Evolutionary analysis of the VP1 gene of Echovirus 30 in Yunnan Province, China / 国际生物医学工程杂志

Objective:To study the genetic characteristics and genetic evolution of echovirus 30 (ECHO30) isolates in Yunnan Province, China.Methods:Virus isolation was performed on nucleic acid-positive samples for hand, foot, and mouth disease pathogen surveillance in Yunnan Province, and VP1 gene sequencing was performed. The sequences of eight ECHO30 isolates from Yunnan Province and the gene sequences of the VP1 region of the ECHO30 reference strain downloaded from GenBank were compared and analyzed using MEGA 5.0 software, and then a phylogenetic tree was constructed to measure the homology of nucleotides and amino acids between the isolates.Results:The ECHO30 virus was distributed in Wenshan, Qujing, Chuxiong, and Kunming in Yunnan Province. The ECHO30 virus was relatively common in Wenshan. ECHO30 isolates belonged to the H2 subtype of the H genotype, which was close to the local reference strain LC120939 in Yunnan Province. On the VP1 gene at site 5, the amino acid change ratio was more active, the amino acids were diverse, and mutations also occurred at sites 54, 156, 258, and so on. Nucleotide and amino acid homology were 84.0% - 100.0% and 98.4% - 100.0%, respectively.Conclusions:ECHO30 isolates from Yunnan Province have certain geographical characteristics and belong to H2 of the H genotype. The nucleotide differences in virus sequences among subtypes are small and have a close genetic relationship.

Molecular characterization of Blastocystis subtypes isolated in the city of Uberaba, Minas Gerais State, Brazil

Abstract INTRODUCTION Blastocystis is an intestinal protozoan that may play a role in the pathogenicity of humans. This study aimed to (i) genetically characterize Blastocystis isolates obtained from human fecal samples and the water supply of the city of Uberaba, Minas Gerais, Brazil, and (ii) to verify the phylogenetic relationship between these isolates. METHODS Blastocystis species present in 26 fecal samples obtained from humans and animals from Uberaba were genetically characterized by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-sequence-tagged sites. All amplicons were partially sequenced and/or defined according to the GenBank classification. RESULTS Polymerase chain reaction amplicons were generated from 21 human isolates and 18 water samples. The subtypes defined were ST1 (53.3%), ST3 (40.0%), and ST2 (6.7%) for human isolates; ST10 (100%) for bovine isolates; and ST5 (50.0%), ST1 (25%), and ST3 (25%) for pigs. Sequencing of polymerase chain reaction products showed a 98%-99% identity for the Blastocystis sequences deposited in GenBank, except for sequences from water samples that showed the identity of algae sequences. Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by ST1, ST5, and ST10, and the other by isolates characterized as ST3 and ST7. Both clades showed human and animal sequences, reinforcing the notion that Blastocystis subtypes are not host-specific. CONCLUSIONS The data showed that Blastocystis subtypes circulating in Uberaba are ST1-ST3, ST5, and ST10, present in both humans and animals, demonstrating that the Blastocystis subtypes are not host-specific; that is, zoonotic transmission is possible.

Review on diagnosis and molecular characterization of Toxoplasma gondii in humans and animals

@#Toxoplasma gondii is an obligate intracellular protozoon which causes toxoplasmosis, an important zoonotic disease that is endemic worldwide. Common sources of T. gondii infection in humans are food or water contaminated with oocysts and raw or undercooked meat with cysts. In animals, common sources of infection include feed, water, or litter contaminated with oocysts. The diagnosis and molecular characterization of T. gondii infection in humans and animals is crucial due to public and veterinary health importance. Various traditional and serological methods have been used in clinical practice for toxoplasmosis diagnosis, but interpreting the results remains a challenge. Several molecular techniques have also been used for the detection and genetic characterization of T. gondii, but primarily in research settings. In this paper, we review the techniques that are currently used for the diagnosis and genetic characterization of T. gondii in humans and animals, along with their advantages and disadvantages. The techniques reviewed have laid the groundwork for the future development of more effective and precise detection and characterization of T. gondii. These advances will contribute to a better understanding of epidemiology, prevention and control of toxoplasmosis. Thus, this review would be of particular interest to clinical physicians, veterinarians and researchers.

Molecular detection of Cercopithifilaria bainae and other tick-borne pathogens in Rhipicephalus sanguineus s.l. isolated from dogs in Midwest Brazil / Detecção molecular de Cercopithifilaria bainae e outros patógenos transmitidos por carrapatos em Rhipicephalus sanguineus s.l. isolados de cães no Centro-Oeste do Brasil

Abstract The aim of the present study was to detect Cercopithifilaria bainae and other tick-borne pathogens and to perform molecular characterization of the tick Rhipicephalus sanguineus s.l. collected from dogs. Ticks (n = 432, including 8 larvae, 59 nymphs, and 365 adults) were sampled from domiciled dogs (n = 73) living in Campo Grande, Mato Grosso do Sul (Midwest Brazil). All ticks were morphologically identified as R. sanguineus. Genomic DNA was extracted in pools (three to five ticks per animal) and was used for definition of R. sanguineus haplotypes (based on 16S rRNA analysis) and pathogen identification (Cercopithifilaria sp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, Babesia vogeli and Rickettsia spp.). Rhipicephal us sanguineus specimens were identified as haplotypes A and B. DNA of Cercopithifilaria bainae (43.83%; 32/73), Ehrlichia canis (24.65%; 18/73), Anaplasma platys (19.17%; 14/73), and Hepatozoon canis (5.47%; 4/73) was detected. The identity of pathogens was confirmed by DNA sequence analysis. The present study confirms the presence of haplotypes A and B of R. sanguineus in the state of Mato Grosso do Sul and its importance as a vector of several pathogens of veterinary concern. Finally, this is the first report to identify C. bainae in ticks in the Midwestern region of Brazil.

Resumo O objetivo do presente estudo foi detectar Cercopithifilaria bainae e outros patógenos transmitidos por carrapatos e realizar a caracterização molecular do carrapato Rhipicephalus sanguineus s.l. coletado em cães. Carrapatos (n = 432, incluindo 8 larvas, 59 ninfas e 365 adultos) foram amostrados de cães domiciliados (n = 73) residentes no município de Campo Grande, Mato Grosso do Sul (centro-oeste do Brasil). Todos os carrapatos foram identificados morfologicamente como R. sanguineus. O DNA genômico foi extraído em pools (três a cinco carrapatos por animal), seguido pela definição de haplótipos (com base no gene 16S rRNA) e pela investigação de patógenos (Cercopithifilaria sp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, Babesia vogeli e Rickettsia spp.). Os espécimes coletados foram identificados como haplótipos A e B de R. sanguineus. Foram detectados DNA de Cercopithifilaria bainae (43,83%; 32/73), Ehrlichia canis (24,65%; 18/73), Anaplasma platys (19,17%; 14/73) e Hepatozoon canis (5,47%; 4/73). A identidade dos patógenos foi confirmada por análise de sequência de DNA. O presente estudo confirma a circulação dos haplótipos A e B de R. sanguineus no estado de Mato Grosso do Sul e sua importância como vetor de vários patógenos de interesse veterinário. Finalmente, este é o primeiro relato de C. bainae em carrapatos na região centro-oeste do Brasil.

Recharacterization of the Canine Adenovirus Type 1 Vaccine Strain based on the Biological and Molecular Properties

Canine adenovirus type 1 (CAV-1) infection results in hepatitis in dogs. In this study, we investigated the biologic and genetic characteristics of the CAV-1 vaccine strain (CAV1V) to improve quality control about CAV vaccine. The identity of CAV1V as CAV-1 was confirmed based on its cytopathic effects and the results of hemagglutination (HA) and immunofluorescence assays, and electron microscopy. The CAV1V strain reached 10(7.5) TCID(50)/mL in MDCK cells at 4 days post-inoculation and exhibited hemmagglutination activity of 256 U using guinea pig erythrocytes. Intranuclear fluorescence in the infected cells was observed and typical adenoviruses were observed in electon microscope. CAV1V strain was identified as a CAV-1 strain by nucleotide sequence analysis. In a comparison of the nucleotide sequences of the fiber genes of several CAV strains, CAV1V showed the highest similarity (99.8%) with the GLAXO strain, which was isolated in Canada. Our biological characterization of CAV1V will facilitate quality control of the canine hepatitis vaccine.

Antigenicity and genetic characterization of HA gene in influenza B/Victoria-lineage virus in Beijing 2017-2018 / 中华实验和临床病毒学杂志

Objective@#To understand the antigenicity and genetic characterization of influenza B virus HA gene in B/Victoria-lineage virus (BV) in Beijing during 2017-2018.@*Methods@#Thirty BV virus strains isolated from MDCK cell culture by 17 laboratories in Beijing were collected. The antigenicity was analyzed by comparing with the vaccine strain recommended by WHO. The total viral nucleic acid was extracted and HA gene was amplified by RT-PCR and sequenced. The phylogenetic tree was constructed by HA and mutant sites were analyzed.@*Results@#Among 30 strains of BV, 23 strains (76.7%) were low-reactive strains, other 7 strains (23.3%) were related to the vaccine. The phylogenetic analysis showed that the HA gene of all 30 strains located in Clade 1A branch. In addition, amino acid mutations occurred in 8 sites, and 6 of them located in the antigen determining region.@*Conclusions@#There was a correlation between the high proportion of low-reactive antigenicity and 6 aa variation in antigenic determinants involved in HA region of BV influenza virus between 2017-2018, which provides an important laboratory basis for the recommendation of BV influenza vaccine.

Occurrence of Cryptosporidium, Enterocytozoon bieneusi and Blastocystis genotypes in Rousettus leschenaultii collected from an urban public park of Hainan Province, China

Objective: To obtain information about the occurrence of Blastocystis spp., Cryptosporidium spp., and Enterocytozoon (E.) bieneusi in fruit bats (Rousettus leschenaultii) collected from an urban public park Hainan Province, China and to analyze the genetic characteristics of the obtained parasites carried by those bats. Methods: On 4th June 2019, ten piles of fresh faecal sample of fruit bats were collected from the Wanlvyuan Gardens in central Haikou, Hainan of China. Blastocystis spp., Cryptosporidium spp., and E. bieneusi were examined by sequencing analysis of the small subunit rRNA gene or internal transcribed spacer (ITS) gene. Results: Among the 10 DNA specimens analyzed, seven (70.0%) were positive for Cryptosporidium spp. and two (20.0%) were positive for E. bieneusi but none of them were positive for Blastocystis. For Cryptosporidium, two novel genotypes were identified which shared 98.2% and 94.4% homology with Cryptosporidium (C.) andersoni Type C, respectively, and were named as C. andersoni Type D (in 6 specimens) and E (in one specimens). The two E. bieneusi-positive isolates were identified as a known zoonotic genotype (PigEbITS7) and a novel genotype (named HNB-I) respectively. Conclusions: The finding of C. andersoni and E. bieneusi genotype PigEbITS7 in fruit bats in Hainan, China suggests that these parasites carried by fruit bats can be transmitted to other animals and humans to cause zoonotic infections.

Isolation and identification of the first D8 genotype measles virus in Liaoning province / 中华实验和临床病毒学杂志

Objective@#To analyze the gene characterization on the first imported D8 genotype measles virus in Liaoning province.@*Methods@#In this study, Vero/Slam cells were used to isolate measles viruses from throat swabs. Fragments of the H gene (1854 nucleotides) and N gene (450 nucleotides) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the PCR products were sequenced and analyzed.@*Results@#The measles virus isolates and World Health Organization (WHO) D8 genotype reference strain (MVi/Manchester.GBR/30.94) belonged to the same branch in the genetic relationship tree. The nucleotide homology of the N and H gene was 98.9%. Phylogenetic trees were constructed with reference strains of the genotype D8 measles virus of China downloaded from GenBank. The result showed that the nucleotide similarities between the measles virus isolated in this study and the D8 genotype measles virus prevalent in Hong Kong from 2012 to 2016 and MVi/LosPatios.COL/11.18/D8 in Columbia was 100%.@*Conclusions@#It is the first time to do surveillance for the D8 genotype measles virus since measles virus surveillance was carried out. It was of great significance to accumulate the bases of measles virus molecular epidemiology in Liaoning province, and helpful to analyze and trace the transmission of measles virus in the whole country and the world.

Characterization analysis of gM, gL genes of varicella zoster virus in six provinces of China / 中华预防医学杂志

Objective@#To analyze the genetic characterization of glycoprotein M(gM.),glycoprotein L(gL) of varicella zoster virus.@*Methods@#According to the program of "Ministry of Science and Technology of China" , Based on the 12 suspected VZV patients monitored in Beijing (1 case), Shanghai (5 cases), Jilin (2 cases), Qinghai (1 case), Guangdong (2 case) and Sichuan (case) in 2007-2015. A total of 12 Vesicle fluid and throat swab samples were collected. Positive samples were identified by Agarose gel electrophoresis and two glycoprotein genes were amplified by polymerase chain reaction (PCR). Nucleotide sequences were determined and analyzed by PCR amplification of VZV positive specimens V-OKA-BK of the domestic varicella attenuated live vaccine and the Varilrix-1 of the imported attenuated live vaccine. Nucleotide sequences of VZV positive specimens, vaccine strains (V-OKA-BK, varilrix-1) and GenBank foreign wild strains (41 strains), parent strains (P-oka), vaccine strains (V-oka, Varilrix, Varivax) were compared using BioEdit and MEGA 5.0.@*Results@#12 specimens were VZV positive. Compared with the vaccine strains and the parent strains, the GM gene of 1 positive specimen had radical mutation at 86686 sites, which resulted in amino acid mutation, 5 positive specimens had base mutation at 87844 sites, and 30 strains of foreign wild strains had the same variation at 87 844 sites. 1 positive specimens of gL gene in 101245 sites had base mutation, and led to amino acid mutation, 6 positive specimens at 101624, 101625, 101626 sites had base of loss and the foreign wild strains in these 3 sites had the same variation. Compared with the vaccine strains, the nucleotide and amino acid homology of gM of 12 VZV positive specimens were 99.2%-100% and 98.2%-100%, respectively, and gL of those were 99.3%-100% and 98.6%-100%, respectively. Compared with 41 strains of foreign wild strains, homology of gM's nucleotides and amino acid were 99.3%-100% and 98.5%-100%, respectively; 99.1%-100% and 98.6%-100% for gL. The results of phylogenetic analysis showed that 7 VZV positive samples were on the same branch with 4 vaccine strains and p-oka strain. Based on gL, 12 VZV positive samples were on the same branch as the vaccine strains and p-oka strain.@*Conclusion@#This study demonstrates that the genes of gM, gL are highly conserved and remain stable immunogen, which may be involved in the attenuation of VZV and need to be further researched.

D-loop haplotype diversity in Brazilian horse breeds

Abstract The first horses were brought to Brazil by the colonizers after 1534. Over the centuries, these animals evolved and adapted to local environmental conditions usually unsuitable for exotic breeds, thereby originating locally adapted Brazilian breeds. The present work represents the first description of maternal genetic diversity in these horse breeds based on D-loop sequences. A D-Loop HSV-I fragment of 252 bp, from 141 horses belonging to ten Brazilian breeds / genetic groups (locally adapted and specialized breeds) were analysed. Thirty-five different haplotypes belonging to 18 haplogroups were identified with 33 polymorphic sites. Haplotype diversity (varying from 0.20 to 0.96) and nucleotide diversity (varying from 0.0039 to 0.0239) was lower for locally adapted than for specialized breeds, with the same pattern observed for FST values. Haplogroups identified in Brazilian breeds are in agreement with previous findings in South American samples. The low variability observed mainly in locally adapted breeds, indicates that, to ensure conservation of these breeds, careful reproductive management is needed. Additional genetic characterization studies are required to support accurate decision-making.

Analysis of genetic characterization on hemagglutinin gene of influenza B virus isolated in Fujian province, 2010-2015 / 中华实验和临床病毒学杂志

Objective@#To understand the epidemiological and virological features of influenza B viruses and the difference between the vaccine strains and epidemic strains, the antigenic and genetic characteristics on hemagglutinin (HA) gene of influenza B viruses circulating in Fujian during 2010-2015.@*Methods@#The representative strains were selected randomly according to the lineage of influenza B viruses isolated from network laboratory in Fujian, 2010-2015. Viral RNA was extracted and gene fragments were amplified by reverse transcription polymerase chain reaction (RT-PCR ) and the PCR products were sequenced. The complete HA gene sequence was obtained and analyzed via bioinformatics.@*Results@#Compared to the vaccine strains recommended by WHO, there were significant changes in genetic and antigenic characteristics on HA gene of B Yamagata lineage viruses from 2010 to 2015, especially in 2010, 2014 and 2015. There were major five amino acid residues substitutions (116, 150, 165, 196 and 202) involved in antigenic determinants, and the variable sites gradually increased as time on over. However, the variability of B Victoria lineage viruses on HA gene was less and there was no obvious trend over time. The results showed that the B Yamagata vaccine strains of 2010 and 2015 recommended by WHO had poor protective effect on influenza virus infection, while the B Victoria vaccine strain still play a satisfactory protective effect on humans in Fujian.@*Conclusions@#With time on, influenza B Yamagata lineage viruses had gradually mutated, causing a poorly match with vaccine strains in part of year, and emerging antigenic drift phenomenon. Strengthening further surveillance of mutations of B influenza virus remains essential to allow for early warning of influenza epidemic.

Análisis genotípico de aislamientos de Mycobacterium tuberculosis en La Habana en 2009 / Genotyping of mycobacterium tuberculosis isolates in Havana, 2009

INTRODUCCIÓN: la búsqueda de alternativas al polimorfismo de la longitud de los fragmentos de restricción (RFLP, siglas en inglés) con la sonda IS 6110 en la genotipificación de Mycobacterium tuberculosis ha propiciado el desarrollo de la tipificación con número variable de repeticiones en tándem de unidades repetitivas interespaciadas de micobacterias (MIRU-VNTR, siglas en inglés). OBJETIVO: evaluar la diseminación de genotipos de M. tuberculosis en La Habana en 2009. MÉTODOS: se estudiaron 80 aislamientos procedentes de unidades de salud durante 2009 y se caracterizaron por tipificación MIRU-VNTR-15. Los genotipos se expresaron como códigos numéricos según el número de copias de cada MIRU-VNTR amplificado, y se analizaron con la herramienta bioinformática en línea MIRU-VNTR plus. Se utilizó MIRU-VNTR-24 como tipificación secundaria en los aislamientos agrupados por MIRU-VNTR-15. RESULTADOS: con MIRU-VNTR-15 se definieron 41 genotipos diferentes; entre ellos, 33 únicos (41,25 por ciento), y ocho que agruparon a 47 aislamientos (58,75 por ciento). La tipificación MIRU-VNTR-24 logró diferenciar sólo el 5 por ciento de éstos, disminuyendo el porcentaje de agrupamiento a 53,75 por ciento. CONCLUSIONES: el elevado agrupamiento encontrado sugirió transmisión reciente, lo que pudo tener influencia en la incidencia de tuberculosis en La Habana en 2009(AU)

INTRODUCTION: the search for alternatives to restriction fragment length polymorphism (RFLP) with the IS6110probe for the genotyping of Mycobacterium tuberculosis has paved the way for the development of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing (MIRU-VNTR). OBJECTIVE: evaluate the spread of M. tuberculosis genotypes in Havana in 2009. METHODS: eighty isolates obtained from healthcare centers during 2009 were examined and characterized by 15-loci MIRU-VNTR typing. The genotypes were expressed as numerical codes according to the copy number of each amplified MIRU-VNTR, and they were analyzed with the online bioinformatic tool MIRU-VNTR plus. 24-loci MIRU-VNTR was used for secondary typing of isolates grouped by 15-loci MIRU-VNTR. RESULTS: forty-one different genotypes were defined with 15-loci MIRU-VNTR. Of these, 33 were single (41.25 percent), whereas 8 clustered 47 isolates (58.75 percent). Only 5 percent of the latter could be differentiated by 24-loci MIRU-VNTR, lowering the percentage of clustering to 53.75 percent. CONCLUSIONS: the high clustering values revealed by the study suggest that transmission was recent. This may have had an influence on the incidence of tuberculosis in Havana in 2009(AU)

Isolation, in vitro propagation, genetic analysis, and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlandia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlandia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlandia and Sao Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.

Prevalence and genetic characterization of Cryptosporidium spp. and Cystoisospora belli in HIV-infected patients / Prevalência e caracterização genética de Cryptosporidium spp. e Cystoisospora belli em pacientes infectados pelo HIV

Cryptosporidium spp. and Cystoisospora belli are monoxenic protozoa that have been recognized as the causative agents of chronic diarrhea in immunocompromised individuals, especially HIV-infected subjects. The objective of this study was to evaluate the frequency of these intestinal protozoa in HIV-positive patients in the Triângulo Mineiro region of Brazil and to correlate the presence of these infections with clinical, epidemiological and laboratory data of the patients. Oocysts were detected in stool samples of 10 (16.9%) of the 59 patients studied, while Cryptosporidium spp. were present in 10.1% (6/59) and C. belli in 6.7% (4/59). The frequency of these parasites was higher among patients with diarrheic syndrome and CD4+ T lymphocyte counts < 200 cells/mm 3 , demonstrating the opportunistic characteristic of these infections. A significant association was observed between the lack of adherence to antiretroviral therapy and the presence of Cryptosporidium spp. and/or C. belli. Parasitism with Cryptosporidium spp. was more frequent in February and April, the months following the period of high rainfall. The same was not observed for C. belli. Genetic characterization of two isolates led to the identification of Cryptosporidium parvum, one of the main species associated with the zoonotic transmission of cryptosporidiosis.

Cryptosporidium spp. e Cystoisospora belli são protozoários monoxenos reconhecidos como agentes causadores de diarréia crônica em indivíduos imunocomprometidos, especialmente aqueles infectados pelo HIV. Os objetivos deste estudo foram o de avaliar a frequência destes protozoários em pacientes HIV - positivos na região do Triângulo Mineiro, Brasil, e correlacionar a presença destas infecções com dados clínicos, epidemiológicos e laboratoriais dos pacientes. Oocistos foram detectados em amostras fecais de 10 (16,9%) dos 59 pacientes estudados, sendo 10.1% (6/59) das amostras positivas para Cryptosporidium spp. e 6,7% (4/59) das amostras positivas para C. belli. A frequência destes parasitos foi maior entre pacientes com síndrome diarreica e contagem de linfócitos T CD4+ < 200 cells/mm 3 , o que demonstra o caráter oportunista destas infecções. Foi observada uma associação significativa entre a falta de aderência à terapia antiretroviral e a presença de Cryptosporidium spp. e/ou C. belli. Parasitismo por Cryptosporidium spp. foi mais frequente em fevereiro e abril, meses subsequentes ao período chuvoso. O mesmo não foi observado para C. belli. A caracterização genética de dois isolados levou à identificação de Cryptosporidium parvum, uma das principais espécies associadas com a transmissão zoonótica da criptosporidiose.

Molecular characterization of rabies virus isolated from non-haematophagous bats in Brazil / Caracterização molecular do vírus da raiva isolado de morcegos não-hematófagos no Brasil

INTRODUCTION: Rabies is an important zoonosis that causes thousands of deaths worldwide each year. Although the terrestrial cycle, mainly transmitted by dogs, is controlled in Brazil, the aerial cycle remains a serious public health issue, besides the economic problem. In the aerial cycle, the haematophagous bat Desmodus rotundus is the main source of infection, where several different species of non-haematophagous bats can be infected and can transmit the virus. METHODS: The aim of this work was to study the epidemiological pattern of rabies using antigenic characterization with monoclonal antibodies and genetic characterization by reverse-transcriptase polymerase chain reaction followed by sequencing and phylogenetic analysis of non-haematophagous bats' and herbivorous animals' central nervous system samples from the western region of the State of São Paulo, Brazil. RESULTS: From 27 samples, 3 antigenic variants were identified: AgV-3, AgV-4, and AgV-6; and from 29 samples, 5 different clusters were identified, all belonging to the rabies virus species. CONCLUSIONS: Although only non-haematophagous bats were evaluated in the studied region, the majority of samples were from antigenic and genetic variants related to haematophagous bats Desmodus rotundus. Samples from the same antigenic variant were segregated in more than one genetic cluster. This study demonstrated the diversity of rabies virus genetic lineages presented and circulating in non-haematophagous bats in the studied region.

INTRODUÇÃO: A raiva é uma importante zoonose responsável por milhares de mortes anualmente em todo o mundo. Embora o ciclo silvestre, onde os cães são os principais transmissores esteja controlado no Brasil, o ciclo aéreo, onde o morcego hematófago Desmodus rotundus é o principal transmissor e diversas espécies de morcegos não hematófagos podem se infectar e transmitir o vírus, permanence como um importante problema econômico e de saúde pública. MÉTODOS: O objetivo deste trabalho foi a caracterização antigênica por meio da utilização de anticorpos monoclonais e a caracterização genética por meio da reação em cadeia pela polimerase pela transcriptase reversa seguida de análise filogenética em morcegos não hematófagos e animais domésticos herbívoros provenientes da região oeste do Estado de São Paulo. RESULTADOS: A análise antigênica de 27 amostras determinou três variantes distintas: Agv-3, AgV-4 e AgV-6; a análise genética de 29 amostras identificou 5 diferentes grupos, todos pertencentes a espécie Rabies virus. CONCLUSÕES: Ainda que apenas amostras de morcegos não hematófagos tenham sido analisadas, a maioria das variantes antigênicas e genéticas identificadas na região estava relacionada com a variante mantida pelos morcegos hematófagos Desmodus rotundus. Amostras de uma mesma variante antigênica segregaram em mais de um clado genético. Este estudo demonstrou a diversidade de linhagens genéticas do vírus da raiva presentes e circulantes em morcegos não hematófagos na região estudada.

Molecular characterization of Trypanosoma cruzi Mexican strains and their behavior in the mouse experimental model / Caracterização molecular de cepas mexicanas de Trypanosoma cruzi e seu comportamento no modelo experimental do camundongo

INTRODUCTION: For a long time, the importance of Chagas disease in Mexico, where many regarded it as an exotic malady, was questioned. Considering the great genetic diversity among isolates of Trypanosoma cruzi, the importance of this biological characterization, and the paucity of information on the clinical and biological aspects of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco. METHODS: Eight Mexican Trypanosoma cruzi strains were biologically and genetically characterized (PCR specific for Trypanosoma cruzi, multiplex-PCR, amplification of space no transcript of the genes of the mini-exon, amplification of polymorphic regions of the mini-exon, classification by amplification of intergenic regions of the spliced leader genes, RAPD - (random amplified polymorphic DNA). RESULTS: Two profiles of parasitaemia were observed, patent (peak parasitaemia of 4.6×10(6) to 10(7) parasites/mL) and subpatent. In addition, all isolates were able to infect 100 percent of the animals. The isolates mainly displayed tropism for striated (cardiac and skeletal) muscle. PCR amplification of the mini-exon gene classified the eight strains as TcI. The RAPD technique revealed intraspecies variation among isolates, distinguishing strains isolated from humans and triatomines and according to geographic origin. CONCLUSIONS: The Mexican T. cruzi strains are myotrophic and belong to group TcI.

INTRODUÇÃO: Durante muito tempo, foi questionada a importância da doença de Chagas no México onde muitos a consideravam um padecimento exótico. Considerando a grande diversidade genética existente, entre os isolados de Trypanosoma cruzi, a importância da caracterização biológica desses e o escasso número de informações sobre os aspectos clínicos e biológicos da doença de Chagas no México, o objetivo deste trabalho foi realizar a caracterização biológica e molecular de isolados de Trypanosoma cruzi originários de diferentes áreas endêmicas deste país, principalmente do Estado de Jalisco. MÉTODOS: Oito cepas mexicanas de Trypanosoma cruzi foram caracterizadas biologicamente e geneticamente (PCR específica para Trypanosoma cruzi, PCR-multiplex, amplificação do espaço não transcrito dos genes do mini-exon, amplificação das regiões polimórficas do gene do mini-exon, classificação pela amplificação de regiões intergênicas dos genes do spliced leader, RAPD - random amplified polymorphic DNA). RESULTADOS: Foram observados dois tipos de parasitemia: patente com picos máximos de parasitemia entre 4,6x10(6) e 10(7) parasitas/mL e subpatente. Além disso, todos os isolados foram capazes de infectar 100 por cento dos animais. Observou-se tropismo predominante pelo músculo estriado (cardíaco e esquelético). As técnicas de PCR do gene do mini-éxon classificaram as oito cepas como TcI e a técnica de RAPD mostrou variação intra-especifica das mesmas, separando as cepas isoladas de humanos daquelas de triatomíneos e por origem geográfica. CONCLUSÕES: As cepas mexicanas de Trypanosoma cruzi são miotrópicas e correspondem ao TcI.

Caracterização e influência de rizóbios isolados de alfafa na germinação e desenvolvimento inicial de plântulas de arroz / Characterization and influence of alfafa rhizobia on germination and early growth of rice seedling

A inoculação de plantas leguminosas com rizóbios é um dos principais métodos biotecnológicos de utilização de micro-organismos em plantas visando à fixação biológica de nitrogênio na agricultura. No entanto, nos últimos anos, vêm sendo observada nesses micro-organismos a capacidade de produção de fitohormônios, principalmente o ácido indol-acético (AIA) e a promoção de crescimento em gramíneas. Dessa forma, os objetivos deste trabalho foram quantificar o ácido indol-acético produzido por rizóbios isolados de alfafa, avaliar o efeito da inoculação desses micro-organismos na germinação de sementes de arroz e realizar a caracterização genética desses isolados. Nove rizóbios isolados de nódulos de alfafa foram avaliados quanto a sua capacidade de produção de equivalentes de AIA e a influência da inoculação desses micro-organismos na germinação e desenvolvimento de plântulas de arroz. Os rizóbios produtores de AIA foram identificados pelo sequenciamento da região do gene 16S do DNAr. A produção de equivalentes ao ácido indol-acético foi observada em todos rizóbios, com valores que variaram de 43,04 a 101,26µg mL-1 em meio de cultura. Com relação à germinação das sementes de arroz, a inoculação com rizóbios acelerou o processo e o crescimento de suas plântulas. Os rizóbios UFRGS Ms58, Ms515, Ms195, Ms205, Ms2010 e 2012 foram identificados como pertencentes à espécie Sinorhizobium meliloti e UFRGS Ms55, Ms72 e Ms75 à espécie Rhizobium sp.

The inoculation of leguminous plants with rhizobia is one of the main methods of biotechnological use of microorganisms in order to obtain biological nitrogen fixation in agriculture. However, in recent years it has been attributed to these microorganisms the ability to produce phytohormones, mainly indole acetic acid (IAA), and to promote the growth in grasses. Thus, the objectives of this study were to quantify the indole acetic acid produced by rhizobia from alfalfa and to evaluate the effect of inoculation of these microorganisms on the germination of rice seed and to perform the genetic characterization of these isolates. Nine rhizobia, from nodules of alfalfa, were evaluated for their ability to produce IAA equivalents and for their influence in inoculating these microorganisms on germination and seedling development of rice. Moreover, these rhizobia producers of IAA were identified by the 16S region of DNAr. The equivalent production of indole acetic acid was observed in all tested isolates, with values ranging from 43.04 to 101.26µg mL-1 in culture medium. Regarding the germination of rice seeds, the inoculation with rhizobia accelerated this germination and its growth. Microorganisms UFRGS Ms58, UFRGS Ms515, UFRGS Ms195, UFRGS Ms205, UFRGS Ms2010 and UFRGS 2012 were identified as belonging to the species of Sinorhizobium meliloti. Microorganisms Ms55 UFRGS, UFRGS Ms75 and UFRG Ms72 were identified as belonging to the species of Rhizobium sp.

Caracterização genotípica dos isolados de Escherichia coli provenientes de frangos de corte / Genotypically characterization of Escherichia coli isolates from poultry

Caracterizaram-se genotipicamente os isolados de Escherichia coli oriundos de fígado de frangos provenientes de dois matadouros avícolas. Foram coletadas 62 amostras de fígados de frangos, sendo 30 macroscopicamente inalterados e 32 com alteração macroscópica e que originaram no descarte da carcaça. Isolaram-se 30 cepas de Escherichia coli pelo método clássico, sendo 21 isoladas de fígados inalterados e nove provenientes de carcaças rejeitadas. Utilizou-se a reação em cadeia de polimerase para verificação de genes de virulência de E. coli, incluindo o gene de resistência sérica (iss) para identificação de E. coli patogênica para aves, o gene para Shiga cytotoxin 1 e 2 (stx) para identificação de E. coli enteroemorrágica, o gene bfpA para identificação de E. coli enteropatogênica e os genes para toxinas LT-I (elt) e ST-I (stI) para identificação de E. coli enterotoxigênica. Identificou-se iss em 83,3 por cento (25/30) dos isolados, sendo 76,2 por cento (16/21) provenientes de fígados de animais hígidos, e detectou-se stx em 13,3 por cento (4/30). Os genes stx e iss foram identificados em três fígados, caracterizando infecção mista. Os genes não foram observados em um isolado de E. coli pelo método clássico. Faz-se necessária a utilização de tecnologias para identificação e prevenção de Escherichia coli nos aviários e matadouros avícolas.

The isolates of Escherichia coli from chicken livers from two slaughterhouses were genotypically characterized in 62 samples. Thirty samples were macroscopically unchanged and 32 demonstrated alterations that led to the disposal of carcass for sanitary inspection. Thirty Escherichia coli strains from 21 unchanged and 9 from carcasses that were rejected were isolated through the classical method. Polymerase Chain Reaction was performed to verify E. coli virulence of the following genes: serum resistance (iss), to identify avian pathogenic E. coli; Shiga cytotoxin 1 and 2 (stx), to identify enterohaemorrhagic E. col; bfpA, to identify enteropathogenic E. coli; LT-I (elt) and ST-I (stI) toxins to identify enterotoxigenic E. coli. Iss gene was identified in 83.3 percent (25/30), being 76.2 percent (16/21) from E. coli isolated strains from healthy animals. stx gene was identified in 13.3 percent (4/30) of E. coli isolates, and in three of these samples was identified as stx and iss, featuring a mixed infection. The genes were not identified in one E. coli isolated from the classic method. Thus, it is necessary to use advanced technologies to identify and prevent Escherichia coli contamination in poultry farms and slaughterhouses.

Characterization of rabies virus isolated from a colony of Eptesicus furinalis bats in Brazil / Caracterização do vírus da raiva isolado de uma colônia de morcegos Eptesicus furinalis, do Brasil

Some bat species have adapted to the expanding human population by acquiring the ability to roost in urban buildings, increasing the exposure risk for people and domestic animals, and consequently, the likelihood of transmitting rabies. Three dead bats were found in the yard of a house in an urban area of Jundiaí city in the state of São Paulo in southeast Brazil. Two of the three bats tested positive for rabies, using Fluorescent Antibody and Mouse Inoculation techniques. A large colony of Eptesicus furinalis was found in the house's attic, and of the 119 bats captured, four more tested positive for rabies. The objectives of this study were to report the rabies diagnosis, characterize the isolated virus antigenically and genetically, and study the epidemiology of the colony.

Algumas espécies de morcegos têm se adaptado ao uso de abrigos em construções urbanas, aumentando a possibilidade de contato desses morcegos com pessoas e animais domésticos e conseqüentemente, o potencial risco de transmissão de raiva. Três morcegos foram encontrados no jardim de uma casa na área urbana da cidade de Jundiaí, Estado de São Paulo, Sudeste do Brasil, dois deles foram positivos para raiva pelas técnicas de imunofluorescência e inoculação em camundongos. Uma grande colônia de E. furinalis foi identificada, vivendo no sótão da casa e 119 morcegos foram encaminhados para diagnóstico de raiva, com mais quatro morcegos positivos. O objetivo desse estudo é apresentar a caracterização genética e antigênica do vírus da raiva isolado desses morcegos e o estudo epidemiológico da colônia.

Microsatellite characterization of Cimarron Uruguayo dogs

Various genetic markers, including microsatellites, have been used to analyze the genetic polymorphism and heterozygosity in canine breeds. In this work, we used nine microsatellite markers to investigate the genetic variability in Cimarron Uruguayo dogs, the only officially recognized native canine breed in Uruguay. DNA from 30 Cimarron Uruguayo dogs from northeastern and southern Uruguay was analyzed. The allelic frequencies for each micro-satellite, the genetic variability and the consanguinity were calculated, as were the polymorphic information content (PIC) and the probability of exclusion (PE). All of the microsatellites studied were polymorphic. FH 2361, FH 2305 and PEZ 03 were the most informative, with PIC values > 0.7, in agreement with results for other canine breeds. The PE values for the markers were within the ranges previously described and were generally greater for microsatellites with higher PIC values. The heterozygosity value (0.649) was considered high since only nine microsatellites were analyzed. Compared with data for other breeds, the results obtained here indicate that Cimarron Uruguayo dogs have high genetic diversity.