Publications by authors named "Angel Cataldi"

88 Publications

Changes in hematological, biochemical, and blood gases parameters in response to progressive inclusion of nitrate in the diet of Holstein calves.

Vet World 2021 Jan 9;14(1):61-69. Epub 2021 Jan 9.

Institute of Pathobiology, National Institute of Agricultural Technology, National Scientific and Technical Research Council, Hurlingham (C1686), Argentina.

Background And Aim: Nitrate (NO) reduces enteric methane emissions and could be a source of non-protein nitrogen in ruminant feeds. Nonetheless, it has a potential toxic effect that could compromise animal health and production. The purpose of this study was to determine the effects of progressive inclusion of NO in the diet on the hematological, biochemical, and blood gases parameters, in turn, the effects on feed intake and live weight gain (LWG) in Holstein calves.

Materials And Methods: Eighteen Holstein heifers and steers (nine animals/treatment) were maintained in individual pens for 45 days. Animals were randomly allocated to either a control or nitrate diet (ND) (containing 15 g of NO/kg of dry matter [DM]). The biochemical parameters and blood gases were analyzed only in the NO group on days: -1, 1, 7, 13, 19, and 25 corresponding to 0, 20, 40, 60, 80, and 100% of the total inclusion of NO in the diet, respectively. In addition, DM intake (DMI) and LWG were evaluated among dietary treatments.

Results: Feeding the ND did not influence DMI or LWG (p>0.05). Methemoglobin (MetHb) and deoxyhemoglobin increased according to the NO concentrations in the diet (p<0.05), while an opposite effect was observed for oxyhemoglobin and carboxyhemoglobin (p<0.05). Hematocrit levels decreased (p<0.05), while albumin, alanine aminotransferase, and gamma-glutamyl transpeptidase concentrations were not modified (p>0.05). However, glucose, urea, aspartate aminotransferase (AST), and retinol concentrations increased (p<0.05) according to the NO concentrations in the diet.

Conclusion: This study confirmed that the progressive inclusion of 123 g of NO/animal/day in the diet could be safe without affecting DMI and LWG of Holstein calves. In turn, a dose-response effect of the MetHb, glucose, urea, AST, and retinol was observed, but these values did not exceed reference values. These results highlighted the importance of using a scheme of progressive inclusion of NO in the diet of calves to reduce the risks of NO toxicity.
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http://dx.doi.org/10.14202/vetworld.2021.61-69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896885PMC
January 2021

Early immune innate hallmarks and microbiome changes across the gut during Escherichia coli O157: H7 infection in cattle.

Sci Rep 2020 12 9;10(1):21535. Epub 2020 Dec 9.

Faculty Veterinary Medicine, Production Animal Health, University of Calgary, HSC 2519, 3330 Hospital Dr. NW, Calgary, AB, T2N 4N1, Canada.

The zoonotic enterohemorrhagic Escherichia coli (EHEC) O157: H7 bacterium causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. Cattle are primary reservoirs and EHEC O157: H7; the bacteria predominately inhabit the colon and recto-anal junctions (RAJ). The early innate immune reactions in the infected gut are critical in the pathogenesis of EHEC O157: H7. In this study, calves orally inoculated with EHEC O157: H7 showed infiltration of neutrophils in the lamina propria of ileum and RAJ at 7 and 14 days post-infection. Infected calves had altered mucin layer and mast cell populations across small and large intestines. There were differential transcription expressions of key bovine β defensins, tracheal antimicrobial peptide (TAP) in the ileum, and lingual antimicrobial peptide (LAP) in RAJ. The main Gram-negative bacterial/LPS signaling Toll-Like receptor 4 (TLR4) was downregulated in RAJ. Intestinal infection with EHEC O157: H7 impacted the gut bacterial communities and influenced the relative abundance of Negativibacillus and Erysipelotrichaceae in mucosa-associated bacteria in the rectum. Thus, innate immunity in the gut of calves showed unique characteristics during infection with EHEC O157: H7, which occurred in the absence of major clinical manifestations but denoted an active immunological niche.
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http://dx.doi.org/10.1038/s41598-020-78752-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726576PMC
December 2020

A Phenotypic Characterization of Two Isolates of a Multidrug-Resistant Outbreak Strain of with Opposite Epidemiological Fitness.

Biomed Res Int 2020 8;2020:4741237. Epub 2020 Apr 8.

Institute of Biotechnology, National Institute of Agricultural Technology (INTA)/IABIMO-CONICET (Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria), Argentina.

Tuberculosis (TB) is an infectious disease, caused by , primarily affecting the lungs. The strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses.
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http://dx.doi.org/10.1155/2020/4741237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168692PMC
January 2021

Analysing nonsynonymous mutations between two Mycobacterium bovis strains with contrasting pathogenic profiles.

Vet Microbiol 2019 Dec 4;239:108482. Epub 2019 Nov 4.

Instituto de Biotecnología, IABIMO, CICVyA/INTA, Argentina. Electronic address:

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a chronic infectious disease that can affect cattle, other domesticated species, wild animals and humans. This disease produces important economic losses worldwide. Two M. bovis strains (04-303 and 534) have been isolated in Argentina. Whereas the 04-303 strain was isolated from a wild boar, the 534 strain was obtained from cattle. In a previous study, six weeks after infection, the 04-303 strain induced 100% mortality in mice. By contrast, mice infected with the 534 strain survived, with limited tissue damage, after four months. In this study we compared all predictive proteins encoded in both M. bovis genomes. The comparative analysis revealed 141 polymorphic proteins between both strains. From these proteins, nine virulence proteins showed polymorphisms in 04-303, whereas five did it in the 534 strain. Remarkably, both strains contained a high level of polymorphism in proteins related to phthiocerol dimycocerosate (PDIM) synthesis or transport. Further experimental evidence indicated that only mutations in the 534 strain have an impact on PDIM synthesis. The observed reduction in PDIM content in the 534 strain, together with its low capacity to induce phagosome arrest, may be associated with the reported deficiency of this strain to replicate and survive inside bovine macrophages. The findings of this study could contribute to a better understanding of pathogenicity and virulence aspects of M. bovis, which is essential for further studies aiming at developing new vaccines and diagnostic techniques for bovines.
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http://dx.doi.org/10.1016/j.vetmic.2019.108482DOI Listing
December 2019

Type VI Secretion System in Pathogenic : Structure, Role in Virulence, and Acquisition.

Front Microbiol 2019 30;10:1965. Epub 2019 Aug 30.

Laboratorio de Escherichia coli, Instituto de Agrobiotecnología y Biología Molecular (IABIMO) INTA-CONICET, Buenos Aires, Argentina.

Bacterial pathogens utilize a myriad of mechanisms to invade mammalian hosts, damage tissue sites, and evade the immune system. One essential strategy of Gram-negative bacteria is the secretion of virulence factors through both inner and outer membranes to reach a potential target. Most secretion systems are harbored in mobile elements including transposons, plasmids, pathogenicity islands, and phages, and is one of the more versatile bacteria adopting this genetic information by horizontal gene transfer. Additionally, is a bacterial species with members of the commensal intestinal microbiota and pathogens associated with numerous types of infections such as intestinal, urinary, and systemic in humans and other animals. T6SS cluster plasticity suggests evolutionarily divergent systems were acquired horizontally. T6SS is a secretion nanomachine that is extended through the bacterial double membrane; from this apparatus, substrates are conveyed straight from the cytoplasm of the bacterium into a target cell or to the extracellular space. This nanomachine consists of three main complexes: proteins in the inner membrane that are T4SS component-like, the baseplate complex, and the tail complex, which are formed by components evolutionarily related to contractile bacteriophage tails. Advances in the T6SS understanding include the functional and structural characterization of at least 13 subunits (so-called core components), which are thought to comprise the minimal apparatus. So far, the main role of T6SS is on bacterial competition by using it to kill neighboring non-immune bacteria for which antibacterial proteins are secreted directly into the periplasm of the bacterial target after cell-cell contact. Interestingly, a few T6SSs have been associated directly to pathogenesis, e.g., roles in biofilm formation and macrophage survival. Here, we focus on the advances on T6SS from the perspective of pathotypes with emphasis in the secretion apparatus architecture, the mechanisms of pathogenicity of effector proteins, and the events of lateral gene transfer that led to its spread.
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http://dx.doi.org/10.3389/fmicb.2019.01965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730261PMC
August 2019

An inhibitory mechanism of action of coiled-coil peptides against type three secretion system from enteropathogenic Escherichia coli.

J Pept Sci 2019 Mar 11;25(3):e3149. Epub 2019 Feb 11.

Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, San Luis, Argentina.

Human pathogenic gram-negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled-coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha-helical structures that play an important role in the protein-protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled-coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS-dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking-molecular dynamic simulations, and quantum-mechanic calculations of the various peptide-protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled-coil domain of the C-terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide-protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad-spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.
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http://dx.doi.org/10.1002/psc.3149DOI Listing
March 2019

Quantification of enterohemorrhagic Escherichia coli O157:H7 protein abundance by high-throughput proteome.

PLoS One 2018 31;13(12):e0208520. Epub 2018 Dec 31.

Institute of Biotechnology, CICVyA, National Institute of Agricultural Technology, Hurlingham, Buenos Aires, Argentina.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To promote a comprehensive insight into the molecular basis of EHEC O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) strain has been analyzed. From shotgun proteomic analysis a total of 2,644 non-redundant proteins of EHEC O157:H7 were identified, which correspond approximately 47% of the predicted proteome of this pathogen. Normalized spectrum abundance factor analysis was performed to estimate the protein abundance. According this analysis, 50 proteins were detected as the most abundant of EHEC O157:H7 proteome. COG analysis showed that the majority of the most abundant proteins are associated with translation processes. A KEGG enrichment analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, the less abundant detected proteins are those related to DNA processes, cell respiration and prophage. Among the proteins that composed the Type III Secretion System, the most abundant protein was EspA. Altogether, the results show a subset of important proteins that contribute to physiology and pathogenicity of EHEC O157:H7.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208520PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312284PMC
May 2019

Novel Effector Protein EspY3 of Type III Secretion System from Enterohemorrhagic Is Localized in Actin Pedestals.

Microorganisms 2018 Oct 27;6(4). Epub 2018 Oct 27.

Instituto de Biotecnología CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Los Reseros y Nicolás Repetto, Hurlingham Buenos Aires 1686, Argentina.

Enterohemorrhagic (EHEC) and enteropathogenic (EPEC) are attaching and effacing (A/E) pathogens, which translocate effector proteins to intestinal enterocytes through a type III secretion system (T3SS). T3SS and most of its effector proteins are encoded in a pathogenicity island called LEE. Recently, new effectors have been located outside the LEE. This study aimed to characterize EspY3, a novel non-LEE encoded T3SS effector of EHEC. EspY3 shares homology with SopD and PipB2 effector proteins of Salmonella's T3SS-1 and T3SS-2, respectively. The presence of recombinant EspY3 in the supernatant samples demonstrated that EspY3 was secreted by the T3SS of EHEC and EPEC. Through infection assays, we demonstrated the translocation of EspY3 into Caco-2 cells by T3SS of EPEC. The subcellular localization of EspY3 was determined in the pedestal region, where its presence generates a significant increase in the size of the pedestals area. The EspY3 effector induced the elongation of polymerized actin pedestals in infected Caco-2 by EPEC. This study confirmed that EspY3 is part of the repertoire of T3SS effectors of EHEC O157:H7, and that it participates in modeling cellular actin during the infection.
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http://dx.doi.org/10.3390/microorganisms6040112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313403PMC
October 2018

Efficacy of a recombinant Intimin, EspB and Shiga toxin 2B vaccine in calves experimentally challenged with Escherichia coli O157:H7.

Vaccine 2018 06 26;36(27):3949-3959. Epub 2018 May 26.

Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Instituto Nacional de Tecnología Agropecuaria, Hurlingham, Argentina. Electronic address:

Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E.coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E.coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine's effect on fecal shedding, groups of calves were immunized with EspB + IntC280, with EspB + IntC280 + BLS-Stx2B, or kept as controls. At 24 days post vaccination calves were challenged with E.coli O157:H7. Shedding of E.coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15 days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E.coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E.coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds.
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http://dx.doi.org/10.1016/j.vaccine.2018.05.059DOI Listing
June 2018

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

Vaccine 2018 03 23;36(13):1728-1735. Epub 2018 Feb 23.

Laboratorio de Fisiopatogenia, Departamento de Fisiología, Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO Houssay-CONICET), Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina. Electronic address:

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.
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http://dx.doi.org/10.1016/j.vaccine.2018.02.060DOI Listing
March 2018

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates.

Rev Argent Microbiol 2017 Oct - Dec;49(4):332-338. Epub 2017 Sep 15.

Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrangolo Hospital, Italia 1750, Florida 1602, Buenos Aires Province, Argentina.

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments.

Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method.

Results: MZ and IPZ showed bacteriostatic activity against M. tuberculosis strains. MIC and MIC to ECO was 4.0μg/ml, while MIC to CLO was 4.0μg/ml and MIC was 8.0μg/ml respectively.

Conclusion: All azole compounds tested in the study showed inhibitory activity against MDR M. tuberculosis clinical isolates.
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http://dx.doi.org/10.1016/j.ram.2017.02.008DOI Listing
February 2019

Immunohistochemical detection of pro-inflammatory and anti-inflammatory cytokines in granulomas in cattle with natural Mycobacterium bovis infection.

Res Vet Sci 2017 Feb 13;110:34-39. Epub 2016 Oct 13.

Departamento de Medicina Interna y Cirugía, Facultad de Veterinaria, Universidad Complutense, Av. Puerta de Hierro s/n, 28040 Madrid, Madrid, Spain.

Cellular immune response was evaluated in lymph nodes and lung with different granulomatous lesions from cattle naturally infected with Mycobacterium bovis. For this purpose, we assessed pro-inflammatory and anti-inflammatory cytokines by immunohistochemical assays. Immunoreaction was observed for all the cytokines analyzed. Fourteen animals displayed advanced stage IV granulomas, with intense immunoreactivity to IFN-γ and TGF-β in areas of caseous necrosis, macrophages and lymphocytes. Seven animals showed stage III granuloma, with high immunoreactivity to IFN-γ (average of 44.5% immunoreactive cells) and moderate to TNF-α and to the anti-inflammatory cytokines IL-10 and TGF-β, in relation to the proliferation of fibroblasts in granuloma periphery We found satellite stage I granulomas in 4 bovines and stage II granulomas in 2 bovines, which exhibited low immunostaining response (-13%). Cytokine expression in stage III and IV granulomas was significant, with predominance of immunoreactivity to IFN-γ, thus suggesting a strong, longstanding local immune response mediated by macrophages and epithelioid cells. In addition, these two stages displayed lower reactivity to IL-10; which suggests a deficit of anti-inflammatory cytokines, suppressed immunity and persistence of the infection. High expression of TGF-β could indicate a chronic process with greater tissue damage and fibrosis. Numerous bacilli observed in necrotic areas in stage III and IV granulomas with low expression of IL-1β suggest failure in the immune response with bacterial multiplication. In this study, evidence of in situ presence of cytokines demonstrates these cytokines are involved in the development and evolution of bovine tuberculosis granulomas.
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http://dx.doi.org/10.1016/j.rvsc.2016.10.006DOI Listing
February 2017

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

PLoS One 2017 3;12(1):e0169422. Epub 2017 Jan 3.

Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Instituto Nacional de Tecnología Agropecuaria, Hurlingham, Argentina.

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169422PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207737PMC
August 2017

Overexpressed Proteins in Hypervirulent Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 Compared to E. coli O157:H7 EDL933 Clade 3 Strain.

PLoS One 2016 23;11(11):e0166883. Epub 2016 Nov 23.

Institute of Biotechnology, CICVyA, National Institute of Agricultural Technology. Hurlingham, Buenos Aires, Argentina.

Escherichia coli O157:H7 is responsible for severe diarrhea and hemolytic uremic syndrome (HUS), and predominantly affects children under 5 years. The major virulence traits are Shiga toxins, necessary to develop HUS and the Type III Secretion System (T3SS) through which bacteria translocate effector proteins directly into the host cell. By SNPs typing, E. coli O157:H7 was separated into nine different clades. Clade 8 and clade 6 strains were more frequently associated with severe disease and HUS. In this study, we aimed to identify differentially expressed proteins in two strains of E. coli O157:H7 (clade 8 and clade 6), obtained from cattle and compared them with the well characterized reference EDL933 strain (clade 3). Clade 8 and clade 6 strains show enhanced pathogenicity in a mouse model and virulence-related properties. Proteins were extracted and analyzed using the TMT-6plex labeling strategy associated with two dimensional liquid chromatography and mass spectrometry in tandem. We detected 2241 proteins in the cell extract and 1787 proteins in the culture supernatants. Attention was focused on the proteins related to virulence, overexpressed in clade 6 and 8 strains compared to EDL933 strain. The proteins relevant overexpressed in clade 8 strain were the curli protein CsgC, a transcriptional activator (PchE), phage proteins, Stx2, FlgM and FlgD, a dienelactone hydrolase, CheW and CheY, and the SPATE protease EspP. For clade 6 strain, a high overexpression of phage proteins was detected, mostly from Stx2 encoding phage, including Stx2, flagellin and the protease TagA, EDL933_p0016, dienelactone hydrolase, and Haemolysin A, amongst others with unknown function. Some of these proteins were analyzed by RT-qPCR to corroborate the proteomic data. Clade 6 and clade 8 strains showed enhanced transcription of 10 out of 12 genes compared to EDL933. These results may provide new insights in E. coli O157:H7 mechanisms of pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166883PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120812PMC
June 2017

High prevalence of clade 8 Escherichia coli O157:H7 isolated from retail meat and butcher shop environment.

Infect Genet Evol 2016 11 5;45:1-5. Epub 2016 Aug 5.

IGEVET-Instituto de Genética Veterinaria (UNLP-CONICET LA PLATA), Facultad de Ciencias Veterinarias UNLP, 60 y 118 s/n, La Plata, Argentina. Electronic address:

Escherichia coli O157:H7 is an enteric pathogen associated with food safety threats and with significant morbidity and mortality worldwide. In Argentina, post-enteric hemolytic uremic syndrome (HUS) is endemic, with >70% of cases associated with E. coli O157 infection. To date the biological basis behind the severity among E. coli O157 infections is unknown. However, single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades, of which clade 8 strains are associated with severe disease cases. The aim of this study was to characterize a collection of 20 STEC O157:H7 strains isolated between 2011 and 2013 from ground beef and different environmental samples from butcher shops of Argentina. All strains harbored the eae, ehxA, fliC, efa, iha, and toxB genes, with stx/stx as the predominant genotype (75%). The XbaI-PFGE analysis showed that the E. coli O157 strains had high genetic diversity. Nine strains were grouped in four XbaI-PFGE clusters, whereas 11 strains showed unique XbaI-PFGE patterns. In contrast, the SNP analysis allowed us to separate the strains in two distinct lineages representing clade 8 (70%) and clade 6 (30%). Our results show the molecular characterization of E. coli O157 strains isolated from ground beef and environmental samples from Argentinean butcher shops.
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http://dx.doi.org/10.1016/j.meegid.2016.08.005DOI Listing
November 2016

Biochemical Characterization of β-Lactamases from Mycobacterium abscessus Complex and Genetic Environment of the β-Lactamase-Encoding Gene.

Microb Drug Resist 2017 Apr 18;23(3):294-300. Epub 2016 Jul 18.

2 Universidad de Buenos Aires , Facultad de Farmacia y Bioquímica, Laboratorio de Resistencia Bacteriana, Buenos Aires, Argentina .

The objectives of this study were to determine the kinetic parameters of purified recombinant Bla and Bla by spectrophotometry, analyze the genetic environment of the bla and bla genes in both species by polymerase chain reaction and sequencing, furthermore, in silico models of both enzymes in complex with imipenem were obtained by modeling tools. Our results showed that Bla and Bla have a similar hydrolysis behavior, displaying high catalytic efficiencies toward penams, cephalothin, and nitrocefin; none of the enzymes are well inhibited by clavulanate. Bla hydrolyzes imipenem at higher efficiency than cefotaxime and aztreonam. Bla and Bla showed that their closest structural homologs are KPC-2 and SFC-1, which correlate to the mild carbapenemase activity toward imipenem observed at least for BlaMmas. They also seem to differ from other class A β-lactamases by the presence of a more flexible Ω loop, which could impact in the hydrolysis efficiency against some antibiotics. A -35 consensus sequence (TCGACA) and embedded at the 3' end of MAB_2874, which may constitute the bla and bla promoter. Our results suggest that the resistance mechanisms in fast-growing mycobacteria could be probably evolving toward the production of β-lactamases that have improved catalytic efficiencies against some of the drugs commonly used for the treatment of mycobacterial infections, endangering the use of important drugs like the carbapenems.
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http://dx.doi.org/10.1089/mdr.2016.0047DOI Listing
April 2017

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis.

Microbiol Immunol 2016 Aug;60(8):552-60

Biotechnology Institute, National Institute of Agricultural Technology (INTA), Hurlingham 1686, Argentina.

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non-synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species-specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia-related genes between M. bovis and M. tuberculosis.
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http://dx.doi.org/10.1111/1348-0421.12402DOI Listing
August 2016

[Interlaboratory test: Isolation of Mycobacterium bovis from granulomatous lesions in bovine].

Rev Argent Microbiol 2016 Apr-Jun;48(2):161-5. Epub 2016 May 26.

Instituto de Biotecnología, CICVyA-INTA, Buenos Aires, Argentina. Electronic address:

Mycobacterium bovis is the causative agent of bovine tuberculosis. The diagnostic laboratory confirmation is made through bacterial isolation. The aim of interlaboratory tests is to assess the performance of each participant in comparison with other of similar capacities. The test objective was to determine the efficiency of isolation of M. bovis. Four laboratories were part of the test and processed 25 blind tissue samples from granulomatous lesions and with previous M. bovis isolation. The laboratory that had the highest proportion of isolates was A (68%), followed by C (60%) and then B and D (both with 52%). The greatest concordance was observed between B-D and B-C laboratories (68%). The differences could be due to specific factors in each laboratory procedures. This type of interlaboratory tests highlights errors in the bacteriology and identifies critical points in the process to detect M. bovis accurately.
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http://dx.doi.org/10.1016/j.ram.2016.03.004DOI Listing
April 2017

Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis.

Tuberculosis (Edinb) 2015 Dec 8;95(6):795-801. Epub 2015 Aug 8.

Laboratorio de Tuberculosis Bovina, Instituto de Biotecnología, CICVyA, INTA-Castelar, Buenos Aires, Argentina. Electronic address:

Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis), responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cell mediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex and poorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (false-positive results) has been crucial to develop a more proper antigen. In the present study, we selected six M. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by in-silico analysis and evaluated them in experimental and natural infection; none of these antigens had been previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animals with both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected with Mycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually induced an IFN-γ response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the most valuable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAP infected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B among TST and MTC specific-PCR positive animals, although this result needs to be proven in further studies with a higher sample size. Our data confirm the feacibility to implement bioinformatic screening tools and suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate their contribution to bTB diagnosis.
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http://dx.doi.org/10.1016/j.tube.2015.07.009DOI Listing
December 2015

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites.

PLoS One 2015 27;10(8):e0136573. Epub 2015 Aug 27.

Instituto de Biotecnología, CICVyA, CNIA, INTA Castelar, Dr. N. Repetto y Los Reseros s/n, (1686) Hurlingham, Provincia de Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136573PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552170PMC
May 2016

Draft Genome Sequences of Escherichia coli O157:H7 Strains Rafaela_II (Clade 8) and 7.1_Anguil (Clade 6) from Cattle in Argentina.

Genome Announc 2015 Jun 11;3(3). Epub 2015 Jun 11.

Escherichia coli O157:H7 is a major etiologic agent of diseases in humans that cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. Here, we report the draft genome sequences of two strains isolated from cattle that had high levels of Shiga toxin 2 and high lethality in mice.
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http://dx.doi.org/10.1128/genomeA.00617-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4463528PMC
June 2015

Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes.

PLoS One 2015 1;10(6):e0127710. Epub 2015 Jun 1.

Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria, Hurlingham, Argentina.

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127710PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452545PMC
March 2016

Inhibition of water absorption and selective damage to human colonic mucosa induced by Shiga toxin-2 are enhanced by Escherichia coli O157:H7 infection.

Int J Med Microbiol 2015 May 5;305(3):348-54. Epub 2015 Mar 5.

Laboratorio de Fisiopatogenia, Departamento de Fisiología, Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO Houssay-CONICET), Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina. Electronic address:

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for a variety of clinical syndromes including bloody and non-bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Although multiple serotypes of STEC have been isolated from hemorrhagic colitis cases, E. coli O157:H7 is by far the most prevalent serotype associated with HUS. Shiga toxin is the major virulence factor of E. coli O157:H7 and is responsible for the more severe symptoms of the infection. However, the mechanisms involved in the pathogenesis of diarrhea mediated by Stx2 are not well known. In this study, we have determined the effects of E. coli O157:H7 strain 125/99 wild type (wt) on the human colonic mucosa mounted in an Ussing chamber. In response to 125/99wt, an inhibition of water absorption across human colonic mucosa was observed. Histological sections showed severe necrosis with detachment of the surface epithelium, mononuclear inflammatory infiltrate and loss of goblet cells after 1h of incubation with 125/99wt. These alterations were not observed with the isogenic mutant strain lacking stx2 or with the filter-sterilized culture supernatant from the 125/99wt strain. These results indicate that the cell damages in human colon are induced by Stx2, and that Stx2 production is increased by the interaction with bacterial cells. Identification of host cell-derived factors responsible for increasing Stx2 can lead to new strategies for modulating STEC infections.
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http://dx.doi.org/10.1016/j.ijmm.2015.02.002DOI Listing
May 2015

Research on bacterial virulence in the developing countries.

Biomed Res Int 2015 2;2015:972187. Epub 2015 Feb 2.

Southern Medical University, Guangzhou, Guangdong, China.

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http://dx.doi.org/10.1155/2015/972187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332757PMC
December 2015

Fitness of drug resistant Mycobacterium tuberculosis and the impact on the transmission among household contacts.

Tuberculosis (Edinb) 2014 Dec 15;94(6):672-7. Epub 2014 Aug 15.

Biotechnology Institute, National Institute of Agricultural Technology (INTA), Hurlingham, Buenos Aires Province, Argentina. Electronic address:

There has been an on-going debate on whether the development of drug resistance in Mycobacterium tuberculosis reduces its relative fitness and its ability to cause disease. The aim of this study was to explore this relationship. For this purpose, we evaluated the in vitro growth of clinical isolates and the transmission of the strains within the patients' households. Clinical and epidemiological data from patients in households, drug-susceptibility and genetic patterns of the isolates were collected. BACTEC MGIT 960™ system with the Epicenter™ software was used to perform fitness experiments and calculate the relative fitness (RF) comparing with the H73Rv reference strain. From 39 households, 124 patients and 388 contacts were included. Concerning transmission, 20 Multi drug-resistant (MDR) and 16 drug sensitive (DS) index cases generated 23 and 28 secondary cases, respectively. An average RF drop of 16.7% was found for MDR strains, but only mutations in rpoB codons 531 were associated with reduced fitness. When the strains were transmitted, their RF tended to decrease, and strains with low RF were less frequently transmitted. Within the limitations of this study, the results showed that the decrease in RF was associated to a limited transmission among the households' contacts.
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http://dx.doi.org/10.1016/j.tube.2014.08.003DOI Listing
December 2014

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

Mem Inst Oswaldo Cruz 2014 Apr 4;109(2):236-45. Epub 2014 Mar 4.

Biotechnology Institute, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Instituto Nacional de Tecnología Agropecuaria, Castelar, Buenos Aires, Argentina.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015263PMC
http://dx.doi.org/10.1590/0074-0276140292DOI Listing
April 2014

Assessment of Mycobacterium bovis deleted in p27-p55 virulence operon as candidate vaccine against tuberculosis in animal models.

Biomed Res Int 2014 21;2014:951978. Epub 2014 Jan 21.

Instituto de Biotecnología, CICVyA-INTA, N. Repetto and De los Reseros, 1686 Hurlingham, Argentina.

A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.
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http://dx.doi.org/10.1155/2014/951978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918748PMC
October 2014

Association between spoligotype-VNTR types and virulence of Mycobacterium bovis in cattle.

Virulence 2014 Feb 7;5(2):297-302. Epub 2014 Jan 7.

Biotechnology Institute; CICVyA-INTA; Castelar, Buenos Aires, Argentina.

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects approximately 5% of Argentine cattle. The aim of this research was to study if it is possible to infer the degree of virulence of different M. bovis genotypes based on scorified observations of tuberculosis lesions in cattle. In this study, we performed association analyses between several parameters with tuberculosis lesions: M. bovis genotype, degree of progression of tuberculosis, and animal age. For this purpose, the genotype was determined by spoligotyping and the degree of bovine tuberculosis gross lesion was quantified with a score based on clinical observations (number, size, and location of granulomas along with histopathologic features). This study was performed with naturally infected cattle of slaughterhouses from three provinces in Argentina. A total of 265 M. bovis isolates were obtained from 378 pathological lesion samples and 192 spoligotyping and VNTR (based on ETR sequences) typing patterns were obtained. SB0140 was the most predominant spoligotype, followed by SB0145. The spoligotype with the highest lesion score was SB0273 (median score of 27 ± 4.46), followed by SB0520 (18 ± 5.8). Furthermore, the most common spoligotype, SB0140, had a median score of 11 ± 0.74. Finally, the spoligotype with the lowest score was SB0145 (8 ± 1.0). ETR typing of SB0140, SB0145, SB0273, and SB0520 did not subdivide the lesion scores in those spoligotypes. In conclusion, SB0273 and SB0520 were the spoligotypes with the strongest association with hypervirulence and both spoligotypes were only found in Río Cuarto at the south of Córdoba province. Interestingly, there is no other report of any of these spoligotyes in Latin America.
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http://dx.doi.org/10.4161/viru.27193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956505PMC
February 2014

Draft Genome Sequence of Mycobacterium bovis 04-303, a Highly Virulent Strain from Argentina.

Genome Announc 2013 Nov 27;1(6). Epub 2013 Nov 27.

School of Computing, UFMS, Campo Grande, Mato Grosso do Sul, Brazil.

Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.
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http://dx.doi.org/10.1128/genomeA.00931-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869323PMC
November 2013