Exploring the cellulolytic activity of environmental mycobacteria.

Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active ß-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.

Bovine campylobacteriosis in heifer: pathogenesis study and insights in the conventional and molecular diagnosis in an experimental bovine model and field cases.

Campylobacter fetus spp. is a bacterium associated to reproductive losses in cattle worldwide. It is a venereal infectious disease known as bovine campilobacteriosis, with high impact mainly in countries with extensive production systems. Here, we show pathogenesis and diagnostic methods for Campylobacter fetus detection in cervico-vaginal mucus (CVM) samples from heifers experimentally infected and field cases from herds with low reproductive performance by campylobacteriosis infection. Bacterial culture, direct immunofluorescence test and qPCR were used as diagnostic methods to evaluate detection of C. fetus. In the experimental model 30 Aberdeen Angus and crossbred heifers and 4 Aberdeen Angus bulls for natural mating were assigned to 3 groups experimentally challenged with C. fetus subsp. fetus (Cff), C. fetus subsps venerealis (Cfv) and C. fetus subsp venerealis biovar intermedius (Cfvi), respectively, and a negative control group, all followed for 9 months. Also, field samples of CVM and aborted fetuses were recollected from seven beef cattle farms. Bacteriological culture had the higher C. fetus detection rate in CVM being the most appropriate, followed by qPCR (with commercial extraction DNA kit), direct immunofluorescence test and qPCR (with in-house extraction DNA method), in both, experimental model and field cases. From experimental model after natural mating, 62.5% and 25% heifers got pregnant from Cff and Cfvi groups, respectively, while from Cfv no pregnancy was detected. The strain more frequently detected was Cfvi, followed by Cff and Cfv. Colonization of Cff in female genital tract with high number of carriers and presence in aborted fetuses was evidenced, suggesting a high risk to bovine reproductive health. Bacteriemia was not detected after genital infection. Given the low detection rate of either test, we suggest the use of both, PCR based methods and bacterial culture could result in higher detection rate in farms with endemic campylobacteriosis.

Bovine campylobacteriosis in bulls: insights in the conventional and molecular diagnosis.

Campylobacter fetus is a gram-negative motile bacterium, with two subspecies relevant to cattle health: C. fetus subsp. venerealis (Cfv) and C. fetus subsp. fetus (Cff). Both subspecies are associated with reproductive losses in cattle. In this study, we evaluated the identification of C. fetus for the diagnosis of bovine campylobacteriosis through bacteriological culture, direct immunofluorescence (DIF) and molecular tests in preputial smegma (PS) samples of three Angus bulls challenged with Cfv, Cfv biovar intermedius (Cfvi) or Cff, respectively, in an experiment imitating the natural infection. Two DNA extraction protocols were tested (in-house thermal extraction and commercial kit). Aspiration and scraping collection for PS were compared by conventional tests. Additionally, bacteremia was also evaluated in blood samples. Bulls were challenged by natural mating with heifers that had been experimentally infected with C. fetus subspecies; which led to infection. The Cfv- and Cfvi-bulls were positive for at least 9 months. Although Cff is not considered a venereal strain, in this study it was transmissible to bull from heifers experimentally infected, as evidenced by its colonization and persistence in the preputial cavity for 5 to 6 months. This finding suggests a potential risk of dissemination within herds. The results obtained by bacteriological culture or direct immunofluorescence (DIF) showed no significant differences, regardless the sampling device used (aspiration with Cassou pipette, metal and plastic scraper). C. fetus qPCR, on the other hand, yielded better results with an in-house DNA extraction method than with a commercial kit (75% vs 66.6%). Furthermore, qPCR diagnosis was more efficient than culture (66.6%) or DIF (56%). Bacteremia in whole blood samples was negative by qPCR and bacteriological culture in all samples. Altogether, this study demonstrated the transmission of Cff from heifers to bull and also showed that PCR-based methods are promising for the diagnosis of Bovine Genital Campylobacteriosis from clinical samples of PS.

Phylogenetic and Multiple-Locus Variable number tandem repeat analysis of Mycobacterium avium subsp. paratuberculosis isolates from Argentina.

Paratuberculosis is a worldwide chronic enteric disease of ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). While MAP has been widely investigated all around the world, little is known about the different strains that circulate in each country. This study describes the genetic diversity of MAP isolates from different bovine and deer herds from Argentina, analyzed by Multiple-Locus Variable number tandem repeat Analysis (MLVA), as well as the phylogenetic relatedness between geographically distant isolates through Whole Genome Sequencing (WGS) and core-genome analysis. A total of 90 MAP isolates were analyzed. The results showed seven different MLVA genotypes, with almost 75% of them belonging to pattern INMV 1, described in all the herds studied. WGS results suggested the presence of a common INMV 1 strain circulating throughout the country. Our results allow confirming the coexistence of different strains in time and space and the mixed infections identified in some animals. These observations suggest the absence of animal monitoring prior to introduction to the herds and the need for a control program in the country. This study represents the first to report WGS of MAP strains in Argentina.

Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles

Abstract The study of outer membrane vesicles (OMVs) became relevant because of theirprobable important role in the transfer of virulence factors to host cells. Campylobacter fetusis mainly a mammal pathogen whose virulence characterization is still limited. The aim of thisstudy was to evaluate and to characterize the secretion of OMVs in this bacterium. By trans-mission electron microscopy, we confirmed the production of OMVs in all the strains assayed.Purified OMVs showed a spherical shape and variable size, although comparable to those ofother gram-negative bacteria. We also confirmed the presence of the S-layer on the surface ofthe OMVs of all the strains assayed with the exception of those derived from the NTCC referencestrain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C.fetus OMVs could contribute to the modulation of the host response and constitute a candidateto be evaluated as an adjuvant of current vaccines used in the veterinary field. This work rep-resents a platform to drive future studies towards the role of these subcellular structures in C.fetus-host interaction.

Resumen El estudio de las vesículas de membrana externa (VME) tomó un rol protagónico, yaque se las ha relacionado con la transferencia de factores de virulencia a la célula hospedadora.Campylobacter fetus es, principalmente, un patógeno de mamíferos cuya virulencia solo hasido caracterizada de forma limitada. El objetivo de este trabajo fue evaluar y caracterizar la secreción de VME en esta bacteria. Mediante microscopía electrónica de transmisión confir-mamos la producción espontánea de VME en todas las cepas estudiadas. Las VME purificadasmostraron una morfología esférica y un tama˜no variable, pero compatible con el reporte deotras bacterias gram negativas. Asimismo, hemos demostrado que estas vesículas conservanla capa S en todas las cepas, menos en la cepa de referencia NCTC y hemos confirmado suinmunorreactividad por dot-blot inmunoblot. Estas VME de C. fetus podrían contribuir a la mod-ulación de la respuesta del hospedador y constituir un buen candidato como adyuvante de lasactuales vacunas empleadas en el campo veterinario. Este trabajo representa una plataformapara impulsar estudios futuros en torno al rol de estas estructuras subcelulares en la interfaseC. fetus-hospedador.

Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles.

The study of outer membrane vesicles (OMVs) became relevant because of their probable important role in the transfer of virulence factors to host cells. Campylobacter fetus is mainly a mammal pathogen whose virulence characterization is still limited. The aim of this study was to evaluate and to characterize the secretion of OMVs in this bacterium. By transmission electron microscopy, we confirmed the production of OMVs in all the strains assayed. Purified OMVs showed a spherical shape and variable size, although comparable to those of other gram-negative bacteria. We also confirmed the presence of the S-layer on the surface of the OMVs of all the strains assayed with the exception of those derived from the NTCC reference strain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C. fetus OMVs could contribute to the modulation of the host response and constitute a candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field. This work represents a platform to drive future studies towards the role of these subcellular structures in C. fetus-host interaction.

Phylogenomic analysis for Campylobacter fetus ocurring in Argentina.

BACKGROUND AND AIM: Campylobacter fetus is one of the most important pathogens that severely affects livestock industry worldwide. C. fetus mediated bovine genital campylobacteriosis infection in cattle has been associated with significant economic losses in livestock production in the Pampas region, the most productive area of Argentina. The present study aimed to establish the genomic relationships between C. fetus strains, isolated from the Pampas region, at local and global levels. The study also explored the utility of multi-locus sequence typing (MLST) as a typing technique for C. fetus. MATERIALS AND METHODS: For pangenome and phylogenetic analysis, whole genome sequences for 34 C. fetus strains, isolated from cattle in Argentina were downloaded from GenBank. A local maximum likelihood (ML) tree was constructed and linked to a Microreact project. In silico analysis based on MLST was used to obtain information regarding sequence type (ST) for each strain. For global phylogenetic analysis, a core genome ML-tree was constructed using genomic dataset for 265 C. fetus strains, isolated from various sources obtained from 20 countries. RESULTS: The local core genome phylogenetic tree analysis described the presence of two major clusters (A and B) and one minor cluster (C). The occurrence of 82% of the strains in these three clusters suggested a clonal population structure for C. fetus. The MLST analysis for the local strains revealed that 31 strains were ST4 type and one strain was ST5 type. In addition, a new variant was identified that was assigned a novel ST, ST70. In the present case, ST4 was homogenously distributed across all the regions and clusters. The global analysis showed that most of the local strains clustered in the phylogenetic groups that comprised exclusively of the strains isolated from Argentina. Interestingly, three strains showed a close genetic relationship with bovine strains obtained from Uruguay and Brazil. The ST5 strain grouped in a distant cluster, with strains obtained from different sources from various geographic locations worldwide. Two local strains clustered in a phylogenetic group comprising intercontinental Campylobacter fetus venerealis strains. CONCLUSION: The results of the study suggested active movement of animals, probably due to economic trade between different regions of the country as well as with neighboring countries. MLST results were partially concordant with phylogenetic analysis. Thus, this method did not qualify as a reliable subtyping method to assess C. fetus diversity in Argentina. The present study provided a basic platform to conduct future research on C. fetus, both at local and international levels.

L-cysteine transporter-PCR to detect hydrogen sulfide-producing Campylobacter fetus.

Phenotypic differences between Campylobacter fetus fetus and C. fetus venerealis subspecies allow the differential diagnosis of bovine genital campylobacteriosis. The hydrogen sulfide production, for example, is a trait exclusive to C. fetus fetus and C. fetus venerealis biovar intermedius. This gas that can be biochemically tested can be produced from L-cysteine (L-Cys). Herein, we report a novel multiplex-PCR to differentiate C. fetus based on the evaluation of a deletion of an ATP-binding cassette-type L-Cys transporter that could be involved in hydrogen sulfide production, as previously described. A wet lab approach combined with an in silico whole genome data analysis showed complete agreement between this L-Cys transporter-PCR and the hydrogen sulfide production biochemical test. This multiplex-PCR may complement the tests currently employed for the differential diagnosis of C. fetus.