Jomar Patrício Monteiro

Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.

Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil.

Haemonchus contortus is the most prevalent nematode in Brazil. The objective of this study was to select 6 populations of H. contortus of known or suspected benzimidazole resistance status and characterize these using quantitative real-time polymerase chain reaction (qPCR) for single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the β-tubulin isotype 1 gene. qPCR was performed using DNA from a pool of 10 adult male H. contortus from a single animal per farm. Faecal egg count reduction test (FECRT) and egg hatch test (EHT) were used to determine the resistance status. Samples were obtained from 6 farms located in 5 counties in the Ceará State: Tauá, Boa Viagem, Quixadá, Santa Quitéria and Solonópole. The inbred-susceptible-Edinburgh (ISE) isolate was used as reference for comparative purposes in the qPCR. Benzimidazole resistance was detected by FECRT on all farms with efficacy values ranging from 0 to 51%. EC50 values as determined by EHT were all above 1.49μg/ml. High frequencies of the resistant SNPs F200Y and F167Y alleles were detected but no resistance was detected at SNP E198A. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations.

Eimeria species in dairy goats in Brazil

The focus of this work is to determine the distribution and identify species of Eimeria parasites of dairy goats in the livestock of the National Goat and Sheep Research Center in Sobral, State of Ceará, Northeast Brazil. Results showed the presence of multiple species in 196 of 215 analyzed samples (91.2%). Fifty five out of these were from kids (28%) and 141 from adult goats (72%). Eight different Eimeria species were identified and their prevalence in the herd was: Eimeria alijevi Musaev, 1970 (26.7%), E. arloingi (Marotel, 1905) Martin, 1909 (20.6%), E. hirci Chevalier, 1966 (18%), E. ninakohlyakimovae Yakimoff & Rastegaieff, 1930 (16.2%), E. jolchijevi Musaev, 1970 (8.7%), E. christenseni Levine, Ivens & Fritz, 1962 (6%), E. caprovina Lima, 1980 (2.8%) and E. caprina Lima, 1979 (1%). Moreover, E. ninakohlyakimovae showed higher prevalence in kids (97%), followed by E. arloingi and E. alijevi (88%). On the other hand, E. alijevi (77%) was more common in adult goats followed by E. hirci (74%) and E. ninakohlyakimovae (70%). The species E. caprina had low frequency in both kids (27%) and adult goats (13%). Data indicated that infection was relatively common among kids and adult goats. The implementation of a routine diagnostic strategy can be useful in maintaining Eimeria populations under monitoring and will enable the determination of its potential impact on dairy goat herds in Northeast Brazil.

SIRT1 deacetylase activity and the maintenance of protein homeostasis in response to stress: an overview.

The present review intends to summarize the, yet preliminary, but very important emerging data underlining the functions exerted by the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT1 on protein homeostasis. The main focus of the discussion is the cooperation between SIRT1 and the heat shock factor 1 (HSF1) responsible for activating the transcription of molecular chaperones, the protein-protective factors that resolve damaged/misfolded and aggregated proteins generated by heat stress or metabolism. SIRT1, a mammalian ortholog of the yeast silent information regulator 2, is a stress activated protein deacetylase that contributes to life-span extension by regulating different cell survival pathways, including replicative senescence, inflammation and resistance to hypoxic and heat stress. Phosphorylation is the major mechanism controlling the level and function of SIRT1 required for normal cell cycle progression and cell survival under stress conditions. Phosphorylated SIRT1 deacetylates and coactivates different substrates, including HSF1. Deacetylated HSF1 binds to the heat shock promoter element found upstream of genes encoding molecular chaperones. Overexpression of SIRT1 in cultured cells also helps them to survive exposure to heat stress. Conversely, its down-regulation accelerates the attenuation of the heat shock response promoting the release of HSF1 from its cognate promoter element. Very recently, in a mouse model for Alzheimers disease, SIRT1 deacetylase activity was also found activating the transcription of α-secretase, the enzyme responsible for inhibiting the formation of aggregates of neuronal β-amyloid plaques. How SIRT1 activity protects cells from the deleterious effects of damaged/misfolded proteins and the implication of these findings on age-related pathologies are discussed.

ASCARIDIASIS IN PEAFOWL PAVO CRISTATUS (PHASIANIDAE) DUE TO ASCARIDIA GALLI SCHRANK, 1788

Twelve white peafowl (Pavo cristatus) affected by an outbreak of an intestinal disease were referred for more detailed examination at the Universidade Estadual de Santa Cruz-BA, Brazil. During the course of the disease, peachicks were severely affected, with enteric signs such as diarrhea plus dehydration, decreased feed intake and progressive weight loss. After examination, 8 of 12 samples (66.6%) presented single or mixed nematode infection and Ascarid eggs were the most frequent finding on fecal examination. Adult peafowl did not present clinical signs even when positive after fecal exam. Morphological analysis, clinical signs, fecal and gross examinations resulted in a diagnosis of ascaridiasis caused by Ascaridia galli Schrank (1788).

Haemonchus contortus β-tubulin isotype 1 gene F200Y and F167Y SNPs are both selected by ivermectin and oxfendazole treatments with differing impacts on anthelmintic resistance

Parasitism by Haemonchus contortus is one of the main limiting factors in small ruminant production in tropical areas. Benzimidazoles (BZ) and macrocyclic lactones (ML) are the most used anthelmintic classes in gastrointestinal nematodes control. There is considerable scientific evidence of a possible relation between the anthelmintic resistance to BZ and ML. This study aimed to characterize the dynamics of anthelmintic resistance in an H. contortus susceptible isolate under selection pressure for BZ and ML alone or in combination and the role of isotype 1 β-tubulin gene SNPs in these situations. A total of 12 Somali sheep were infected with 5000 third stage larvae of H. contortus Inbred-Susceptible Edinburgh (ISE) isolate. Once infection was established, animals were distributed in three groups (n=4), each treated with crescent doses of oxfendazole (OXF), ivermectin (IVM) and oxfendazole plus ivermectin (IVMOXF). An additional control group with untreated animals was maintained during the entire experiment. After each treatment, eggs were collected and real-time PCR was performed to identify single nucleotide polymorphisms (SNPs) F167Y, F200Y and E198A, in addition to egg hatch test (EHT) for BZ and larval development test (LDT) for ivermectin resistance. All treatments led to increased resistance allelic frequencies at SNPs F200Y and F167Y (p <0.05). In vitro results showed increased phenotypic resistance against both anthelmintic classes in groups IVM and IVMOXF while group OXF only developed resistance against BZ. Finally, we provide evidence that while isotype 1 β-tubulin gene SNPs may have some involvement with ML resistance, the presence of these β-tubulin SNPs alone are not sufficient to develop ML resistance.

Coccidia of gallinaceous meat birds in Brazil

Coccidiosis is a disease that limits the production and marketing of gallinaceous birds in North America, especially quails, pheasants and chukar partridges. Virtually no research has been conducted in South America on the causative agents of diseases among these birds, including coccidia. The aim of this work was to make first observations on Eimeria spp. in the chukar partridge Alectoris chukar and the grey quail Coturnix coturnix, which are reared for meat in Brazil. Fecal and tissue samples were collected from commercial farms and were examined for oocysts, gross and microscopic lesions or endogenous stages. From this examination, it was found that partridges raised in Brazil did not have any visible infection. However, grey quails presented mild infection and two Eimeria species that had previously been described in other birds were identified.