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BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

Background Trypanosoma cruzi, the etiologic agent of Chagas disease, is currently divided into six discrete typing units (DTUs), named TcI-TcVI. TcII is among the major DTUs enrolled in human infections in South America southern cone, where it is associated with severe cardiac and digestive symptoms. Despite the importance of TcII in Chagas disease epidemiology and pathology, so far, no genome-wide comparisons of the mitochondrial and nuclear genomes of TcII field isolates have been performed to track the variability and evolution of this DTU in endemic regions. Results In the present work, we have sequenced and compared the whole nuclear and mitochondrial genomes of seven TcII strains isolated from chagasic patients from the central and northeastern regions of Minas Gerais, Brazil, revealing an extensive genetic variability within this DTU. A comparison of the phylogeny based on the nuclear or mitochondrial genomes revealed that the majority of branches were shared by both sequences. The subtle divergences in the branches are probably consequence of mitochondrial introgression events between TcII strains. Two T. cruzi strains isolated from patients living in the central region of Minas Gerais, S15 and S162a, were clustered in the nuclear and mitochondrial phylogeny analysis. These two strains were isolated from the other five by the Espinhaço Mountains, a geographic barrier that could have restricted the traffic of insect vectors during T. cruzi evolution in the Minas Gerais state. Finally, the presence of aneuploidies was evaluated, revealing that all seven TcII strains have a different pattern of chromosomal duplication/loss. Conclusions Analysis of genomic variability and aneuploidies suggests that there is significant genomic variability within Minas Gerais TcII strains, which could be exploited by the parasite to allow rapid selection of favorable phenotypes. Also, the aneuploidy patterns vary among T. cruzi strains and does not correlate with the nuclear phylogeny, suggesting that chromosomal duplication/loss are recent and frequent events in the parasite evolution. Electronic supplementary material The online version of this article (10.1186/s12864-018-5198-4) contains supplementary material, which is available to authorized users.

ESAT-6, CFP-10 and EspC are virulence factors that have been extensively assayed for bovine and human tuberculosis diagnosis due their potent T-cell inducing activities. While polymorphisms of ESAT-6 and CFP-10 were analyzed, with the description of CFP-10 variants in M. tuberculosis, this fact has not been explored in M. bovis field isolates. The coding sequences of esxA (ESAT-6), esxB (CFP-10) and mb3645c (EspC) from 58 M. bovis strains exhibiting genomic variability (spoligotyping) were analyzed. Two genes –esxA and esxB – remained invariant while mb3645c exhibited one synonymous polymorphism (G to A mutation, position 66bp) in one isolate, compared to M. bovis AF2122/97 reference strain. All isolates exhibited a synonymous nucleotide polymorphism simultaneously (G to A mutation, position 255bp), compared to M. tuberculosis H37Rv reference strain. This study confirms the high conservation for ESAT-6, CFP-10 and EspC in local M. bovis field isolates and reinforce the use of these three antigens in the diagnosis of bovine tuberculosis. Further studies should be performed to globally confirm these findings. Fil: Encinas, Micaela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marfil, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Garbaccio, Sergio Gabriel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Barandiaran, Soledad. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Huertas, Pablo Sebastián. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morsella, C.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Macías, A.. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Patología Animal; Argentina Fil: Magnano, Gabriel Gustavo. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Patología Animal; Argentina Fil: Zapata, L.. Universidad Nacional de Río Cuarto. Facultad de Agronomía y Veterinaria. Departamento de Patología Animal; Argentina Fil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Paolicchi, Fernando Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina Fil: Zumárraga, Martín José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Eirin, Maria Emilia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Background Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates. Results In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype. Conclusions The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

Background Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin. Methods Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots. Results The manual and automated quantification showed strong, positive correlation (r2 = 0.959, p

Six sets of teneral Glossina palpalis gambiensis (Diptera: Glossinidae) were fed on mice infected with six different isolates of Trypanosoma brucei gambiense (each mouse was infected with one of the isolates), previously isolated from patients in the sleeping sickness focus of Bonon, Cote d’Ivoire and in Makoua, Congo. All the tsetse flies were dissected 42 days post-infection and midgut and salivary glands were examined for trypanosomes by microscopical examination. No infection was observed with the reference stock whereas each of the five recently isolated trypanosome isolates was able to infect tsetse flies, with rates of infection varying between 9.7 and 18.2% depending on the isolate. Three isolates displayed only immature infections with 9.7, 17.3 and 18% of the flies showing trypanosomes in their midgut. One isolate gave both immature (12.1%) and mature infections (6.1%). Finally, the last isolate involved only mature infections in 9.7% of the Glossina species examined. These substantial differences in the cyclical transmission of T. b. gambiense in the same fly species could have important implications for the epidemiology of the transmission of Human African Trypanosomiasis.

Avian infectious bronchitis is an economically important viral disease of poultry resulting in reduced performance, reduced egg quality and quantity, significantly increased susceptibility to concurrent infections and a costly vaccination program. The disease is caused by infectious bronchitis virus (IBV) which is in the family 'Coronaviridae'. IBV is highly infectious and spreads rapidly through horizontal transmission by aerosol or ingestion. In an earlier study sentinel chickens were employed to determine the incidence of IBV in Ontario layers. IBV isolates recovered from the sentinel birds were not vaccine related as shown by genetic analysis of the S1 region. Five of these IBV field isolates were selected for further characterization. Analysis of tracheal lesion scores for each isolate revealed statistically significant differences (p

Single-strand conformational polymorphism (SSCP) analysis of polymerase chain reaction (PCR) amplified products is a sensitive technique for rapid scanning of mutations occurring in single stranded DNAs. It is based on the fact that single stranded DNAs differing by a single base substitution may form different conformers. The conformational changes can be detected as the alterations in the electrophoretic mobility in non-denaturating polyacrylamide gels. The SSCP method was applied by us for differentiation of infectious bursal disease virus (IBDV) isolates. Field isolates were analyzed for variations in 422-bp fragment of VP2 gene. The single stranded DNAs, obtained by treatment of PCR products by alkali denaturing buffer (ADB), were electrophoretically analyzed in 8 % polyacrilamide gel. The SSCP analysis showed the difference between analyzed IBDV field isolates and confirmed our previous results of isolate differentiation carried out by restriction analysis using Cfo I, Sac I, Ssp I, Stu I and Taq I enzymes (Lojkić et al. (2003) Avian Dis. 47: 59).

Infectious bursal disease viruses (IBDVs) in 26 IBDV-positive bursa samples collected in Croatia during the period 1996-2000 and in two commercially available vaccines were differentiated by the presence or absence of the CfoI, SacI, SspI, StuI, and TaqI restriction sites in the 422-bp fragment of segment A of the VP2 gene (nt 732-1153). The fragments from 14 (54%) field isolates were TaqI+ StuI+ SspI+ and SacI- CfoI-, indicating their very virulent (vv) character. The presence of CfoI restriction site in 10 (38%) field isolates is uncommon for vvIBDV strains. It was detected in only the 88180 vvIBDV strain. Nevertheless, these isolates can be classified as vv strains according to TaqI+ StuI+ SspI+ SacI- restrictions. Two SacI+ StuI+ CfoI+ TaqI- SspI- field isolates (8%) could be classified as non-vvIBDVs. The StuI+ restriction is common to vvIBDV strains. However, the StuI recognition sequence is present in the F52/70 classic European and 002-73 attenuated strains as well. The SacI+ CfoI+ StuI- SspI- restrictions and the lack of the TaqI restriction at nt position 832 show that the IBDV in GUMBOKAL IM-SPF vaccine corresponds to the attenuated and/or vaccine strains. The TaqI restriction at nt position 875 suggests that the IBDV in GUMBOKAL SPF vaccine could belong to the mild strains.

Reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction fragment-length polymorphism (RFLP) analysis was tested in order to determine its accuracy and sensitivity for differentiation of infectious bursal disease virus (IBDV) strains. Two commercial vaccine strains and two Croatian field isolates were analysed. RT-PCR assay was used for amplification of a variable part of VP2 gene (422 bp) encoding for major neutralizing epitopes. Amplified VP2 fragments were further characterized by RFLP analysis using the SacI and TaqI restriction endonucleases. Both field isolates showed RFLP pattern similar to highly virulent European and Asian strains. Both vaccine strains had RFLP profiles different from that of the field isolates. Vaccinal strain 2512 had RFLP pattern similar to mild vaccinal strain pbg-98, and strain VMG-91 was similar to other classical vaccinal strains of intermediate pathogenicity., Procijenjena je točnost i osjetljivost metode temeljene na razlici dužina restrikcijskih odsječaka DNA (RFLP, restriction fragment lenght polymorphism) za razlikovanje sojeva virusa zarazne bolesti burze (VZBB). Lančanom reakcijom polimerazom (PCR, polymerase chain reaction) uz prethodnu reverznu transkripciju (RT, reverse transcription) umnožen je dio promjenljivog dijela VP2 gena koji kodira glavne virusne neutralizacijske epitope. Nastali produkti veličine 422 parova baza (bp, base pair) pocijepani su restrikcijskim endonukleazama SacI i TaqI. Restrikcijski profili nastali enzimskim cijepanjem uspoređeni su s podatcima navedenim u banci gena. Metoda je testirana na dva komercijalna cjepiva proizvedena u Hrvatskoj (Gumbokal® SPF, soj 2512 i Gumbokal® IM SPF, soj VMG 91) te dva hrvatska terenska izolata VZBB (IBDV/96/01 i IBDV/ 97/02). Oba su terenska izolata pokazala RFLP profil jednak vrlo virulentnim europskim i azijskim sojevima VZBB. Oba cijepna soja imala su različit RFLP profil od terenskih izolata. Cijepni soj 2512 ima RFLP profil jednak blagim cijepnim sojem pbg-98, a VMG 91 je sličan poznatim intermedijarnim sojevima VZBB.

Reverse transcription-polymerase chain reaction (RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis was tested in order to determine its accuracy and sensitivity for differentiation of infectious bursal disease virus (IBDV) strains. Two commercial vaccines produced in Croatia and two Croatian field isolates were analysed. RT-PCR assay was used for amplification of a variable part of VP2 gene (422 bp) encoding for major virus-neutralizing epitopes. Amplified VP2 fragments were further characterized by RFLP analysis using the SacI and TaqI restriction endonucleases. Both field isolates showed RFLP pattern similar to highly virulent European and Asian strains. Both vaccine strains had RFLP profiles different than that of the field isolates. Vaccinal strain 2512 had RFLP pattern similar to mild vaccinal strain pbg-98, and strain VMG-91 was similar to other classical vaccinal strains of intermediate pathogenicity.

Accumulation of tebuconazole by germlings of wild-type isolates of Botrytis cinerea Pers. ex Fr. was characterized by a high initial level of intracellular tebuconazole during the first 10 min of incubation and a subsequent gradual release of the fungicide into the surrounding medium. This transient accumulation pattern indicates that the efflux of the fungicide has an inducible character. Accumulation of tebuconazole by laboratory-generated mutants resistant to sterol demethylation inhibitors (DMIs) was significantly less, suggesting that efflux capacity in these mutants is correlated with resistance. Accumulation of tebuconazole by field isolates with a relatively low sensitivity to DMI fungicides was transient in time and accumulation levels did not differ significantly from those of the wild-type isolates tested. These results suggest that reduced accumulation of tebuconazole in fungal mycelium may account for resistance in laboratory-generated mutants but not in field isolates of B. cinerea with a relatively low sensitivity to the fungicide. Various chemicals such as carbonyl cyanide 3-chlorophenylhydrazone, copper sulfate, fluazinam and BAS 490F enhanced accumulation of tebuconazole by a DMI-sensitive and a DMI-resistant isolate. This enhanced accumulation is ascribed to inhibition of the energy-dependent efflux of the fungicide.

Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992). Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b). We have studied the diversity of this SERP region in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa. The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced. Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of an anti-blood stage malaria vaccine.