In Vitro Evaluation of the Combinatorial Effect of Naringenin and Miltefosine against Leishmania amazonensis.

Leishmania amazonensis causes a clinical form called diffuse cutaneous leishmaniasis (DCL) with challenges to treatment, like low efficiency and drug toxicity. Therefore, it is necessary to investigate new therapies using less toxic leishmanicidal compounds, such as flavonoids like naringenin, and their combination with conventional drugs, such as miltefosine. Antileishmanial dose/response activity, isobologram, calculation of dose reduction index (DRI), and fractional inhibitory concentration index (FICI) tests were performed on in vitro assays using reference promastigote forms of L. amazonensis (IFLA/BR/67/PH8) to assess the combinatorial effect between naringenin and miltefosine. The in vitro results of isobologram, DRI, and FICI calculations showed that the combination of the compounds had an additive effect and was able to reduce the half maximal inhibitory concentration (IC50) of miltefosine in the promastigote forms of the parasite compared to the treatment of the drug alone. This study demonstrated in vitro the viability of a combination action of the flavonoid with the treatment with miltefosine, opening space for further investigations on the association of natural compounds with the drugs used for the treatment of L. amazonensis.

Molecular detection of Theileria annulata, Theileria mutans and Theileria velifera but no evidence of Theileria parva infected or vaccinated cattle in Nigeria despite extensive transboundary migrations.

The extensive livestock management system predominant in Nigeria necessitates active disease surveillance for the early detection and prompt control of transboundary animal diseases. Theileriae are obligate intracellular protozoa which infect both wild and domestic bovidae throughout much of the world causing East Coast Fever (Theileria parva), Tropical or Mediterranean theileriosis (Theileria annulata) or benign theileriosis (Theileria mutans; Theileria velifera). This study aimed to detect and characterize Theileria spp. infecting cattle in Nigeria using conventional PCR and sequencing approach. Five hundred and twenty-two DNA samples obtained from different cattle blood samples were subjected to PCR targeting the 18S rRNA gene of piroplasmida and specifically, the p104 kDa and Tp1 genes for the evidence of infection or vaccination respectively, with T. parva. A total of 269 out of 522 (51.5%) of the cattle tested PCR- positive for DNA of piroplasmida. Nucleotide sequence and phylogenetic analyses showed that the cattle were infected with T. annulata, T. mutans and T. velifera. Piroplasmida DNA was associated with sex (ꭓ2 = 7.2; p = 0.007), breed (ꭓ2 = 115; p = 0.000002) of animals and the state where the samples were collected (ꭓ2 = 78.8; p = 0.000002). None of the samples tested positive for T. parva DNA or showed evidence of vaccination (Tp1 gene). This is the first report on the molecular detection and characterization of T. annulata in the blood of cattle from Nigeria. Continuous surveillance of Nigerian cattle for East Coast Fever (ECF) is encouraged considering the recent report of the disease in cattle in the neighboring country, Cameroon, where unregulated transboundary cattle movement into Nigeria has been observed.

Hemotropic mycoplasmas (hemoplasmas) in free-ranging bats from Southern Brazil.

Hemotropic mycoplasmas (hemoplasmas) are bacteria distributed worldwide and affect domestic and wildlife animals and human beings. Hemoplasmas have been described infecting hematophagous and non-hematophagous bats; however, transmission risk and zoonotic potential in vampire bats remain to be fully established. This study aimed to evaluate the presence of hemotropic mycoplasma species in free-ranging bats from this area using a universal PCR protocol for hemoplasmas. Accordingly, ten blood samples were collected from six male common vampire bats (Desmodus rotundus), two male hairy-legged vampire bats (Diphylla ecaudata), and two female non-hematophagous Pallas's mastiff bats (Molossus sp.) from the Curitiba's region, Paraná State, Southern Brazil. A total of eight (8/10) blood samples were positive byconventional PCR; five (5/6) Desmodus rotundus, two (2/2) Diphylla ecaudata, and one (1/2) Molossus sp. bats. The analyses of the partial sequence of the 16S rDNA gene suggest that the hemoplasma detected in Desmodus rotundus in South Brazil has a high identity compared to the hemoplasma circulating in vampire bats from Central and South America.

Uncommon Ehrlichia canis infection associated with morulae in neutrophils from naturally infected dogs in Brazil.

Ehrlichia and Anaplasma species are the most common tick-borne disease (TBD) pathogens in dogs worldwide. Ehrlichia canis, the aetiological agent of the Canine Monocytic Ehrlichiosis (CME), is known to replicate within the cytoplasm of mononuclear cells into clusters of organisms called morulae. However, detection of morulae in neutrophils is commonly observed in dogs infected by Ehrlichia ewingii or Anaplasma phagocytophilum. We report uncommon clinical cases of canine ehrlichiosis presenting morulae compatible with E. ewingii and A. phagocytophilum in dogs from two distinct regions of Brazil. Eight dogs were admitted to two veterinary teaching hospitals from Brazil, showing clinical or haematological signs suggestive of TBD. Blood or peritoneal fluid was withdrawn for haematological and cytologic analysis. All samples were evaluated by PCR assays for Ehrlichia and Anaplasma using genus-specific primers for dsb, 16S rRNA and groEL genes, followed by sequencing. Samples were also evaluated by nested PCR assays for the 16S rRNA gene of E. ewingii and groEL gene of A. phagocytophilum and Anaplasma platys. Seven dogs revealed thrombocytopenia, six dogs had monocytosis and five presented lymphopenia and anaemia. All dogs showed morulae structures compatible with Ehrlichia spp. in neutrophils and were PCR-positive for the dsb and 16S rRNA gene fragments of Ehrlichia, with sequences showing 100% identity with multiple E. canis sequences deposited in the GenBank™. Sequencing of 16S rRNA and groEL gene fragments from one PCR-positive dog showed 100% identity with A. platys. Overall, our data suggest that in endemic regions for E. canis, that is Brazil, the presence of morulae in neutrophils may indicate infection by this bacterium. Herein, morulae were also found in neutrophils present in the peritoneal fluid of a dog. Also, this is the first report of E. canis and Hepatozoon canis co-infection in neutrophils from naturally infected dogs confirmed by DNA sequencing.

First molecular screening of Plasmodium species in ungulates from Southern Brazil.

OBJECTIVE: Despite malaria epidemiology has been extensively studied in primates, few studies were conducted in ungulates. After half a century without descriptions of Plasmodium spp. in deer since its first identification, recent research has rediscovered Plasmodium on ungulates in Africa, Asia, North America and South America, including Central Brazil. Here, a captive herd was evaluated in southern Brazil using light microscopy and PCR. DNA samples were tested for fragment amplification of two Plasmodium spp. genes: mitochondrial cytochrome b and small subunit ribosomal RNA. RESULTS: All analyses were negative. However, the tests were performed on samples that were collected at a single time point, and parasitemia may fluctuate over the parasite's life cycle. Thus, the possibility of occult infection cannot be ruled out. Despite the negative results of all of the methods applied, it cannot be categorically stated that these animals are free from Plasmodium sp. infection. Further monitoring and/or multiple sequential sampling may improve the success rate of detecting parasites. Moreover, although this survey of Plasmodium represents the first molecular study on ungulate malaria parasites from Southern Brazil, further analysis of samples from different ungulate species is important for characterizing the epidemiology of Plasmodium of these mammals in this region.