Comparison of conjunctival swabs and blood samples in dogs for molecular and serological detection of Leishmania infantum infection in Colombia.

BACKGROUND OBJECTIVES: Surveillance of canine leishmaniasis in Colombia is restricted to the appearance of visceral leishmaniasis cases in humans, and is mainly performed by serological tests. This requires blood sampling by veterinarians or technicians according to Colombian laws. The aim of this study was to evaluate the utility of conjunctival swabs in the molecular detection of Leishmania in dogs from the municipality of Ovejas, Sucre. METHODS: The present study was cross-sectional and descriptive. The collection source of samples and information was primary. Blood samples and conjunctival swabs from 121 dogs were analysed by PCR-ITS1 to detect Leishmania spp. Positive samples were used to amplify a conserved region of the Leishmania infantum kinetoplast minicircle. Performance of both sample types was calculated by proportion of positive samples of each type and the degree of agreement between them was determined by Cohen's kappa (κ) agreement index. RESULTS: Leishmania infection was detected in 17.4% (21/121) of blood samples and in 16.5% (20/121) of conjunctival swabs. In total, 28.1% (34/121) of the canines were infected, of which 11.8% (4/34) were infected with L. infantum in the conjunctival swabs and 5.9 % (2/34) in the blood samples. The agreement between blood and conjunctiva was medium (κ = 0.207) by PCR-ITS1 amplification. INTERPRETATION CONCLUSION: The use of conjunctival swab as a non-invasive sample could be used as an alternative method for surveillance of canine leishmaniasis.

Eye to eye: classification of conjunctival sac polystomes (Monogenea: Polystomatidae) revisited with the description of three new genera Apaloneotrema n. g., Aussietrema n. g. and Fornixtrema n. g.

Polystomes (Monogenea: Polystomatidae) of freshwater turtles are currently represented by five genera, namely Neopolystoma, Polystomoides, Polystomoidella, Uropolystomoides and Uteropolystomoides. These parasites can infect the urinary, oral and/or the conjunctival sac systems of their hosts, showing strict site specificity. A recent phylogenetic study showed that the two most diverse genera within chelonian polystomes, i.e. Neopolystoma and Polystomoides, are not monophyletic. Furthermore, polystomes infecting the conjunctival sacs of their host, except for one species, formed a robust lineage. A fusiform egg shape has been reported for conjunctival sac polystomes and it was assumed that this characteristic could be a good character for the systematics of polystomes. Our objective in the present work was, therefore, to study more in depth the morphology of polystomes collected from the conjunctival sacs of chelonians to find characters defining a putative new genus. To achieve this objective, more specimens were collected in 2018 and 2019 from turtles sampled in North Carolina and Florida (USA) to extend taxon sampling for the phylogenetic analysis. Morphological characters of relevant polystome specimens were re-examined from several collections from Asia, Australia, Europe, South Africa, South America and North America. Based on a Bayesian tree inferred from the analysis of four concatenated genes, namely 12S, 18S, 28S and COI, polystomes found in the conjunctival sacs were grouped in three distinct lineages, the first one including a single species infecting an Australian pleurodire turtle; the second one including eleven species infecting cryptodire turtles of South America, North America and Asia; and the last one including a single species infecting a softshell cryptodire turtle of North America. Based on observations of live specimens by Dr. Sylvie Pichelin and our morphological analysis, the conjunctival sac polystomes from Australian turtles are small, cannot extend their body significantly, have a spherical ovary and egg, have a large genital bulb and possess latero-ventral vaginae at the level of the testis. Based on observations of live specimens and morphological analysis of whole mounted specimens, polystomes of the second lineage share the following morphological characteristics: the ability to stretch out and double their length, a long oval ovary, a separate egg-cell-maturation-chamber, fusiform to diamond-shaped eggs with acute tips, small genital bulb and vaginae peripheral on the side of the body at the level of the testis. The polystome species of the third lineage occupies a basal position, has the ability to stretch out and possess an elongated ovary, a large fusiform egg with rounded tips, a small genital bulb and small latero-ventral vaginae at the level of the ovary. These three distinct conjunctival sac polystome lineages are herein described as separate new genera, Aussietrema, Fornixtrema and Apaloneotrema, respectively.

Morphological and phylogenetic characterisation of Unicauda tavaresii n. sp. (Myxosporea: Myxobolidae): a parasite of the circumorbital tissue of the eye of two characiform fishes from the Amazon region of Brazil.

Myxozoans of the family Myxobolidae are common parasites in fish. The diversity and ecology of the species of the genus Unicauda are poorly known, which hampers the understanding of the distribution and prevalence of this group of parasites. In the present study, cysts containing parasites whose morphology was consistent with the genus Unicauda were found in the circumorbital region of the ocular conjunctiva of the freshwater fish Moenkhausia grandisquamis Müller & Troschel, 1845 (Characiformes: Characidae) and Triportheus angulatus Spix & Agassiz, 1829 (Characiformes: Triportheidae). The spores have an oval body and long caudal appendage, with a mean total length of 65.2 ± 5.9 µm and width of 5.2 ± 0.7 µm, with two oval and symmetrical polar capsules of 4.9 ± 0.5 µm in length and 1.4 ± 0.2 µm in width, containing polar filaments with five or six coils. An integrated comparative analysis of the morphological characteristics of this parasite and partial sequences of the SSU rDNA gene supported the identification of a new species of histozoic parasite of the genus Unicauda found in fish from the Tocantins River basin, in the eastern Brazilian Amazon region. The new species was denominated by Unicauda tavaresii n. sp.

Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples.

Multiple polymerase chain reaction (PCR)-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Towards harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA quantitative polymerase chain reaction (qPCR) and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10-1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82-1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR, support its selection as the L. (Viannia) in research laboratories, as a first step towards harmonization of research protocols in the region.

Partial regression of large anterior scleral staphyloma secondary to rhinosporidiosis after corneoscleral graft - a case report.

BACKGROUND: Rhinosporidiosis is a rare chronic infection of the mucous membranes caused by the Rhinosporidium seeberi. Approximately 15% of cases of rhinosporidiosis are ocular, occurring mainly in the tarsal conjunctiva. There are only 11 cases of scleral melt with staphyloma formation associated with bulbar conjuctival oculosporidiosis and none of them was associated with partial regression of the scleral ectasia after a corneoscleral tectonic graft. CASE PRESENTATION: a 13-year-old girl with a progressively increasing black mass in the upper nasal part above the cornea of the left eye. The biomicroscopy revealed an oval, bluish mass measuring 10x10x5 mm with congestion of the overlying conjunctiva. Conjunctival biopsy showed sporoblasts of Rinosporidium seeberi. Treatment was conducted by conjunctival resection and tectonic corneoscleral graft (13x13mm) over the staphyloma. Within 1 year of follow-up the patient presented a partial staphyloma reduction, 9x9x2.5 mm, and the patch detached from the lesion. A novel surgical approach was done reducing the corneal patch and no recurrence was seen after 9 months. CONCLUSIONS: This case is one of the largest anterior scleral staphylomas secondary to rhinosporidiosis described in the literature. Scleral anterior staphyloma partial regression is an unusual outcome after a tectonic corneoscleral graft. Infection resolution and graft covering of thinned area contributed to scleral reepithelization.

Serological and molecular diagnostic tests for canine visceral leishmaniasis in Brazilian endemic area: one out of five seronegative dogs are infected.

Euthanasia of infected dogs is one of the measures adopted in Brazil to control visceral leishmaniasis (VL) in endemic areas. To detect infected dogs, animals are screened with the rapid test DPP® Visceral Canine Leishmaniasis for detection of antibodies against K26/K39 fusion antigens of amastigotes (DPP). DPP-positives are confirmed with an immunoenzymatic assay probing soluble antigens of promastigotes (ELISA), while DPP-negatives are considered free of infection. Here, 975 dogs from an endemic region were surveyed by using DPP, ELISA and real-time PCR (qPCR) for the diagnosis of VL. When DPP-negative dogs were tested by qPCR applied in blood and lymph node aspirates, 174/887 (19·6%) were positive in at least one sample. In a second sampling using 115 cases, the DPP-negative dogs were tested by qPCR in blood, lymph node and conjunctival swab samples, and 36/79 (45·6%) were positive in at least one sample. Low-to-moderate pairwise agreement was observed between all possible pair of tests. In conclusion, the official diagnosis of VL in dogs in Brazilian endemic areas failed to accuse an expressive number of infected animals and the impact of the low accuracy of serological tests in the success of euthanasia-based measure for VL control need to be assessed.

Detection of Leishmania infantum DNA in conjunctival swabs of cats by quantitative real-time PCR.

Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis.

Value of the oral swab for the molecular diagnosis of dogs in different stages of infection with Leishmania infantum.

This study was based on the need to employ a sensitive and specific method with samples that could be easily collected for diagnosing dogs infected with Leishmania infantum. To this end, we used real time-PCR (qPCR) to assess the value of the oral swab (OS) in detecting infected sick dogs (SD; n=62), including, for the first time, the analysis of apparently healthy infected dogs (AD; n=30), both from endemic areas for visceral leishmaniasis (VL). For comparison, we also evaluated the performance of the conjunctival swab (CS), blood (BL), lymph node (LN) and serology. We detected the presence of Leishmania DNA in the oral cavity in 62 out of the 92 dogs studied. The OS positivity (67.4%) was equivalent to the CS (68.5%) (p>0.05), higher than BL (52.2%) (p≤0.05), and lower than LN (84.8%) (p≤0.05). OS and CS performed well in SD dogs (82.3% and 83.9%, respectively) but not in AD dogs (36.7% for both samples). BL showed the lowest positivity (52.2%) and provided equivalent results between AD (60.0%) and SD (48.4%) dogs (p>0.05). LN yielded the highest positivity (84.8%), and it was also higher in the SD population (93.5%) compared to the AD population (66.7%) (p≤0.05). Parasite load was high in LN, moderate in OS and CS, and low in BL, showing the relationship between the levels of parasitism and the positivity rates found in these samples. Serology was positive in 82.2% of the SD group and in 70% of the AD dogs (p>0.05). Among the 20 seronegative dogs, seven (35%) were positive in either OS or CS, and 12 (60%) were positive when both noninvasive samples were jointly considered. The OS/CS combination resulted in a significant increase of positivity (p≤0.05) for the AD dogs (from 36.7% to 63.4%), as well as OS/serology (80%) and OS/CS/serology (83.4%). For the SD population, positivity reached up to 95.2% with the same combinations, showing that combination of samples and/or tests is required for the identification of dogs infected with L. infantum and that the OS and CS combination based on qPCR notably improves the detection of both AD and SD dogs. In conclusion, OS proved to be a suitable sample for the molecular diagnosis of infected dogs with clinical signs of VL, but not for dogs with inapparent infection. For these, we recommend the combination of OS results with CS and/or serology in order to reach relevant positivity for L. infantum. Finally, another advantage of using OS or both noninvasive samples is the increased likelihood of diagnosing seronegative dogs.

Detection of canine visceral leishmaniasis by conjunctival swab PCR.

INTRODUCTION: Conjunctival swab PCR was evaluated as a tool to diagnose visceral leishmaniasis in dogs. METHODS: Conjunctival swab PCR was compared to indirect immunofluorescence antibody test and blood PCR. RESULTS: Indirect immunofluorescence was significantly correlated with conjunctival swab PCR (p < 0.05), but not with blood PCR (p > 0.05). In addition, conjunctival swab PCR was significantly associated with presence of clinical symptoms (p < 0.05), whereas blood PCR was associated with absence of clinical symptoms (p < 0.05). CONCLUSIONS: Results indicate that conjunctival swab PCR is useful in epidemiological surveys of canine visceral leishmaniasis.

Conjunctival swab PCR to detect Leishmania spp. in cats.

The relevance of the dog as a source of visceral leishmaniasis infection is known, but the role of cats as reservoir hosts for leishmaniasis is not yet fully clear. This study assessed the efficacy of conjunctival swab PCR (CS-PCR) in the detection of cats infected by Leishmania spp. The results were seven (13.5%) cats positive for Leishmania spp. in the PCR, in 52 cats tested from Pirassunuga-SP and Ilha Solteira-SP. From the city of Pirassununga - SP 28.6% (2/7) were positive and from the city of Ilha Solteira - SP 11.1% (5/45) were positive. The results showed that CS-PCR was capable of detecting cats infected by this protozoan. Conjunctival swab samples proved easier to perform in cats, which might facilitate studies on the frequency and distribution of feline leishmaniasis.

Detection of Leishmania infantum in 4 different dog samples by real-time PCR and ITS-1 nested PCR.

The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil, which involves the elimination of infected dogs, the main animal reservoir host of the disease. The aim of the present study was to evaluate a sensitive real-time PCR method for Leishmania infantum detection in 4 different clinical samples of dogs, including the noninvasive conjunctival swab (CS) sample. The results of real-time PCR were compared with those obtained using internal transcribed spacer 1 nested PCR. Animals were divided into 2 groups based on the absence or presence of CVL clinical sings. The CS associated with real-time PCR, using primers addressed to kinetoplast DNA minicircles, was able to detect L. infantum infection in 96.7% of dogs without clinical signs and in 100% of the symptomatic animals, demonstrating the importance of these procedures for diagnosing CVL.

Detecção de formas amastigotas em exame parasitológico de esfregaço obtido a partir de suabe conjuntival de cães com leishmaniose visceral / Detection of amastigote forms in a parasitological exam of smears made from conjuntival swab in dogs with visceral leishmaniasis

Leishmaniose é uma enfermidade multissistêmica cujas manifestações clínicas são extremamente variáveis. Em cães sinais clínicos oftálmicos são relativamente frequentes, ainda que outros sinais sistêmicos não sejam identificados. Atualmente, o diagnóstico da doença baseia-se em métodos parasitológicos, sorológicos e moleculares, mas, até o momento, a identificação de formas amastigotas desse parasito em esfregaços feitos a partir de suabes conjuntivais não é empregada rotineiramente. Valendo-se de cães sorologicamente positivos para leishmaniose, portadores (G1) ou não (G2) de alterações oftálmicas, este estudo avaliou a viabilidade do esfregaço a partir de suabe conjuntival como método de diagnóstico para a enfermidade. O exame suprarreferido foi positivo em 60% dos animais do G1 e 38,1% do G2, no entanto não houve diferença estatisticamente significativa em relação à positividade nos dois grupos (P=0,2167). Os dados apontam para uma tendência de os cães com leishmaniose e com sinais oftálmicos serem positivos ao exame parasitológico de esfregaço a partir de suabe conjuntival, podendo esse método ser útil no diagnóstico parasitológico da leishmaniose canina.

Leishmaniasisis is a multisystemic disease with varying clinical presentations. In dogs, alterations in the eyes are commonly observed even in animals with no systemic signs. The diagnosis of leishmaniasis is currently based on parasitological, serological and molecular methods, although the identification of amastigote forms of this parasite in conjunctival swabs is not a routine technique in clinical practice. Serologically positive dogs for leishmaniasis presenting (G1) or not (G2) ocular alterations were enrolled in this study to evaluate the conjunctival swab as a method for the diagnosis of this disease. The parasitological evaluation of the swabs disclosed 60% positivity for dogs in group 1, whereas only 38.1% of dogs in group 2 were positive. However, no significant difference was documented between the two groups (P=0.2167). Our data suggest a tendency for dogs with ocular signs to be tested positive in the conjunctival swab exam, thus, this method is useful in the diagnosis of canine leishmaniasis.

Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil.

BACKGROUND: We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively. METHODOLOGY/PRINCIPAL FINDINGS: Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups. CONCLUSIONS: The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.

Detecção de formas amastigotas em exame parasitológico de esfregaço obtido a partir de suabe conjuntival de cães com leishmaniose visceral / Detection of amastigote forms in a parasitological exam of smears made from conjuntival swab in dogs with visceral leishmaniasis

Leishmaniose é uma enfermidade multissistêmica cujas manifestações clínicas são extremamente variáveis. Em cães sinais clínicos oftálmicos são relativamente frequentes, ainda que outros sinais sistêmicos não sejam identificados. Atualmente, o diagnóstico da doença baseia-se em métodos parasitológicos, sorológicos e moleculares, mas, até o momento, a identificação de formas amastigotas desse parasito em esfregaços feitos a partir de suabes conjuntivais não é empregada rotineiramente. Valendo-se de cães sorologicamente positivos para leishmaniose, portadores (G1) ou não (G2) de alterações oftálmicas, este estudo avaliou a viabilidade do esfregaço a partir de suabe conjuntival como método de diagnóstico para a enfermidade. O exame suprarreferido foi positivo em 60% dos animais do G1 e 38,1% do G2, no entanto não houve diferença estatisticamente significativa em relação à positividade nos dois grupos (P=0,2167). Os dados apontam para uma tendência de os cães com leishmaniose e com sinais oftálmicos serem positivos ao exame parasitológico de esfregaço a partir de suabe conjuntival, podendo esse método ser útil no diagnóstico parasitológico da leishmaniose canina.(AU)

Leishmaniasisis is a multisystemic disease with varying clinical presentations. In dogs, alterations in the eyes are commonly observed even in animals with no systemic signs. The diagnosis of leishmaniasis is currently based on parasitological, serological and molecular methods, although the identification of amastigote forms of this parasite in conjunctival swabs is not a routine technique in clinical practice. Serologically positive dogs for leishmaniasis presenting (G1) or not (G2) ocular alterations were enrolled in this study to evaluate the conjunctival swab as a method for the diagnosis of this disease. The parasitological evaluation of the swabs disclosed 60% positivity for dogs in group 1, whereas only 38.1% of dogs in group 2 were positive. However, no significant difference was documented between the two groups (P=0.2167). Our data suggest a tendency for dogs with ocular signs to be tested positive in the conjunctival swab exam, thus, this method is useful in the diagnosis of canine leishmaniasis.(AU)

The use of conjunctival swab samples for PCR screening for visceral leishmaniasis in vaccinated dogs.

The polymerase chain reaction (PCR) has been shown to provide a rapid and sensitive technique for Leishmania detection. The aim of this study was to evaluate the technique of noninvasive conjunctival swabs (CS) as a sampling method for molecular screening for visceral leishmaniasis (VL) in a group of 42 police dogs, all of them vaccinated against VL, and to compare the results with those obtained by serological tests. The serological assays were performed independently by three laboratories. Laboratories 1 and 2 were private laboratories and laboratory 3 was the National Reference Laboratory. The first serological screening performed by laboratory 1 showed 15 reactive dogs and 4 indeterminate. Laboratory 2 confirmed only 3 reactive dogs and 2 indeterminate. Laboratory 3 confirmed 7 reactive dogs and 3 indeterminate. The PCR diagnosis using the CS procedure was performed on all 42 animals and was able to detect Leishmania DNA in 17 dogs. The PCR assay confirmed all the cases that were simultaneously reactive in the serological tests by two laboratories. The results showed that the CS technique was a sensitive and practical method for sample collection, thus allowing reliable diagnostic tests through PCR.

The use of conjunctival swab samples for PCR screening for visceral leishmaniasis in vaccinated dogs / O uso de amostras de swab conjuntival para triagem por PCR da leishmaniose visceral em cães vacinados

The polymerase chain reaction (PCR) has been shown to provide a rapid and sensitive technique for Leishmania detection. The aim of this study was to evaluate the technique of noninvasive conjunctival swabs (CS) as a sampling method for molecular screening for visceral leishmaniasis (VL) in a group of 42 police dogs, all of them vaccinated against VL, and to compare the results with those obtained by serological tests. The serological assays were performed independently by three laboratories. Laboratories 1 and 2 were private laboratories and laboratory 3 was the National Reference Laboratory. The first serological screening performed by laboratory 1 showed 15 reactive dogs and 4 indeterminate. Laboratory 2 confirmed only 3 reactive dogs and 2 indeterminate. Laboratory 3 confirmed 7 reactive dogs and 3 indeterminate. The PCR diagnosis using the CS procedure was performed on all 42 animals and was able to detect Leishmania DNA in 17 dogs. The PCR assay confirmed all the cases that were simultaneously reactive in the serological tests by two laboratories. The results showed that the CS technique was a sensitive and practical method for sample collection, thus allowing reliable diagnostic tests through PCR.

A PCR (do inglês Polymerase Chain Reaction) tem demonstrado ser uma técnica rápida e sensível para detecção de Leishmania. O objetivo deste estudo foi avaliar a técnica não invasiva do swab conjuntival na identificação por PCR de animais infectados em um grupo de 42 cães policiais, todos vacinados contra a Leishmaniose Visceral (VL), e comparar os resultados com aqueles obtidos pelos testes sorológicos. Os ensaios sorológicos foram realizados independentemente por três laboratórios. Os laboratórios 1 e 2 eram privados. O laboratório 3 era o Laboratório de Referência Nacional. A primeira triagem sorológica realizada pelo laboratório 1 apresentou 15 cães reativos e 4 indeterminados. O laboratório 2 confirmou apenas 3 cães reativos e 2 animais indeterminados. O laboratório 3 confirmou 7 cães reativos e 3 cães foram classificados como indeterminados. O diagnóstico pela PCR, utilizando o procedimento do swab conjuntival, foi realizado em todos os 42 animais e foi capaz de detectar DNA de Leishmania em 17 cães. A PCR confirmou todos os casos simultaneamente reativos nos testes sorológicos de dois laboratórios. Os resultados demonstraram que o swab conjuntival é um método sensível e prático para coleta de amostra, permitindo um diagnóstico consistente através da PCR.

PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples.

The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs. We concluded that the use of CS for regular screenings of dogs by PCR should be considered.

Evaluation of the conjunctival swab for canine visceral leishmaniasis diagnosis by PCR-hybridization in Minas Gerais State, Brazil.

The visceral leishmaniasis (VL) in Brazil is caused by Leishmania chagasi (L. infantum) and dogs are considered to be the main domestic reservoir. The epidemiological control involves the elimination of infected dogs. Therefore, the correct diagnosis is very important in order to avoid the disease transmission or unnecessary culling of dogs. Recently, an antileishmanial vaccine for dogs was licensed and commercialized in Brazil. Vaccinated dogs test positive in the conventional serological tests, rendering these assays useless for control programs involving vaccinated animals. The Polymerase Chain Reaction (PCR) is an attractive alternative to the diagnosis in this context; but non-invasive samplings have great importance because they are simpler, painless and less resisted by dog-owners. This study aimed at evaluating the conjunctival swab (CS) for canine VL diagnosis by the PCR-hybridization procedure. Two groups of 23 seropositive dogs were used. CS samples were obtained from both eyes of each animal. The DNA extraction from CS was performed by the phenol chloroform method in group 1 and by boiling in group 2. In addition, blood was collected from each animal so that 30 microl was spotted onto filter paper (FP) and 1.0 ml was treated to obtain the buffy coat (BC). The DNA extraction from the BC and FP was accomplished by identical procedures in both groups using commercial kits. The PCR positivities for both groups 1 and 2 were, respectively: 73.9% and 52.2% (CS), 13% and 30.4% (BC), 8.7% and 17.4% (FP). The hybridization step increased the positivities for: 91.3% and 65.2% (CS), 21.7% and 34.8% (BC), 30.4% and 43.5% (FP), respectively. The highest frequency of positivity was obtained by the association between CS and DNA extraction by phenol chloroform. This approach can be very useful for diagnosis of canine leishmaniasis and could be applied to the follow-up and regular screening of vaccinated dogs.

Humoral immune response in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival route.

The aim of this study was to compare systemic humoral immune responses in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival and intravenous routes. Twenty nine heifers separated in three experimental groups were studied: Group 1 (n=10 animals) and Group 2 (n=9 animals) were inoculated with 10(8) of N. caninum tachyzoites by conjunctival and intravenous routes at 5th month of gestation, respectively; Group 3 (n=10 animals) were non-inoculated control animals. An indirect fluorescent antibody test (IFAT) and western immunoblotting (IB) were used to analyze the humoral immune response. All animals from Group 1 developed N. caninum specific antibody responses after conjunctival inoculation recording the highest antibody titer (mean+/-SE: 160+/-49.9) at 6th month of gestation. There were statistical differences between humoral immune responses found in Group 1 and 2 being higher in the second one at 6.5th, 8.5th and 9th months of gestation (P<0.05). Interestingly, all heifers from Group 1 reverted to seronegative status at the end of gestation. No increase in antibody was detected in the uninfected control group. Same pattern of N. caninum antigens was recognized by sera from heifers inoculated by conjunctival route and heifers inoculated by intravenous route. Recognized antigens were 116, 92, 84, 77, 45, 40, 25-26 and 17-18 kDa. The conjunctival instillation of N. caninum tachyzoites in pregnant heifers induces specific systemic antibodies. Further work is needed in order to clarify the consequences of this novel experimental route of infection not only on the fetus but also on the dam.

Mast cells in the eyes of Calomys callosus(Rodentia: Cricetidae) infected by Toxoplasma gondii.

The mast cell is a powerful effector cell for the innate immune system, acting through the secretion of several distinct mediators. Few studies have demonstrated the relationship between mast cells and toxoplasmosis. In this study, mast cells were investigated in two experimental Toxoplasma infections using Calomys callosus (Rodentia: Cricetidae) as the host. Animals were inoculated either intraperitoneally or via the conjunctiva with tachyzoites of Toxoplasma gondii (RH strain) and sacrificed after 5 days or 24 h, respectively. Enucleated eyes were processed for histological and ultrastructural analysis. Neither experimental infection altered the localization of mast cells compared to control eyes, but they did lead to an accumulation in some tissues as well as to their activation. There was a significant increase in the number of mast cells within 5 days and 24 h after infection. The ocular lesions were characterized by the presence of tachyzoites, inflammatory cells and vasodilatation in the iris and retina. In conclusion, mast cells were mobilized in these experimental infections, suggesting that they play an important role in the host inflammatory response after infection with T. gondii.