An overview on the molecular diagnosis of animal leptospirosis.

The most common presentation of animal leptospirosis is the subclinical and silent chronic form, that can lead to important reproductive disorders. The diagnosis of this chronic form remains a challenge. The aim of the present study is to gather and critically analyse the current information about molecular tools applied to animal leptospirosis diagnosis, particularly the silent chronic presentation of the infection. Regarding clinical specimens, samples from urinary tract were the most used (69/102, 67·7%), while few studies (12/102, 11·8%) investigated samples from reproductive tract. Concerning the molecular methods applied, the most used is still the conventional polymerase chain reaction (PCR) (46/102, 45%), followed by real-time PCR (38/102, 37·2%). The lipL32 gene is currently the most common target used for Leptospira detection, with 48% of studies applying this genetic marker. From all the studies, only few (21/102, 20·5%) performed gene sequencing. According to the majority of authors, current evidence suggests that lipL32-PCR is useful for an initial screening for Leptospira DNA detection in animal clinical samples. Posteriorly, if DNA sequencing could be performed on positive lipL32-PCR samples, we encourage the use of secY gene as a genetic marker. The molecular methods appear as the most important tools for the diagnosis of the chronic silent leptospirosis on domestic animals, reinforcing its evident impact not only on animal reproduction but also on a One Health context.

In vitro activity of carvacrol, cinnamaldehyde and thymol combined with antifungals against Malassezia pachydermatis.

Malassezia pachydermatis is an important opportunistic agent of dermatitis and otitis in dogs. M. pachydermatis is generally treated with topical therapies using combinations of antifungal, antimicrobial and anti-inflammatory agents. We investigated the in vitro activities of carvacrol (CRV), cinnamaldehyde (CIN) and thymol (THY) alone and in combination with antifungal agents (fluconazole, itraconazole, ketoconazole, clotrimazole, miconazole, terbinafine and nystatin) against M. pachydermatis. The assays were performed according to the Clinical and Laboratory Standards Institute (CLSI), using Sabouraud dextrose broth and checkerboard microdilution. The mean fractional inhibitory concentration index (FICI) showed primary synergies for the combinations carvacrol+nystatin, thymol+nystatin, and carvacrol+miconazole (80%). In conclusion, the results obtained indicate that the phytochemicals tested showed relevant in vitro anti-M. pachydermatis activity. Future in vivo experiments are needed to elucidate the safety and therapeutic potential of these combinations.

First record of the anisakid nematode Anisakis nascettii in the Gervais' beaked whale Mesoplodon europaeus from Brazil.

Anisakid nematodes are parasites with an indirect life cycle, involving many hosts, including cetaceans that act as definitive hosts. Identification at species level is crucial for a better understanding of the epidemiology and ecology of these parasites, but an accurate diagnosis based only on morphology is difficult. In Brazil, genetic characterization of anisakids is still rare, with Anisakis typica and A. physeteris being the only two species identified by genetic markers until now. The definitive hosts of A. nascettii are Mesoplodon grayi, M. bowdoini, M. layardii and M. mirus. Geographical locations of A. nascettii include the coasts of New Zealand, South Africa and Europe. In this study, a nematode was collected from a Gervais' beaked whale, Mesoplodon europaeus, stranded in Ceará State, north-east Brazil. Genetic analysis based on 18S rDNA, internal transcribed spacer (ITS) region and mtDNA cox2 gene revealed 100% identity with Anisakis sp. U94365, 99% with Anisakis sp. (MP-2005 EU718477 A. nascettii) and 99% with A. nascettii DQ116431, respectively. Phylogenetic analyses of ITS and cox2 sequences using both neighbour-joining and maximum-likelihood methods provided strong support for a clade with only A. nascettii specimens. This study demonstrated for the first time the identification of A. nascettii in the Brazilian Atlantic Coast, revealing a wider geographical distribution of this parasite worldwide and extending the range of its definitive hosts to a new Mesoplodon species, M. europaeus, the Gervais' beaked whale.

Echinococcus canadensis (G7) and Echinococcus granulosus sensu stricto (G1) in swine of southern Brazil.

The cystic echinococcosis (CE) is an important zoonotic disease caused by the parasite Echinococcus spp. In Brazil, this parasite is present in Rio Grande do Sul (RS) state, border with Argentina and Uruguay, causing several damages to human and animal health. This study aimed to identify Echinococcus spp. in hydatid cysts of swine and evaluate the similarity of the genotypes through the phylogenetic analysis. A total of 3,101,992 swine were slaughtered in the central/northern region of RS/Brazil, during 2008-2012. Five isolates were characterized as hydatid cyst by molecular analysis, based on the mitochondrial gene cytochrome c oxidase subunit I (cox-I). The genotypes E. granulosus sensu stricto (G1) (n=2) and E. canadensis (G7) (n=3) were identified in the hydatid cysts. The swine represents a potential intermediate host for different genotypes of Echinococcus spp., besides it can contribute to the perpetuation of the parasite's life cycle in rural areas.

Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences.

Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum.

In vitro susceptibility of fluconazole-susceptible and -resistant isolates of Malassezia pachydermatis against azoles.

OBJECTIVES: The first aim of this study was to evaluate the in vitro efficacies of fluconazole, ketoconazole, itraconazole and voriconazole on M. pachydermatis growth inhibition. This study also evaluated M. pachydermatis azole cross-resistance, comparing wild clinical isolates and the same isolates with in vitro-induced fluconazole resistance. METHODS: Two techniques were used: (1) a broth microdilution method based on protocol M27-A3 from the Clinical and Laboratory Standards Institute to determine the minimum inhibitory concentration (MIC) and (2) the Fekete-Forgács method to induce fluconazole resistance in vitro. The isolates were divided into two groups: group 1 included fluconazole-susceptible clinical isolates (n=30) and group 2 contained the same isolates with in vitro-induced fluconazole resistance (n=30). RESULTS: The two groups exhibited differences in susceptibility (p<0.001). Group 1 isolates were susceptible to azoles: ketoconazole (MIC 0.01-1.0 µg/mL), itraconazole (MIC 0.01-1.0 µg/mL), voriconazole (MIC 0.01-4.0 µg/mL), and fluconazole (MIC 0.01-4.0 µg/mL). Group 2 isolates demonstrated a wider range of MICs to azoles: ITZ (MIC 0.06-64.0 µg/mL), KTZ (MIC 0.25-32.0 µg/mL), VRZ (MIC 2.0-128.0 µg/mL), and FLZ (MIC 64.0-128.0 µg/mL). CONCLUSIONS: It was shown that FLZ-resistant M. pachydermatis isolates exhibit cross-resistance to other azoles, reinforcing the importance of susceptibility tests as a guide for the therapeutic prescription of antifungals in medical and veterinary mycology.

Systemic tizanidine hydrochloride (Zanaflex) partially decreases experimental postoperative pain in rats.

IMPLICATIONS: Tizanidine was given systemically to rats with experimental postoperative pain. Partial pain relief was obtained, but the effect was not clinically significant. Although systemic tizanidine is effective in relieving experimental neuropathic pain, it is not as effective for postoperative pain.

Systemic tizanidine hydrochloride (Zanaflex) relieves thermal hyperalgesia in rats with an experimental mononeuropathy.

UNLABELLED: We sought to determine whether tizanidine, an alpha2-agonist, relieved thermal hyperalgesia in rats with surgically induced neuropathic pain. We used a Sprague-Dawley rat model in which a chronic constriction of the sciatic nerve caused the rats to develop postural changes, mechanical allodynia, and thermal hyperalgesia. Thermal hyperalgesia was verified through paw withdrawal latency (PWL). PWL was tested before surgery, after surgery, and after injections with tizanidine (0.5, 1.0, or 2.0 mg/kg) or normal saline. Ambulatory and total movements were evaluated by placing the rats in activity cages. Thermal hyperalgesia was induced in all rats after surgery. Tizanidine, but not saline, caused a significant improvement in PWL (P < 0.05), with complete reversal of thermal hyperalgesia at all doses on postoperative Day 6. Rats who received tizanidine 2 mg/kg maintained complete reversal of thermal hyperalgesia through postoperative Day 9. Some sedation was observed with tizanidine 2 mg/kg, but not with smaller doses. We conclude that tizanidine effectively reversed thermal hyperalgesia in a rat model. IMPLICATIONS: This study was conducted to determine whether tizanidine could attenuate the thermal hyperalgesia that occurs in rats with surgically induced chronic constriction of the sciatic nerve. Tizanidine was effective in reducing sensitivity to heat, as measured by paw withdrawal latency, and did not cause sedation at smaller doses.