Determination of investigation of the link between human and animal Brucella isolates in Iran using multiple-locus variable number tandem repeat method comprising 16 loci (MLVA-16)

ABSTRACT Background: Epidemiological studies are important tools to assess the diversity of Brucella isolates and to estimate their epidemiological relationship among isolates from different geographical origins. In this study the MLVA16 (multiple-locus variable number tandem repeat analysis based on 16 loci) was employed to investigate the diversity of Brucella spp. Isolated from humans and animals for epidemiological purposes and to determine the most common Brucella genotypes in Iran. Methods: We designed a molecular-based study to evaluate the potential reservoirs of human brucellosis. After isolation and identification of 54 Brucella spp human and animal specimens from three regions of Iran, bacterial genomic DNA was extracted MLVA with three panel was used for the genotyping of isolates. The size of PCR products were analyzed and converted to repeat unit numbers using a published allele numbering system and data set was imported into Bionumerics. Results: Three isolates (5.55%) were identified as Brucella abortus and 51 (94.44%) as Brucella melitensis. Two isolates of Brucella abortus were from humans and one from an animal. Thirty-four Brucella melitensis isolates were from humans and 17 from animals. Using MLVA16-genotyping, 54 isolates with genetic similarity coefficient of 80% were divided into 46 genotypes and 22 genotypes were represented by a single isolate, while 4, 2, 1 and 2 genotypes were represented by 2, 3, 4 and 7 isolates, respectively. The most prevalent genotype was represented by 14 isolates. There were two other frequent genotypes each represented by seven isolates, among which only one was restricted to a geographic region. Discriminatory power for each locus was determined in this study and panel 2B shows the high discretionary power [Bruce04 (0.837), Bruce30 (0.806), Bruce 09 (0.787), Bruce 07 (0.772), Bruce16 (0.766)]. Conclusion: MLVA16 analysis of 54 Brucella isolates showed high level polymorphism in their genotypes. Only two genotypes, each observed in seven isolates, were related to one another and only one of these genotypes were found in to two separate regions.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

DRD4 genotyping may differentiate symptoms of attention-deficit/hyperactivity disorder and sluggish cognitive tempo

Objective: Studies to reduce the heterogeneity of attention-deficit/hyperactivity disorder (ADHD) have increased interest in the concept of sluggish cognitive tempo (SCT). The aim of this study was to investigate if the prevalence of two variable-number tandem repeats (VNTRs) located within the 3′-untranslated region of the DAT1 gene and in exon 3 of the dopamine D4 receptor (DRD4) gene differ among four groups (31 subjects with SCT but no ADHD, 146 individuals with ADHD but no SCT, 67 subjects with SCT + ADHD, and 92 healthy controls). Methods: We compared the sociodemographic profiles, neurocognitive domains, and prevalence of two VNTRs in SCT and ADHD subjects versus typically developing (TD) controls. Results: The SCT without ADHD group had a higher proportion of females and lower parental educational attainment. Subjects in this group performed worse on neuropsychological tests, except for psychomotor speed and commission errors, compared to controls. However, the ADHD without SCT group performed significantly worse on all neuropsychological domains than controls. We found that 4R homozygosity for the DRD4 gene was most prevalent in the ADHD without SCT group. The SCT without ADHD group had the highest 7R allele frequency, differing significantly from the ADHD without SCT group. Conclusion: The 7R allele of DRD4 gene was found to be significantly more prevalent in SCT cases than in ADHD cases. No substantial neuropsychological differences were found between SCT and ADHD subjects.

Evidence of in utero infection by Mycobacterium avium subsp. paratuberculosis using Multiple-Locus Variable-number tandem-repeat Analysis: first report in Argentina / Evidência de infecção intra-uterina por Mycobacterium avium subsp. paratuberculosis usando Multiple-Locus Variable-number tandem-repeat Analysis: primeiro relato na Argentina

A pregnant heifer with an advanced clinical stage of paratuberculosis was reported in a herd in Argentina. Thus, the animal was euthanized and samples of organs of the cow and its fetus was taken and cultured for bacteriology in specific medium. Tissues were analyzed by histopathology (hematoxylin-eosin and Ziehl-Neelsen staining). Histopathological analysis of the cow's samples revealed the presence of lesions consistent with paratuberculosis, and Ziehl-Neelsen staining revealed the presence of acid-fast bacilli, whereas the fetal tissues showed absence of lesions but the presence of acid-fast bacilli by Ziehl-Neelsen staining. After growing in specific medium, colonies in tissues from both cow and fetus were positive for IS900-PCR, confirming the presence of Mycobacterium avium subsp. paratuberculosis (MAP). Finally, the isolates were typed by Multiple-Locus Variable-number tandem-repeat Analysis (MLVA), which confirmed the epidemiological link between them. This study is the first in Argentina to report the detection of MAP that shares an identical MLVA type in a pregnant cow and its fetus. The results of this study are consistent with previous reports and highlight the intra-uterine transmission of MAP as an important source of infection within herds.(AU)

Uma novilha prenha em estado clínico avançado de paratuberculose foi observada em um rebanho bovino na Argentina. O animal foi eutanasiado e foram colhidas amostras dos seus órgãos e dos órgãos feto as quais foram cultivadas para bacteriologia em meio específico. Os tecidos foram examinados por histopatologia (coloração de hematoxilina-eosina e Ziehl-Neelsen). Na histopatologia das amostras colhidas da novilha foram observadas lesões compatíveis com paratuberculose e a coloração de Ziehl-Neelsen revelou a presença de bacilos álcool-ácido resistentes, nos tecidos fetais não foram observadas lesões, porém a coloração de Ziehl-Neelsen revelou a presença de bacilos álcool-ácido resistentes. Após o crescimento em meio específico, as colônias foram positivas para o teste IS900-PCR nos tecidos de ambos, vaca e feto, confirmando a presença de Mycobacterium avium subsp. paratuberculosis. Por fim, os isolados foram tipados por Multiple-Locus Variable-number tandem-repeat Analysis, confirmando a relação epidemiológica entre eles. Este estudo relata a primeira detecção de Mycobacterium avium subsp. paratuberculosis na Argentina em que houve o compartilhamento de um tipo idêntico de MLVA em uma vaca prenhe e no seu feto. Os resultados deste estudo são consistentes com relatos anteriores e destacam a transmissão intra-uterina de Mycobacterium avium subsp. paratuberculosis como importante fonte de infecção nos rebanhos de bovinos.(AU)

Identification of Brucella abortus biovar 4 of bovine origin in Colombia / Identificación de Brucella abortus biovar 4 de origen bovino en Colombia

The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These isolates are included in the collection of the Germplasm Bank of Microorganisms of Animal Health Interest -Bacteria and Virus (BGSA-BV). The identification was carried out through conventional methods such as macro and microscopic morphological descriptions, enzymatic activity, biochemical profile, substrate use and sensitivity to dyes. Complementary genotypic characterization was carried out using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis-Erytritol (AMOS-ERY-PCR), RFLP-IS711, by southern blot hybridization, as well as by the multiple locus variable number of tandem repeat analysis (MLVA) using the ery gene and the insertion sequence IS711 and variable number of tandem repeats (VNTR) as molecular markers. The results of the phenotypic and molecular characterization allowed to identify twelve isolates as B. abortus biovar 4 as well as to differentiate field from vaccine strains. This is the first study on the phenotypic and molecular identification of B. abortus isolates in Colombia. It was concluded that the phenotypic and molecular identification of twelve isolates as B. abortus biovar 4 could be achieved using conventional and molecular techniques with enough resolution power. The identification of these isolates to the biovar level in taxonomic and epidemiological terms will allow the use of this genetic resource as reference strains in future research. This finding constitutes the basis for identifying biotypes not previously reported in the country that might be useful to support brucellosis survey programs in Colombia.

El objetivo de este estudio fue identificar 12 aislamientos de Brucella abortus de origen bovino procedentes del departamento de Narino, Colombia, hasta la descripción de biovar. Estos aislamientos conforman la colección del Banco de Germoplasma de Microorganismos de Interés en Salud Animal, Bacterias y Virus. La identificación se hizo mediante métodos convencionales, como la descripción morfológica macro y microscópica de actividad enzimática, de perfiles bioquímicos, de utilización de sustratos y de sensibilidad a colorantes. Se hizo una caracterización genotipica complementaria mediante PCR múltiple para Brucella abortus, Brucella melitensis, Brucella ovisy Brucella suis-eritritol (AMOS-ERY-PCR); RFLP-/S7II; hibridación Southern blot y análisis multi-locus de repeticiones en tándem de número variable (MLVA), empleando como marcadores moleculares el gen ery, la secuencia de inserción /S711 y el número variable de repeticiones en tándem (VNTR). Los resultados de la caracterización fenotípica y molecular permitieron identificar 12 aislamientos de campo como B. abortus biovar 4 y diferenciar cepas de campo de cepas vacunales. Este es el primer estudio de identificación fenotípica y molecular de aislamientos de B. abortus en Colombia. Por su importancia taxonómica y epidemiológica, la identificación de estos aislamientos hasta el nivel de biovar permitirá disponer de recursos genéticos que se pueden emplear como cepas de referencia en futuras investigaciones. Estos resultados pueden considerarse como una base para la identificación de biotipos no reportados en el país y podrán ser utilizados en programas de monitoreo y vigilancia de la brucelosis bovina en Colombia.

Genotipificación de aislamientos del complejo Mycobacterium tuberculosis mediante MIRU-VNTR, Cali, Colombia, 2013-2015 / MIRU-VNTR genotyping of Mycobacterium tuberculosis in a population of patients in Cali, Colombia, 2013-2015

Resumen Introducción. La tuberculosis continúa siendo uno de los problemas de salud más importantes a nivel mundial y, con la infección por el virus de la inmunodeficiencia humana (HIV), constituye la principal causa de muerte por infecciones. En el 2016, se notificaron 6,3 millones de casos nuevos de la enfermedad. Objetivo. Describir los patrones genéticos determinados mediante la genotipificación del número variable de repeticiones en tándem de unidades repetitivas interespaciadas de micobacterias (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats, MIRU-VNTR) en la población de estudio y compararlos con los hallados en otros estudios locales e internacionales. Materiales y métodos. Mediante MIRU-VNTR, entre el 2013 y el 2015 se hizo la genotipificación de 105 muestras de ADN extraídas del esputo o de aislamientos en cultivo de M. tuberculosis provenientes de pacientes residentes en Cali con diagnóstico de tuberculosis pulmonar. La amplificación de 24 loci MIRU-VNTR se hizo por medio de la reacción en cadena de la polimerasa (PCR). Los amplicones resultantes se visualizaron por electroforesis en geles de agarosa (2 %) teñidos con SYBR Safe™. La asignación de los alelos se hizo con un análisis gráfico con el programa GelAnalyzer 2010. Los resultados obtenidos se analizaron con el algoritmo UPGMA y se compararon con las bases de datos internacionales MIRU-VNTRplus y SITVITWEB. Resultados. Se genotipificaron por completo 62 de las muestras y se obtuvieron 58 perfiles diferentes de MIRU-VNTR. Al comparar con las bases de datos internacionales, su distribución por linajes fue la siguiente: 54,8 % para el LAM, 25,8 % para el Haarlem, 14,5 % para el S, 3,2 % para el Beijing y 1,6 % para el Cameroon. Los patrones MIRU-VNTR correspondieron a 20 tipos internacionales de MIRU (MIRU International Types, MIT) diferentes, y los más frecuentes fueron el MIT 190 y el MIT 110, con 22,6 y 6,5 %, respectivamente. Conclusión. Estos resultados confirmaron hallazgos previos sobre el predominio de los linajes LAM y Haarlem en la ciudad y la presencia de los MIT encontrados en otra ciudad de Colombia.

Abstract Introduction: Tuberculosis continues to be one of the main public health problems in the world. Together with the HIV infection, it is one of the main causes of death due to infections worldwide. In 2016, 6.3 million new cases of the disease were reported. Objective: To describe the genetic patterns determined by genotyping using variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) in the study population and compare them with other studies carried out in Cali, Colombia, and the world. Materials and methods: We genotyped a total of 105 DNA samples extracted from sputum or culture isolates of the Mycobacterium tuberculosis complex, which were obtained from pulmonary tuberculosis diagnosed patients over the period 2013-2015, in Cali. We performed PCR amplification of 24 loci by MIRU-VNTR on the DNA extracted from the samples. The amplicons were visualized in agarose gel electrophoresis (2%) with SYBR Safe™ staining. Then, the alleles were designated by graphical analysis using the GelAnalyzer 2010 software. These results were analyzed using the UPGMA logarithm and compared with the registers from the MIRU-VNTR plus and SITVITWEB databases. Results: We genotyped 62 of the samples completely and we obtained 58 different MIRU-VNTR profiles. By comparing with the international databases, we determined the following distributions per lineage: LAM, 54.8%; Haarlem,25.8%; S, 14.5%; Beijing, 3.2%, and Cameroon, 1.6%. The MIRU-VNTR patterns corresponded to 17 different MITs; the most frequent were MIT 190 and MIT 110, with 22.6% and 6.5%, respectively. Conclusions: These results demonstrated previous observations about the predominance of the LAM and Haarlem lineages in the city, and the presence of the MITs found in another city of Colombia.

Comparative study of RFLP-IS6110 and MIRU-VNTR from Mycobacterium tuberculosis isolated in the state of Minas Gerais, Brazil

Abstract DNA genotyping of Mycobacterium tuberculosis has been widely applied in the understanding of disease transmission in many countries. The purpose of this study was to genotype the strains of M. tuberculosis isolated in patients with new tuberculosis (TB) cases in Minas Gerais, as well as to compare the similarity, discriminatory power, and agreement of the clusters between the IS6110 Restriction Fragment Length Polymorfism (RFLP) and 12 loci Variable Number Tandem Repeat - Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) techniques. It was observed that 32% (66/204) of the isolated strains in the RFLP-IS6110 and 50.9% (104/204) of the isolated strains in the MIRU-VNTR presented a similarity of equal to or above 85%. The RFLP-IS6110 and MIRU-VNTR proved to contain a high discriminatory power. The similarity index resulting from the RFLP showed no recent transmission. Good agreement was observed between the techniques when clusters were detected; however, the best epidemiological relationship was found when using the RFLP-IS6110.

Persistence of the same genetic type of Mycoplasma hyopneumoniae in a closed herd for at least two years / Persistencia del mismo tipo genético de Mycoplasma hyopneumoniae por al menos 2 anos en una piara sin reposición externa de animales

Two cross-sectional studies were carried out in 2013 and 2015 monitoring for Mycoplasma hyopneumoniae presence in a swine farm. In these studies, the genetic diversity of M. hyopneumoniae was assessed in clinical specimens using a Multiple Locus Variable-number tandem repeat Analysis (MLVA) targeting P97 R1, P146 R3 and H4 loci. The samples from August 2015 showed the MLVA profile prevalent in June 2013, therefore it can be concluded that a same genetic type of M. hyopneumoniae can persist for at least two years in a closed herd. In addition, the nested PCR reactions implemented in this study showed to be useful for MLVA typing in non-invasive clinical samples.

Dos estudios transversales fueron realizados en los anos 2013 y 2015 monitorizando la presencia de Mycoplasma hyopneumoniae en una piara. En esos estudios la diversidad genética de M. hyopneumoniae fue evaluada a partir de muestras clínicas utilizando un análisis multilocus de regiones repetidas en tándem (MLVA) de los loci P97 R1, P146 R3 y H4. Las muestras colectadas en agosto del 2015 mostraron el perfil de MLVA prevalente en junio del 2013, por lo tanto, se puede concluir que el mismo tipo genético de M. hyopneumoniae puede persistir por al menos 2 años en una piara sin reposición externa de animales. Además, las reacciones de PCR anidadas implementadas en este estudio mostraron ser útiles para la tipificación por MLVA a partir de muestras clínicas no invasivas.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Detection, quantification and genetic variability of Mycoplasma hyopneumoniae from apparently healthy and pneumonic swine / Detecção, quantificação e variabilidade genética de Mycoplasma hyopneumoniae a partir de suínos pneumônicos e aparentemente saudáveis

Molecular differences among Mycoplasma hyopneumoniae strains present in pneumonic lungs of swine have been largely studied. However, no comparative studies concerning the strains present in apparently healthy pigs have been carried out. This study aimed to detect, quantify and perform molecular analysis of M. hyopneumoniae strains in pig lungs with and without pneumonic lesions. The detection of M. hyopneumoniae was performed using multiplex PCR (YAMAGUTI, 2008), real-time PCR (STRAIT et al., 2008) and multiple VNTR amplification (VRANCKX et al., 2011). Molecular characterization of the strains was achieved by analysis of the VNTR copy number in P97R1, P146R3, H2R1 and H4. M. hyopneumoniae was detected in samples from healthy and pneumonic pigs and the amount of M. hyopneumoniae positive samples detected varied with the type of assay. The greater number of positive samples was identified by the multiple VNTR amplification combined with capillary electrophoresis. Using real-time PCR, 4.9*104 M. hyopneumoniae genome copies/mL was detected in apparently healthy lungs. A mean quantity of 3.9*106 M. hyopneumoniae genome copies/mL was detected in pneumonic lungs. The analysis of VNTR copy number demonstrated a high genetic variability of the M. hyopneumoniae strains present in apparently healthy and pneumonic lungs. Strains having 3 VNTR copy number in P97R1, were detected only in pneumonic lungs and strains having 40 and 43 VNTR copy number in P146R3 were detected only in apparently healthy lungs. Despite the genetic variability of M. hyopneumoniae, predominant strains in the swine farms could be identified...

As diferenças moleculares entre as estirpes de Mycoplasma hyopneumoniae presentes em pulmões de suínos com pneumonia tem sido estudadas. Porém, estudos comparativos relativos as estirpes presentes nos suínos aparentemente saudáveis não foram levados a cabo. O objetivo do estudo foi a detecção, quantificação e analise molecular de M. hyopneumoniae nos pulmões suínos com e sem lesões pneumônicas. Para a detecção de M. hyopneumoniae usaramse o PCR Multiplo (YAMAGUTI, 2008), o PCR a Tempo Real (STRAIT et al., 2008) e a amplificação de múltiplo VNTR (VRANCKX et al., 2011). A caracterização molecular das estirpes foi realizada mediante a análise do número de copias de VNTR em P97R1, P146R3, H2R1 e H4. O M. hyopneumoniae foi detectado em amostras de suínos saudáveis e pneumônicos e a quantidade de M. hyopneumoniae nas amostras positivas variou com o tipo de ensaio. O maior número de amostras positivas foi identificado pela amplificação de múltiplas VNTR combinado com a eletroforese de capilares. Usando o PCR a Tempo Real, 4.9*104 copias de genoma/mL de M. hyopneumoniae foram detectadas em pulmões aparentemente saudáveis. Uma quantidade média de 3.9*106 copias de genoma/mL de M. hyopneumoniae foi detectada em pulmões pneumônicos. A análise do número de copias de VNTR demonstrou uma elevada variabilidade...

Genotipos de cepas de Leptospira spp. aisladas de perros en Buenos Aires, Argentina / Genotypes of Leptospira spp. strains isolated from dogs in Buenos Aires, Argentina

La leptospirosis es una enfermedad infecciosa de amplia distribución global; endémica en Argentina. El objetivo de este estudio fue obtener los perfiles genéticos de las cepas de Leptospira spp. aisladas de casos clínicos de perros provenientes de la provincia de Buenos Aires, empleando el análisis de repeticiones en tándem de número variable en múltiples locus [multiple-locus variable-number tandem repeats analysis (MLVA)]. Fueron genotipificadas por MLVA ocho cepas aisladas de perros. Se obtuvo un perfil idéntico al de Leptospira interrogans serovar Canicola Hond Utrecht IV en las cepas denominadas Dogy y Mayo. Las cepas denominadas Bel, Sarmiento, La Plata 4581 y La Plata 5478 mostraron un perfil idéntico al genotipo de L. interrogans serovar Portlandvere MY 1039. La cepa denominada Avellaneda presentó un perfil idéntico al genotipo L. interrogans serovar Icterohaemorrhagiae RGA, y la cepa denominada SB mostró un perfil idéntico al genotipo de L. interrogans serovar Pomona Baires y similar al serovar Pomona Pomona. Sería de gran utilidad incluir un mayor número de cepas provenientes de distintas poblaciones caninas de diversas provincias de Argentina a fin de conocer los perfiles de las cepas circulantes en el país. La información así obtenida contribuirá al control de la leptospirosis en la población canina

Leptospirosis is an infectious disease of wide global distribution, which is endemic in Argentina. The objective of this study was to obtain the genetic profiles of Leptospira spp. strains isolated from clinical cases of dogs in the province of Buenos Aires by the multiple-locus variable-number tandem repeat analysis (MLVA). Eight isolated canine strains were genotyped by MLVA, obtaining the identical profile of Leptospira interrogans serovar Canicola Hond Utrecht IV in the strains named Dogy and Mayo. The strains named Bel, Sarmiento, La Plata 4581 and La Plata 5478 were identical to the profile of the genotype of L. interrogans serovar Portlandvere MY 1039.The strain named Avellaneda was identical to the genotype profile of L. interrogans serovar Icterohaemorrhagiae RGA and the strain named SB had the same profile as the L. interrogans serovar Pomona Baires genotype and was similar to the profile of serovar Pomona Pomona genotype. It would be useful to include a larger number of isolates from different dog populations in various provinces of Argentina and to characterize the genetic profiles of the strains circulating in the country. The information obtained will be useful for the control of leptospirosis in the dog population

Analysis of genetic diversity and differentiation of sheep populations in Jordan

Background Genetic diversity of sheep in Jordan was investigated using microsatellite markers (MS). Six ovine and bovine MS located on chromosomes 2 and 6 of sheep genome were genotyped on 294 individual from ten geographical regions. Results The number of alleles per locus (A), the expected heterozygosity (He) and observed heterozygosity (Ho) were measured. Overall A, He and Ho were 12.67, 0.820 and 0.684, respectively. On the other hand, genetic distances undoubtedly revealed the expected degree of differentiation among the studied populations. The finding showed closeness of three populations from south (Maan, Showbak and Tafeilah) to each other. Populations from the middle regions of Jordan (Karak, Madaba, Amman, AzZarqa and Mafraq) were found to be in one cluster. Only two populations of the middle region were an exception: AlSalt and Dead Sea. Finally, sheep populations from Irbid were located in separated cluster. It was clear that the studied predefined populations were subdivided from four populations and would be most probably accounted as ancestral populations. These results indicate that number of population is less than the predefined population as ten based on geographical sampling areas. Conclusions The possible inference might be that geographical location, genetic migration, similar selection forces, and common ancestor account for population admixture and subdivision of Awassi sheep breed in Jordan. Finally, the present study sheds new light on the molecular and population genetics of Awassi sheep from different regions of Jordan and to utilize the possible findings for future management of genetic conservation under conditions of climate changes and crossbreeding policy.

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.

Mycobacterium tuberculosis belonging to family LAM and sublineage RD Rio: common strains in Southern Brazil for over 10 years

A sublineage of Mycobacterium tuberculosis called RD Rio was described in 2007. Although only recently described, this strain may have been present previously in the population, and its identification in clinical isolates will elucidate bacterial transmission dynamics and host-pathogen interactions. This study evaluated the clonal diversity of the RD Rio sublineage in clinical isolates from Rio Grande-RS obtained between 1998 and 2001. Among the 45 samples analyzed by the MIRU-VNTR method, there were six clusters with two samples each and 33 orphan strains with unique pattern. The strains were distributed across several different lineages including LAM (34.04%), X (14.89%), Haarlem (12.77%), UgandaI (10.64%), S (4.26%), NEW-1 (2.13%) and Cameroon (2.13%); 14.89% of the strains matched to multiple lineages. RD Rio strains were present in 28.9% of the samples and 81.25% of the identified strains belonged to the LAM family. The high clonal diversity observed in this study is a constant feature in this region. The RD Rio sublineage has been in Rio Grande-RS since 1998. The continued monitoring of RD Rio in clinical isolates will enhance the understanding of its epidemiological significance.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Análise molecular de mycobacterium tuberculosis isolados de pacientes atendidos em Goiânia, Goiás, por meio do RFLP-IS6110 e do 15 loci MIRU-VNTR / Molecular analysis of Mycobacterium tuberculosis isolates from patients in Goiânia, Goiás, by IS6110-RFLP and VNTR-15loci miru

Atualmente, o controle da tuberculose é dependente de vários fatores tais como rápido diagnóstico, terapia adequada e meios de evitar futuras transmissões. Assim, a caracterização de linhagens de Mycobacterium tuberculosis por tipagem molecular por meio da técnica de RFLP-IS6110 representa uma contribuição primordial e tem sido amplamente utilizada nos estudos de genotipagem para que sejam traçadas cadeias de transmissão. No entanto, por causa da complexidade desta técnica e da dificuldade de interpretação dos dados, outras técnicas têm sido propostas. Entre elas, destaca-se o estudo do número variável de unidades repetitivas (MIRU-VNTR) indicado como novo padrão de genotipagem. O presente estudo teve como objetivo analisar as técnicas de RFLP-IS6110 e 15 loci MIRU-VNTR em isolados de pacientes atendidos no município de Goiânia, Goiás. Para isso, foram caracterizados geneticamente os isolados a fim de se estabelecerem possíveis ligações epidemiológicas entre os casos da doença. Também fez-se a comparação entre os resultados encontrados pelas duas técnicas e o cenário do Brasil. Os dados demonstraram que a técnica de 15 loci MIRU-VNTR discriminou mais os isolados que a técnica de RFLP-IS6110. Não foi encontrada associação epidemiológica entre os pacientes estudados. Os resultados validaram o uso da técnica 15 loci MIRU-VNTR para tipagem molecular de M. tuberculosis por apresentar maior poder discriminatório, boa eficiência para caracterizar geneticamente os isolados em Goiânia-GO, podendo, portanto, ser um método usado em estudo epidemiológico isolado ou em conjunto com outras técnicas...

Molecular analysis of Mycobacterium tuberculosis isolated from patients in Goiânia, Brazil, using RFLP-IS6110 and 15 loci MIRU-VNTR techniques.Currently, tuberculosis control is dependent on several factors, such as rapid diagnosis, appropriate therapy and measures to prevent future transmission. Thus, the characterization of strains of Mycobacterium tuberculosis by molecular typing using RFLP-IS6110, provides a major contribution, and has been widely used in genotyping studies in order to trace transmission pathways. However, due to the complexities of the technique and data interpretation, other techniques have been proposed. Among them, the study of the variable number of repeat units (MIRU-VNTR) has been indicated as a new standard method. This study aimed to apply and compare the RFLP-IS6110 and 15 loci MIRU-VNTR techniques for the analysis of isolates from tuberculosis patients treated in Goiânia, Brazil, in order to establish possible molecular epidemiological links between cases of the disease, and also to compare the results found by both techniques against the wider situation in Brazil. The results showed that 15 loci MIRU-VNTR discriminated between the isolates better than the RFLP-IS6110. No epidemiological link was observed among the patients studied. The results validate the use of the 15 loci MIRU-VNTR technique for molecular typing of M. tuberculosis, as it showed greater discriminatory power with good efficiency to genetically characterize the isolates in Goiânia, Goiás. This can be used in epidemiological studies alone or in conjunction with other molecular techniques...

Isolation of Leptospira borgpetersenii in synanthropic Didelphis albiventris in Jaboticabal, São Paulo, Brazil / Isolamento de Leptospira borgpetersenii em Didelphis albiventris sinantrópicos em Jaboticabal, São Paulo, Brasil

Leptospirosis is a waterborne disease and, therefore, stands out for the possibility of environmental contamination, the cross transmission between domestic and wild animals and humans. Opossum species are important reservoirs of this disease making them potential pathogen spreaders. Aiming to verify the presence of Leptospira spp. and the antibodies against Leptospira spp. in the Campus of São Paulo State University, in Jaboticabal, São Paulo, Brazil, freeliving wild life opossum (Didelphis albiventris) were captured for blood and urine sampling. Serological analysis was performed Microscopic Agglutination Test (MAT). Aliquots of urine were seeded in media Ellinghausen-McCullough- Johnson-Harris (EMJH) and Fletcher without antibiotics. The samples in which there was growth of leptospires were forwarded to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were genotyped using Multiple Locus Variable number tandem repeat Analysis (MLVA). Of the 15 analyzed animals, nine (60.0%) were reactant to Patoc serovar. The pathogenic specie Leptospira borgpetersenii was isolated and identified in three Didelphis albiventris. The isolation findings of pathogenic specie Leptopsira borgpetersenii in the urine culture of three Didelphis albiventris in a university campus are a major discovery in the area of preventive veterinary medicine and public health and open a discussion about the important role of free-living wild animals as reservoirs of this agent to domestic animals and humans, a condition that serves as a warning for the improvement of health practices...

A leptospirose é uma zoonose de veiculação hídrica e, portanto, se destaca pela possibilidade de contaminação ambiental, o que facilita a transmissão cruzada entre animais domésticos, selvagens e humanos. Espécies de gambás são importantes reservatórios dessa enfermidade, tornando-os potenciais disseminadores do agente. Com o objetivo de verificar a presença de Leptospira spp. e de anticorpos contra Leptospira spp. no Campus da Universidade Estadual Paulista, em Jaboticabal, foram capturados gambás (Didelphis albiventris) de vida livre para a colheita de amostras de sangue e de urina. As análises sorológicas foram efetuadas pela técnica de Soroaglutinação Microscópica (SAM). Alíquotas de urina foram semeadas nos meios Ellinghausen-McCullough-Johnson-Harris (EMJH) e Fletcher sem antibióticos. As amostras que apresentaram crescimento de espiroquetas foram levadas ao Laboratório de Leptospirose do Instituto de Patobiologia, no Instituto Nacional de Tecnologia Agropecuaria, Buenos Aires, Argentina e foram genotipadas com a técnica de Múltiplos Locus de Números Variáveis de Repetição em Tandem (MLVA). Dos 15 animais examinados pela SAM, nove (60,0%) foram reagentes à sorovariedade Patoc. Foi isolada e identificada a espécie patogênica Leptospira bosrpetersenii de três Didelphis albiventris. Os achados de isolamento da espécie patogênica Leptospira borgpetersenii na cultura de urina de três Didelphis albiventris são um grande descobrimento para as áreas da medicina veterinária preventiva e da saúde pública e reforçam a discussão sobre o importante papel dos animais selvagens de vida livre como reservatórios desse agente para animais domésticos e seres humanos, situação que serve de alerta para melhorias nas práticas sanitárias...

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.

Multiple-locus variable-number tandem repeat analysis of reference strains used for the diagnosis of leptospirosis in Argentina / Multiple-locus variable-number tandem repeat analysis de cepas de referencia usadas para el diagnóstico de leptospirosis en Argentina

Leptospirosis is a worldwide zoonosis caused by a spirochete that belongs to the genus Leptospira. In the last years, new methods, such as the PCR-based multiple-locus variable-number tandem repeat analysis (MLVA), have been developed for the genotyping of leptospires. In the present work, the MLVA patterns for all reference strains used in Argentina for bovine, ovine, porcine, equine, caprine and canine leptospirosis diagnosis, as well as in human and wild animal diagnosis, were obtained. MLVA results are presented in such a way that they can be readily used for the identifcation of these strains by the simple and direct comparison of agarose gels. Making the use and interpretation of the MLVA for leptospires typing easier will help increase the use of this method as a routine procedure for human and animal diagnosis, for epidemiological studies, vaccine control and other applications.

La leptospirosis es una zoonosis de distribución global causada por una espiroqueta perteneciente al género Leptospira. En los últimos años se han desarrollado nuevos métodos para la genotipifcación de las leptospiras, entre ellos el denominado multiple-locus variable-number tandem repeat analysis (MLVA). En este trabajo se obtuvieron los patrones de MLVA de todas las cepas de referencia utilizadas en la Argentina para el diagnóstico de leptospirosis en bovinos, ovinos, porcinos, equinos, caprinos y perros, y que también son utilizadas en el diagnóstico de leptospirosis en humanos y en animales salvajes. Los resultados del MLVA se muestran de manera tal que pueden ser fácilmente utilizados para la identifcación de estas cepas por simple comparación visual de geles de agarosa. Al facilitar el uso y la interpretación del MLVA para la tipifcación de leptospiras, se ayudará a difundir la utilización rutinaria de este método en el diagnóstico humano y animal, en estudios epidemiológicos y para el control de vacunas, entre otras aplicaciones.

Bacillus anthracis: una mirada molecular a un patógeno célebre / Bacillus anthracis: a molecular look at a famous pathogen

Bacillus anthracis es un bacilo gram positivo del grupo Bacillus cereus, que posee un genoma extremadamente monomórfco y comparte gran similitud fsiológica y de estructura genética con B. cereus y Bacillus thuringiensis. En este artículo se describen nuevos métodos moleculares para la identifcación y tipifcación de B. anthracis, basados en repeticiones en tándem de número variable o en diferencias genéticas detectadas por secuenciación, desarrollados en los últimos años. Los aspectos moleculares de los factores de virulencia tradicionales, cápsula, antígeno protector, factor letal y factor edema se describen en profundidad, junto con factores de virulencia recientemente propuestos, como los sideróforos, petrobactina y bacilibactina, la adhesina de la capa S y la lipoproteína MntA. También se detalla la organización molecular de los megaplásmidos pXO1 y pXO2, incluyendo la isla de patogenicidad de pXO1. El esqueleto genético de estos plásmidos se ha encontrado en otras especies relacionadas, probablemente debido a eventos de transferencia lateral. Finalmente, se presentan los dos receptores celulares del antígeno protector, ANTXR1/TEM8 y ANTXR2/CMG2, esenciales en la interacción del patógeno con el hospedador. Los estudios moleculares realizados en los últimos años han permitido aumentar enormemente el conocimiento de los diferentes aspectos de este microorganismo y su relación con el hospedador, pero a la vez han abierto nuevos interrogantes sobre este notorio patógeno.

Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identifcation and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The molecular aspects of traditional virulence factors: capsule, protective antigen, lethal factor and edema factor are described in depth, together with virulence factors recently proposed, such as the siderophores petrobactin and bacillibactin, the S-layer adhesin and the MntA lipoprotein. It is detailed the molecular organization of megaplasmids pXO1 and pXO2, including the pathogenicity island of pXO1. The genetic skeleton of these plasmids has been observed in related species, and this could be attributed to lateral gene transfer. Finally, the two anthrax toxin protective antigen receptors, ANTXR1/TEM8 and ANTXR2/CMG2, essential for the interaction of the pathogen with the host, are presented. The molecular studies performed in recent years have greatly increased knowledge in different aspects of this microorganism and its relationship with the host, but at the same time they have raised new questions about this noted pathogen.