmaniasis, an incurable multisystemic disease that leads to death in symptomatic dogs, when not treated. This parasite causes visceral, cutaneous, and mucosal leishmaniasis in people in the Mediterranean Basin, North Africa, South America, and West Asia. This disease is mostly unknown by veterinarians outside the endemic areas, but the disease is expanding in the Northern Hemisphere due to travel and climate change. New methodologies to study the epidemiology of the disease have found new hosts of leishmaniasis and drawn a completely new picture of the parasite biological cycle. Canine leishmaniasis diagnosis has evolved over the years through the analysis of new samples using novel molecular techniques. Given the neglected nature of leishmaniasis, progress in drug discovery is slow, and the few drugs that reach clinical stages in humans are unlikely to be commercialised for dogs, but several approaches have been developed to support chemotherapy. New-generation vaccines developed during the last decade are now widely used, along with novel prevention strategies. The implications of the epidemiology, diagnosis, treatment, and prevention of canine leishmaniasis are fundamental to public health.

Urea, salts, and Tween 20 influence on adsorption of IgG and Leishmania rNTPDase2 to nitrocellulose

Lateral flow immunochromatography is a widely used technique for immunological assays. Construction of test and control lines is mostly done by antigen adsorption to nitrocellulose membranes, a process not fully understood. This study aimed to evaluate the influence of urea, salts, and Tween 20, on adsorption. The performance of canine IgG in water and in buffer containing urea and salts (pH 8.3) were compared to observe if the interferents would lead to protein stripping when challenged with increasing concentrations of Tween 20 in the lateral flow buffer. Immobilization of the rLiNTPDase2, an antigen for Canine Leishmaniasis diagnosis, was evaluated and compared to the rLbNTPDase2 by the same method. There were no differences between adsorption coefficients of IgG in water and in buffer, but high salt and urea concentrations seems to stabilize and enhance IgG immobilization. Adsorption performance between canine IgG and rNTPDases had different patterns, but was highly similar between rNTPDases, indicating that protein identity may have an important role. Also, low concentrations of Tween 20 in the flow solution may aid the maintenance of rNTPDase2 on the strips. Our results bring insights about protein adsorption and perspectives about the influence of urea, salts and Tween 20 on this process.

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

BackgroundLeishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia.MethodsUsing in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers.ResultsThe results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples.ConclusionsThe results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.Graphical

Comparative Study of Real-Time PCR (TaqMan Probe and Sybr Green), Serological Techniques (ELISA, IFA and DAT) and Clinical Signs Evaluation, for the Diagnosis of Canine Leishmaniasis in Experimentally Infected Dogs.

Canine leishmaniasis (CanL) diagnosis is not fully resolved. Currently, two specific methodologies are in continuous development, the detection of the parasite DNA or RNA in target organs and the detection of specific antibodies against Leishmania sp. For a correct diagnosis, it has been shown that the joint use of this type of test is necessary. In this work, a Sybr Green and a TaqMan Probe based on real time PCRs (qPCR) was performed for the detection of Leishmania sp. in order to correlate the results with clinicopathological and serological evaluations (IFA, ELISA and DAT) to propose an optimal biological sample to be used to detect the parasite in both early and late stages of the infection. A total of four samples were processed: conjunctival swabs, popliteal lymph node aspirates, bone marrow aspirates, and peripheral blood from experimentally infected dogs belonging to a larger study. Our results indicated that a single non-invasive sample (conjunctival swab) and the application of both types of qPCR would be reliable for determining Leishmania infection as well as the disease stage in dogs, thus avoiding bone marrow, lymph node aspirate or blood samples collection.

Serodiagnosis of canine leishmaniasis using a novel recombinant chimeric protein constructed with distinct B-cell epitopes from antigenic Leishmania infantum proteins

Visceral leishmaniasis (VL) is an important public health problem in the world, and control measures are insufficient to avoid the spread of this neglected disease. Dogs are important domestic reservoirs of Leishmania parasites in countries where VL is a zoonosis, representing a major source of infection between sand fly vectors and humans. In this context, a precise diagnosis of canine leishmaniasis (CanL) could help to reduce the number of human cases. Distinct approaches for the diagnosis of CanL have used recombinant proteins in serological assays. However, variable results of the antigens have been found, mainly to diagnosis asymptomatic cases. The present study used bioinformatics to select specific B-cell epitopes of four Leishmania infantum proteins, which had previously been proven to be antigenic in VL, aiming to produce a novel chimeric protein and to evaluate it for the diagnosis of CanL. Seven B-cell epitopes were identified and used to construct the chimera, which was analyzed in a recombinant format through an ELISA assay against a canine serological panel. A soluble Leishmania antigenic extract (SLA) was used as an antigen control. Results showed 100 % sensitivity and specificity for chimera, while when using SLA the values were 26.0 % and 96.4 %, respectively. The performance of chimera was compared with a commercial kit using asymptomatic and symptomatic dog sera, and the data showed that no false-negative result was found when the recombinant protein was used. However, when using the commercial kit, 40.0 % and 16.0 % of the false-negative results were found, respectively. In conclusion, the recombinant chimera showed an antigenic potential to be evaluated in new studies against a larger serological panel for the diagnosis of CanL.

Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs.

The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.

Seasonal dynamics of phlebotomine sand flies and autochthonous transmission of Leishmania infantum in high-altitude ecosystems in southern Spain

Leishmaniasis is a vector-borne disease transmitted by sand flies. A dozen species have been involved in the transmission of Leishmania infantum in the Mediterranean region. Climate change may alter sand fly distribution at particular altitudes and latitudes. The objective of this study was to interrogate the existence of stable populations of sand flies in high-altitude ecosystems and evaluate if these populations are enough to support autochthonous transmission of leishmaniasis. These altitudinal conditions can be found in Sierra Nevada (southern Spain). Therefore, we have determined the sand fly population dynamics in different biotopes located at elevations above 1,300 m a.s.l. and searched for evidence of leishmaniasis transmission. Five collecting sites above 1,300 m a.s.l. containing large livestock concentrations were selected. Sand flies were caught using CDC light traps from May to November, annually from 2008 to 2013, and these were morphologically identified. Association between sand fly density or presence and temperature/humidity was estimated by linear and logistic regression, respectively. Leishmania infantum detection in female sand flies was performed by PCR. Diagnosis of canine leishmaniasis (CanL) was carried out by indirect immunofluorescence and PCR. A total of 2,973 specimens of 5 sand fly species were collected from June to October. Phlebotomus perniciosus was the most frequent (100%), abundant (80.1%) and densest species (9.8 sand flies/trap). The minimum temperature on the day of capture was the most important variable factor for sand fly presence and P. perniciosus density. An increase in altitude showed a negative effect over the sand fly diversity and activity period, driving changes in seasonal dynamics similar to those reported by latitudinal changes. CanL prevalence was 23%, a similar rate to previous surveys carried out on randomly selected dogs from towns in southern Spain. A successful host-vector-pathogen network was found at this altitude characterised by 9.9% L. infantum infection rate in non-blood fed P. perniciosus and Phlebotomus ariasi females and high CanL prevalence that entails an increase in the leishmaniasis risk area driven by sand fly colonization.

Diagnosis of Canine Leishmaniasis using Ear Blood PCR in Region of Brazil

Diagnosis of Canine Leishmaniasis using Ear Blood PCR in Region of Brazil

The serological diagnosis of canine Leishmaniasis by using ELISA in Nineveh province

The serological diagnosis of canine Leishmaniasis by using ELISA in Nineveh province

Accidental and late parasitological diagnosis of Leishmania sp. in a dog from a low disease transmission area of Brazil: A case report

Canine Leishmaniasis diagnosis must be fast and accurate since dogs are urban reservoirs of the disease and earlier therapeutic intervention is more clinically effective. However, this still represents a challenge, particularly in low transmission areas. The present report describes the difficulties of clinical suspicion and the late diagnosis of a dog infected with Leishmania sp.

Geographical variation in clinical signs and prevalence of Leishmania sp. infection among dogs in Fortaleza, Ceará State, Brazil

Background: Leishmaniosis are anthropozoonosis caused by protozoa of the genus Leishmania, representing a complex of diseases with significant diversity epidemiological and clinical spectrum and can affect 40% of the canine population. Infected dogs may be asymptomatic or may develop canine leishmaniasis (CL), a severe and progressive disease associated with the appearance of clinical signs. Serological tests are frequently used for screening of dogs, however, these techniques present limitations in terms of reproducibility and specificity. In this way, information on the geographical distribution and prevalence of CL is essential to the implementation of appropriate control measures. Therefore, the aim of this study was to determine geographical variations in clinical signs and prevalence of Leishmania sp. infections from dogs in Fortaleza, Ceara state, Brazil. Materials, Methods & Results: Bone marrow samples of 2829 domestic dogs were collected by puncture for parasitological diagnosis of canine leishmaniasis (CL), being considered positive by the presence of Leishmania sp. All dogs were examined and clinical signs were classified as score 0: no clinical signs, score 1: skin lesions, score 2: visceral signs and score 3: skin lesions + visceral signs. Kruskal-Wallis (P <0.05) was used to compare the scores and parasitological diagnosis among the seven regions of Fortaleza. So, it was observed that 72% of dogs were positive and the regions I and V had the highest prevalence (78.3% and 80% respectively), no significant differences among the seven regions. Asymptomatic dogs represented 55% of infected dogs, and the main clinical sign, among symptomatic dogs (45%), was skin lesion, visualized in 43% of dogs. Furthermore, 70% of all dogs had ectoparasites. Discussion: These results show that, despite of the campaign against the disease, there is no great progress in the control of visceral leishmaniasis in the urban areas like Fortaleza, which present an easy access in the fight against the disease. The diagnostic in dogs based in the Indirect Fluorescent Antibody Test (IFAT) associated to others factors, like irregular serological inquiry and no-treated performer personal, perhaps made possible the permanence of susceptibility animals in this area, beyond of the high number of asymptomatic and no-identified dogs. In this study, it was observed that 55% of dogs were asymptomatic, without showing evidence of risk of transmission to human population, escaping from controlling methods. From this we can imply that most of these dogs to LC are asymptomatic and the prevalence did not differ significantly in different regions of Fortaleza, requiring equal attention throughout the city. Moreover, our results demonstrate that Fortaleza city has favorable areas for the maintenance of the parasite cycle, with a high risk of transmission to human and canine population, and it requires special attention because of the large number of asymptomatic cases. Therefore, it is necessary to control the population of asymptomatic dogs with more efficient controlling methods, with the change of methodology of serological screening raising the number of detected dogs, reducing the diagnostic time and the use of diagnostic techniques with levels of specificity higher.

Sensitivity and specificity of an in-clinic point-of-care PCR test for the diagnosis of canine leishmaniasis

Canine leishmaniasis is an important infectious disease worldwide. Although commonly used, antibody tests are often falsely negative, and in such cases direct detection of the pathogen, such as PCR, is necessary. However, PCR is only performed in specialized laboratories and not available in all localities. The aim of this study was to compare the sensitivity and specificity of an in-clinic point-of-care (ICPOC) PCR for the diagnosis of canine Leishmania spp. infection to those of a well characterized reference real-time PCR. In this study, 515 samples from 251 dogs (201 EDTA blood samples, 244 conjunctival swabs, 19 lymph node aspirates, and 51 bone marrow aspirates) were collected prospectively and analysed for the presence of Leishmania DNA using an ICPOC test. The results were compared to those of a reference real-time PCR for identification of Leishmania kinetoplast minicircle DNA. Sensitivity and specificity with 95% confidence interval (CI 95%) were determined.Specificity was 100% for all samples examined. Sensitivity was 57.1% (CI 95%, 34.0–78.2) in bone marrow aspirates, 58.8% (CI 95%, 32.9–81.6) in lymph node aspirates, 46.9% (CI 95%, 32.5–61.7) in conjunctival swabs, and 10.0% (CI 95%, 3.3–21.8) in blood. The ICPOC PCR was easy to perform and was reliable in the case of positive test results. A negative result, however, did not exclude infection and therefore requires further diagnostics.

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26–52% sensitivity) although their performance was increased in the canine sera (48–91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This “Mix” resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.

Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope.

BackgroundLeishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies.Methodology/Principal findingsSeventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1–28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67–81 and 122–135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%).Conclusions/SignificanceOur work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.