Evaluation of Cytopathological Techniques for the Diagnosis of Canine Visceral Leishmaniosis with Lymph Node Samples.

The identification of the parasite in cytological smears of lymph node aspirates is a widely applied technique for the direct diagnosis of Leishmania spp. infection, especially in endemic areas. Although very specific, this method has limited sensitivity, and improving the technique would be highly desirable. This study aimed to evaluate the efficacy of conventional smear cytology (SC), liquid-based cytology (LBC), cell block (CB) stained with haematoxylin and eosin (HE) and immunocytochemistry (ICC), and formalin-fixed paraffin wax-embedded tissue immunohistochemistry (FFPE-IHC) compared with serology and polymerase chain reaction for the diagnosis of canine visceral leishmaniosis (CVL) in lymphoid tissue. The use of a preservative medium and centrifugation for cytological samples reduced the number of unsatisfactory artefacts/background. Moreover, LBC allowed excellent cellular preservation and the application of ancillary techniques, such as CB and ICC. SC was the most accurate morphological diagnostic method (45.0%). CB-ICC alone or associated with SC demonstrated significantly higher sensitivity (70.0% and 72.0%, respectively) when compared with SC alone (34.00%). CB-ICC was found to be more effective in the detection of infected animals with mild clinical signs, similar to FFPE-IHC. The specificity and positive predictive value were similar between all methods. Finally, the detection limit for CB-ICC and SC + CB-ICC was identical (18.46 amastigotes/mm2). Our study suggests that CB-ICC is a promising tool for improvement of the cytopathological diagnosis of CVL and may be applied in routine epidemiological screening.

Vaccine effectiveness and use of collar impregnated with insecticide for reducing incidence of Leishmania infection in dogs in an endemic region for visceral leishmaniasis, in Brazil.

Although a national programme for control of visceral leishmaniosis (VL) is being run in Brazil, the disease continues to spread. This programme is essentially based on culling infected dogs from endemic regions. Thus, there is an urgent need to develop other control measures against VL to deter its advance. Here, a subunit vaccine, a recombinant vaccine, an insecticide-impregnated collar and the associations between these measures were evaluated for reducing the incidence of Leishmania infection in dogs. This was through a cohort study conducted in an endemic region of Brazil, considering the incidence and time of total exposure over a period of 1 year. The incidence of VL was estimated by means of serological and molecular diagnostic tests, 180 and 360 days after the application of the control measures. The estimates of the effectiveness (EF) were not significant in any cohort. The EF of the subunit vaccine, the recombinant vaccine and the collar were 26.4%, 32.8% and 57.7% and the upper limit of the 95% confidence interval for EF were 63.7%, 67.9% and 82.5%, respectively. In conclusion, under the conditions of this study, none of the immunogens for VL control was sufficiently effective to protect dogs against infection. On the other hand, use of collars impregnated with insecticide seems to constitute a method with better prognosis, corroborating other studies in this field.

Dog skin parasite load, TLR-2, IL-10 and TNF-α expression and infectiousness.

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

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.

Performance of a real time PCR for leishmaniasis dignosis using a L. (L.) infantum hypothetical protein as target in canine samples

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese.

OBJECTIVE: Analyze the infectivity and storage resistance of cysts of the ME-49 strain of Toxoplasma gondii in artificially infected bovine milk and homemade fresh cheese. METHODS: Pasteurized bovine milk was infected with 10 cysts/ml of the ME-49 strain of T.gondii and inoculated in different groups of mice, immediately or after storage at 4 degrees C for 5, 10 and 20 days. Homemade fresh cheese was prepared with artificially infected milk, and also tested in groups of mice, using the same storage process. Infection was identified by the presence of cysts in the brain or serological testing in challenged mice after 5 weeks, confirmed by Western Blot and histology. RESULTS: The infectivity of cysts of the ME-49 strain of T.gondii was maintained in the milk even after storage for 20 days at refrigerator temperatures. Cysts were also able to survive the production process of homemade fresh cheese and storage for a period of 10 days in the same conditions. CONCLUSIONS: These data demonstrated that milk and dairy products could be an important source of T.gondii in human contamination, reinforcing the importance of milk pasteurization before any processing or ingestion.

Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese

Objetivo: Analisar a infecciosidade e a resistência de cistos de T. gondii em leite e queijo fresco caseiro, pela infecção artificial de leite bovino. Métodos: O leite bovino pasteurizado foi infectado artificialmente com 10 cistos/ml de T.gondii cepa ME49 e inoculado em grupos de camundongos, imediatamente ou após ser estocado por 5, 10 e 20 dias a 4oC. Preparou-se queijo fresco caseiro com leite infectado, sendo testado em grupos de camundongos, utilizando a mesma conservação. A infecção foi detectada pela presença de cistos no cérebro dos camundongos desafiados ou testes sorológicos após cinco semanas, também confirmada por Western Blotting e histologia. Resultados: A infecciosidade dos cistos da cepa ME49 de T.gondii foi mantida mesmo quando armazenado no leite até 20 dias de conservação em condições de refrigeração a 4oC. Os cistos resistiram ao processo de fabricação do queijo e eram infectantes após um período de 10 dias nas mesmas condições. Conclusões: Os achados mostraram que o leite e seus derivados podem ser uma importante fonte de contaminação humana pelo T.gondii, reforçando a importância da pasteurização do leite antes de qualquer processamento ou ingestão

The prevalence and avidity of Toxoplasma gondii IgG antibodies in pigs from Brazil and Peru.

Raw or inadequately cooked pork is an important source of Toxoplasma gondii infection, and the infection rate in animals used as human food, is an important risk predictor. The prevalence of this infection was estimated in 396 sera from 5-month old pigs obtained at abattoirs in São Paulo, Brazil (300) and Lima, Peru (96). The seroprevalence was higher in pigs from Peru (32.3%) as compared to Brazil (9.6%), as detected by ELISA and Western blot. Hemagglutination gave poor resolution which was not useful for the diagnosis of T. gondii infection. Specific antibody avidity is correlated with infection time, as shown in experimentally infected piglets. Using an arbitrary cut-off of 50% avidity index, Brazilian pigs were found to be more recently infected than Peruvian pigs. Pork should be considered a significant source of human T. gondii infection both in Brazil and Peru. Avidity assays could help in the detection of the time of T. gondii infection in pigs, allowing preventive management.