Publications by authors named "Martin Zumarraga"

42 Publications

Tuberculosis in wild South American sea lions Otaria flavescens stranded in Chubut, Argentina.

Dis Aquat Organ 2020 Nov 19;142:33-40. Epub 2020 Nov 19.

Consejo Nacional de Investigaciones Científicas y Técnicas, Centro para el Estudio de Sistemas Marinos (CESIMAR-CONICET), U9120, Chubut, Argentina.

Pinniped tuberculosis, commonly caused by Mycobacterium pinnipedii, is a zoonotic disease reported in free-living and captive otariid species of the southern hemisphere. Currently, data concerning pinniped tuberculosis in South America are scarce, reinforcing the need for further studies of the disease in free-ranging pinnipeds. In this study, we investigated the presence of tuberculosis in South American sea lions Otaria flavescens (SASLs) stranded along the Chubut coastline (Argentina). Necropsies were performed in 9 SASLs, and tissue samples were collected for histopathology, bacteriology, and molecular diagnosis. Four SASLs showed enlarged tracheobronchial lymph nodes (TBLNs) with multifocal to coalescing granulomas. In these animals, a direct IS6110-PCR amplification confirmed the presence of a Mycobacterium tuberculosis complex member in TBLNs (n = 4) and lungs (n = 2), but the agent could not be further identified. In one SASL, Mycobacterium murale was isolated from lungs without lesions. This study confirms the presence of tuberculosis in SASLs from Chubut, where tourist activities promote close interaction with the animals, generating a potential risk to human health. Further research is currently focusing on addressing the prevalence of tuberculosis in wild SASLs, to assess the risk for public health and develop management strategies to avoid human infection.
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http://dx.doi.org/10.3354/dao03520DOI Listing
November 2020

Prevalence of paratuberculosis in dairy cattle in ecuador.

Int J Mycobacteriol 2020 Jan-Mar;9(1):1-6

One Health Research Group, Facultad de Ciencias de la Salud, Universidad de Las Américas, Quito, Ecuador, Departamento de Tuberculosis, Instituto de Biomedicina Jacinto Convit, Universidad Central de Venezuela, Caracas, Venezuela.

Background: Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic infectious contagious disease of the intestinal tract of ruminants that are also associated with Crohn's disease in humans. The existence of paratuberculosis in Ecuador is virtually unknown; hence, the present study was performed to gain insight into the prevalence of this disease.

Methods: Three dairy cattle farms in different geographic regions in Ecuador were investigated for the infection with MAP, and 600 blood samples, 200 of each cattle herd, were processed with an indirect enzyme-linked immunosorbent assay. Fecal samples of the seropositive cows were processed for culture on modified Löwenstein-Jensen medium.

Results: One hundred and fifty bovines (25%) resulted seropositive and we confirmed with culture the presence of MAP in 4.7% (7/150) of the seropositive cows. Approximately 20% of the fecal samples of seropositive cows yielded nontuberculous mycobacteria (NTM) species including M. avium subsp. avium, a NTM species closely related to MAP.

Conclusions: The seroprevalence of paratuberculosis in this first study for Ecuador is high (25%). We discuss a possible interference of NTM species, isolated from fecal samples, with the diagnosis of paratuberculosis. With this report, a baseline study, we confirm for the first time the presence of paratuberculosis in Ecuador, and we provide the necessary information for future studies and control of this disease.
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http://dx.doi.org/10.4103/ijmy.ijmy_175_19DOI Listing
April 2021

Epidemiological Study of Infection in Buffalo and Cattle in Amazonas, Brazil.

Front Vet Sci 2019 10;6:434. Epub 2019 Dec 10.

Center for Comparative Epidemiology, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.

Bovine Tuberculosis (BTB) is an endemic disease in about one hundred countries, affecting the economy causing a decrease in productivity, condemnation of meat, and damaging the credibility on international trade. Additionally, the major causative agent for BTB can also infect humans causing a variety of clinical presentations. The aim of this study was to determine BTB prevalence and the main risk factors for the prevalence in cattle and buffalos in Amazonas State, Brazil. Tissue samples from 151 animals (45 buffalo and 106 cattle from five herds with buffalo only, 22 herds with cattle only, and 12 herds with buffalo and cattle) were obtained from slaughterhouses under State Veterinary Inspection. were isolated on Stonebrink medium. The positive cultures were confirmed by polymerase chain reaction (PCR) testing. The apparent herd and animal prevalence rates were 56.4 and 5.40%, respectively. Regarding animal species, the apparent prevalence rates were 3% in cattle and 11.8% in buffalo. Generalized Linear Mixed Models (GLMM) with random effect were used to assess the association with risk factors on the prevalence. Species (buffalo), herds size (>100 animals) and the presence of both species (buffalo and cattle) in the herd were the major risk factors for the infection by in the region. The findings reveal an urgent need for evidence-based effective intervention to reduce BTB prevalence in cattle and buffalo and prevent its spread to the human population. Studies are needed to understand why buffalo are more likely to be infected by than cattle in Amazon. Recommendations for zoning, use of data from the inspection services to generate information regarding BTB focus, adoption of epidemiological tools, and discouragement of practices that promote the mixing of cattle and buffalo, were made.
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http://dx.doi.org/10.3389/fvets.2019.00434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914675PMC
December 2019

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

Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil.

Vet Med Sci 2020 02 30;6(1):133-141. Epub 2019 Sep 30.

Center for Comparative Epidemiology, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA.

The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.
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http://dx.doi.org/10.1002/vms3.203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036311PMC
February 2020

Surveillance and characterization of drug-resistant isolated in a reference hospital from Argentina during 8 years' period.

Int J Mycobacteriol 2019 Jul-Sep;8(3):223-228

Tuberculosis and Mycobacterioses Laboratory, Dr. Cetrángolo Hospital, Vicente López, Argentina.

Background: Argentina is considered a country with a middle tuberculosis (TB) incidence. However, according to the last national epidemiological report released in 2018, since 2013, the trends are steadily increasing. The aims of this study were to determine the drug-resistance (DR), multi-DR and extensively DR (MDR/XDR-TB), and rifampicin resistance (RIF-R) burden as a part of the local TB diagnosis (June 2010-August 2018); to detect the mutations associated to isoniazid (INH) and RIF-R and their geographical distribution; and to analyze the lineage relationship among the genetic patterns of the isolates circulating in the community.

Methods: Respiratory and extrapulmonary specimens were processed by Ziehl-Neelsen stain and cultured on specific media. Drug-susceptibility testing of isolates was performed by the MGIT 960 and a colorimetric micro-method. Mutations conferring DR were detected by Genotype and DNA sequencing.

Results: The study showed a DR-TB prevalence of approximately 20% of the isolated strains, while M/XDR-TB-and particularly RIF-R-affected more than 5.0% of the total amount of cases. DR geographical distribution revealed isolates carrying mutations in the inhA gene promoter region only constrained to three districts where it was also registered two same family relatives' cases with the infrequent rpoB S522 L/Q mutation. The fact that most DR/MDR-TB isolates were not grouped in genetic clusters suggested that these cases may mostly have occurred due to endogenous reactivation rather than recently transmission.

Conclusion: According to the obtained results, it would be convenient, in highly MDR-TB suspected individuals, to confirm phenotypically, the INH and RIF susceptibility detected by molecular tests.
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http://dx.doi.org/10.4103/ijmy.ijmy_94_19DOI Listing
January 2020

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

J Vet Med Sci 2019 Oct 28;81(10):1400-1408. Epub 2019 Aug 28.

Embrapa Beef Cattle, Campo Grande, MS, 79106-550, Brazil.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.
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http://dx.doi.org/10.1292/jvms.19-0214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863732PMC
October 2019

Bayesian Assessment of the Accuracy of a PCR-Based Rapid Diagnostic Test for Bovine Tuberculosis in Swine.

Front Vet Sci 2019 26;6:204. Epub 2019 Jun 26.

Veterinary Population Medicine Department, Center for Animal Health and Food Safety, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, United States.

Infection with the causes a disease referred to as bovine tuberculosis (bTB), which affects a wide range of mammal hosts. Many countries have implemented control and eradication plans that have resulted in variable levels of efficacy and success. Although bTB is a notifiable disease in Argentina, and a control plan that targets cattle herds has been in place for decades, is still prevalent in cattle, swine, and certain wild species. The aim of the paper here was to assess the sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV) of PCR from tissue, which is a test for rapid detection in swine. Bacteriological culture was also performed for comparison purposes. A Bayesian approach was applied to estimate the accuracy of the diagnostic tests, PCR and bacteriological culture, in 266 swine samples with bTB-like lesions recovered during routine official inspections at slaughterhouses. A one-population model, assuming conditional dependence between test results, and incorporating prior information on the performance of the tests obtained from the literature, was used to estimate the tests Se and Sp. The accuracy of the combined (in parallel) application of both tests was also estimated. The Se of the PCR (82.9%) was higher than the Se of the bacteriological culture (79.9%), whereas the Sp of both tests was similar (88.5 and 89.0%, respectively). Furthermore, when both techniques were assessed in parallel, the Se of the diagnostic system increased substantially (Se = 96.6%) with a moderate Sp loss (Sp = 78.8%; PPV = 92.8%; NPV = 89%). Results suggest that the PCR, or the combined application of bacteriological culture and PCR, may serve as an accurate diagnostic tool to confirm bTB in swine samples. Results here will help the design and implementation of effective surveillance strategies for the disease in swine of Argentina and other settings in which the disease is prevalent.
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http://dx.doi.org/10.3389/fvets.2019.00204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608602PMC
June 2019

Prevalence and species diversity of nontuberculous mycobacteria in drinking water supply system of Bahía Blanca City, Argentina.

Int J Mycobacteriol 2019 Apr-Jun;8(2):138-145

Department of Biology, Biochemistry and Pharmacy, Southern National University, San Juan 670, Bahía Blanca, Argentina.

Background: There is evidence that tap water is the vehicle through which nontuberculous mycobacteria (NTM) infect or colonize the human body. The objective of this study was to determine the presence and diversity of NTM in the water distribution system of Bahía Blanca city, Argentina (sites S2/S3) and in the dike that supplies water to it (S1).

Methods: Culture-dependent method, biochemical tests, and molecular method (16S rRNA sequencing gene) were combined to detect and identify NTM.

Results: NTM were isolated in 51.6% (64/124) of all the samples analyzed. Mycobacterium gordonae was the most frequently isolated organism (15/64) in all samples analyzed, followed by Mycobacterium peregrinum and Mycobacterium frederiksbergense. Significant differences were found in the residual chlorine values between sampling S2 and S3. In both sites, maximum counts were recorded but they did not correlate with low chlorine values. A concentration higher than 500 colony-forming unit/L of NTM was never found, which can be attributed to the negative effect caused by decontamination methods being a point to consider for the recovery of NTM. In 46.9% (30/64) of samples, both methods coincided in the identification, and the obtained sequences presented ≥99% identity. Identification at the species level was achieved in 50% (32/64) of the isolates. Nearly 17.2% (11/64) of the isolates showed a similarity <99%.

Conclusions: It should be taken into account that sequencing of the 16S rRNA gene and biochemical tests are useful for the identification of several species, but it is necessary to incorporate other genes (hsp 65 and rpo B) to obtain accurate identification.
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http://dx.doi.org/10.4103/ijmy.ijmy_39_19DOI Listing
November 2019

[Species diversity of non-tuberculous mycobacteria isolated from aquatic environments of General Pico city, Province of La Pampa (Argentina)].

Rev Argent Microbiol 2019 Jul - Sep;51(3):259-267. Epub 2018 Dec 19.

Instituto Nacional de Tecnología Agropecuaria (CICVyA/INTA), Hurlingham, Buenos Aires, Argentina.

Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.
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http://dx.doi.org/10.1016/j.ram.2018.08.005DOI Listing
March 2020

Detection of Viable in Lungs and Livers Sold in Butchers' Shops in Buenos Aires, Argentina.

Foodborne Pathog Dis 2018 12 18;15(12):758-762. Epub 2018 Oct 18.

Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de Biotecnología, Buenos Aires, Argentina.

Although is the major etiological agent of tuberculosis in bovines, it can infect other mammalians. Previously reported cases of tuberculosis caused by in cats from the Autonomous City of Buenos Aires (CABA) led to the conclusion that the main source of infection for these felines was the ingestion of raw bovine lungs. Thus, for this study, we collected samples of bovine viscera from butchers' shops of the Greater Buenos Aires (GBA) and the CABA to assess presence and viability of these mycobacteria in bovine lungs (including the lymph nodes) and livers. We analyzed 216 different samples and obtained 5 isolates of (4 from lungs and 1 from liver) by culture analysis. We also confirmed the presence of different isolates by polymerase chain reaction, spoligotyping, and MIRU-VNTR assays. The results obtained in this work emphasizes the need of social education for food hygiene, and to change the habit of feeding pets with raw viscera, which carries the risk of epizootic and zoonotic transmission. Moreover, control and eradication programs of bovine tuberculosis should be strengthened and improved.
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http://dx.doi.org/10.1089/fpd.2018.2467DOI Listing
December 2018

Utilization of molecular and conventional methods for the identification of nontuberculous mycobacteria isolated from different water sources.

Int J Mycobacteriol 2018 Jan-Mar;7(1):53-60

Laboratory of Mycobacteria, Faculty of Veterinary Sciences, National University of La Pampa, General Pico, La Pampa, Argentina.

Background: The environment is the nontuberculous mycobacteria (NTM) reservoir, opportunistic pathogens of great diversity and ubiquity, which is observed in the constant description of new species capable of causing infection. Since its introduction, molecular methods are essential for species identification. Most comparative studies between molecular and conventional methods, have used isolated strains from clinical samples.

Methods: The aim of this study was to evaluate the usefulness of molecular methods, especially the hsp65-PRA (PCR-Restriction Enzyme Analysis), and biochemical tests in the identification of NTM recovered from water of different origins, using the sequencing of 16S rRNA and hsp 65 genes as assessment methods of the previous ones. Species identification was performed for all 56 NTM isolates what were recovered from 32 (42.1%) positive water samples, using conventional phenotypic methods, hsp65-PRA, partial sequencing of 16S rRNA and sequencing of hsp 65 genes.

Results: Phenotypic evaluation and hsp65-PRA were concordant with 23 (41.1%) isolates. Also, the PRA was concordant with 16 (28.6%) and 27 (48.2%) isolates, with the partial sequencing of 16S rRNA and sequencing of hsp 65 genes, respectively. It is considered that the 19.6% (n = 11) could not be identified.

Conclusion: Identification of NTM environmental isolates to the species level, especially when they are pigmented and fast-growing, both the analysis of the restriction patterns obtained by PRA and the sequencing of the 16S rRNA and hsp 65 genes are insufficient by themselves. Although they are demanding and time-consuming, biochemical tests are very useful to support data obtained by molecular methods.
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http://dx.doi.org/10.4103/ijmy.ijmy_192_17DOI Listing
March 2019

Draft Genome Sequences of Two Strains Isolated from Beef Cattle in Paraguay.

Genome Announc 2017 Jul 13;5(28). Epub 2017 Jul 13.

Embrapa Beef Cattle, Campo Grande, Mato Grosso do Sul, Brazil.

This work reports the draft genome sequences of the strains M1009 and M1010, isolated from the lymph nodes of two infected cows on a beef farm in Paraguay. Comparative genomics between these strains and other regional strains may provide more insights regarding epidemiology in South America.
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http://dx.doi.org/10.1128/genomeA.00616-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511916PMC
July 2017

Mycobacterioses in dogs and cats from Buenos Aires, Argentina.

J Vet Diagn Invest 2017 Sep 9;29(5):729-732. Epub 2017 Jun 9.

Department of Infectious Diseases, School of Veterinary Medicine, University of Buenos Aires, Buenos Aires, Argentina (Barandiaran, Martínez Vivot, Falzoni, Marfil, Pérez Tort, Duchene).

Mycobacterioses can produce nonspecific clinical signs in dogs and cats that make diagnosis difficult. Furthermore, the full characterization of mycobacterial agents is not always possible or practical. We characterized mycobacteria detected through cytology in 12 dogs and 7 cats with generalized clinical signs from the province of Buenos Aires in Argentina. In dogs, molecular testing confirmed the presence of Mycobacterium avium subsp. hominissuis (MAH) in 8 cases and M. fortuitum in 1 case. All dogs were Miniature Schnauzers, suggesting that this breed may be more susceptible to M. avium than other dog breeds. The cat isolates were 2 M. bovis, 1 M. fortuitum, and 1 MAH. Mycobacterial interspersed repetitive unit-variable-number tandem repeat patterns suggested possible links with cattle, swine, and humans studied previously in Argentina. The results show that pets may act as susceptible hosts with the potential risk of transmitting the infection to humans and other animals.
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http://dx.doi.org/10.1177/1040638717713795DOI Listing
September 2017

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

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

[Mycobacterium bovis in wildlife of the dairy regions of Santa Fe (Argentina)].

Rev Argent Microbiol 2015 Jul-Sep;47(3):174-82. Epub 2015 Sep 14.

Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Rafaela, Santa Fe, Argentina.

Control eradication campaigns of bovine tuberculosis based on the «test and slaughter» approach were successful in many countries and regions; however, in some areas the infection persists and one of the main reasons is Mycobacterium bovis infection in wild life species. Argentina has applied the same approach since 1999, achieving progress in dairy cattle herds. Nonetheless, the wildlife role has never been investigated. The objective of this study was to determine if wildlife from the Santa Fe dairy area is infected with M. bovis. Wildlife species having a positive tuberculin skin test were captured in five dairy farms. Ninety five wildlife mammals were captured; M. bovis was recovered from 7 possums (Didelphys albiventris), from one fox (Lycolapex gimnocercus) and from one rat (Rattus norvegicus). None of the animals exhibited macroscopic lesions. The most frequently isolated M. bovis spoligotypes were types 34 (4 isolates) and 12 (3 isolates). Spoligotype 34 is the most frequently isolated type in Argentine cattle. The role of D. albiventris as spillover host of M. bovis is discussed in this study.
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http://dx.doi.org/10.1016/j.ram.2015.04.005DOI Listing
February 2016

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis.

Braz J Microbiol 2015 Jun 1;46(2):619-26. Epub 2015 Jun 1.

Instituto Nacional de Tecnología Agropecuaria, National Agricultural Technology Institute, Buenos Aires, Argentina, Institute of Biotechnology, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute, Buenos Aires, Argentina.

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.
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http://dx.doi.org/10.1590/S1517-838246220131206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507559PMC
June 2015

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

Braz J Microbiol 2015 Jun 1;46(2):557-64. Epub 2015 Jun 1.

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

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.
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http://dx.doi.org/10.1590/S1517-838246220140283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507551PMC
June 2015

Mycobacterium bovis infection in a horse with granulomatous enterocolitis.

J Vet Diagn Invest 2015 Mar 12;27(2):203-5. Epub 2015 Feb 12.

Department of General and Systemic Pathology, Veterinary Faculty, National University of Rosario, Casilda, Argentina (Sarradell, Biscia)Department of Veterinary Population Medicine (Alvarez, Perez), College of Veterinary Medicine, University of Minnesota, St. Paul, MNVeterinary Diagnostic Laboratory (Wunschmann, Armien), College of Veterinary Medicine, University of Minnesota, St. Paul, MNBiotechnology Institute, Centre of Agronomy and Veterinary Sciences, National Agricultural Technology Institute (INTA), Hurlingham, Buenos Aires, Argentina (Zumarraga).

A 2-year-old dappled Percheron horse had a wasting condition that did not respond to antibiotic treatments and ultimately resulted in death. Thickening of the wall of the large colon and enlargement of the mesenteric lymph nodes were observed at postmortem examination, along with the presence of pinpoint whitish foci in the liver. Microscopic examination of affected tissues revealed diffuse chronic granulomatous enterocolitis, granulomatous mesenteric lymphadenitis, and multifocal granulomatous hepatitis. The DNA extracted from paraffin-embedded intestinal and lymph node samples was analyzed using both a polymerase chain reaction (PCR) assay and PCR-restriction endonuclease analysis and demonstrated the presence of Mycobacterium bovis.
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http://dx.doi.org/10.1177/1040638715571359DOI Listing
March 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

Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis.

Nature 2014 Oct 20;514(7523):494-7. Epub 2014 Aug 20.

1] Department of Archaeological Sciences, University of Tübingen, Ruemelinstraße 23, 72070 Tübingen, Germany [2] Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen 72070, Germany [3] Max Planck Institute for Science and History, Khalaische Straße 10, 07745 Jena, Germany.

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.
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http://dx.doi.org/10.1038/nature13591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550673PMC
October 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

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

PLoS One 2014 11;9(3):e91023. Epub 2014 Mar 11.

Embrapa Gado de Corte, Campo Grande, MS, Brazil.

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091023PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949733PMC
May 2015

Mycobacterium intracellulare infection in a capybara (Hydrochoerus hydrochaeris).

J Zoo Wildl Med 2013 Dec;44(4):1098-101

Laboratorio de Histopatología y Citología, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, R. P. Kreder 2805, 3080, Esperanza, Santa Fe, Argentina.

This report describes the first case of Mycobacterium intracellulare infection with typical granulomatous lesions of mycobacteriosis in a capybara (Hydrochoerus hydrochaeris). The individual was a captive-bred young female, part of the control group of an experimental study on stress. Multiple granulomatous lesions were detected in a mesenteric lymph node of this young female. Mycobacterial infection was confirmed by bacteriologic culture and molecular identification methods. Clinical lesions were characterized by histopathology.
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http://dx.doi.org/10.1638/2013-0017R1.1DOI Listing
December 2013

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

First evaluation in Argentina of the GenoType® MTBDRplus assay for multidrug-resistant Mycobacterium tuberculosis detection from clinical isolates and specimens.

Rev Argent Microbiol 2012 Oct-Dec;44(4):283-9

Laboratorio de Referencia del Programa de Control de la Tuberculosis de la Provincia de Buenos Aires, Hospital Dr. Antonio Cetrángolo, Italia 1750, Florida (1602) Buenos Aires.

Tuberculosis (tB) and multidrug and extensively drug-resistant (dR) tB are important public health problems that are spreading worldwide. The aims of this study were to determine the sensitivity and specificity of the genotype® mtBdRplus assay from smear-positive clinical specimens and isolates and to explore its possible application in routine work. Clinical samples were previously decontaminated using naoH-n-acetyl-l-cystein or naoH-Clna hypertonic solution for Ziehl-neelsen staining and cultures. The leftover sediments of smear-positive samples were stored at -20 °C, 70 of which were selected to be included in this study according to their dR profile. thirty dR Mycobacterium tuberculosis isolates were also assessed. Sequencing was used as gold standard to detect mutations conferring isoniazid (InH) and rifampicin (RIF) resistance. Valid results were obtained in 94.0 % of the samples and 85.5 % (53/62) of the InH-R samples were properly identified. mutations in the katGS315t gene and inhA C-15t gene promoter region were present in 59.7 % (37/62) and 25.8 % (16/62) of the InH-R samples, respectively. the system could also identify 97.7 % (41/42) of the RIF-R samples; the mutations found were rpoBS531l (66.7 %, 28/42), d516V (19.0 %, 8/42), H526Y and S531p/W (4.8 %, 2/42 each one), and S522l/Q (2.4 %, 1/42). a 98.8 % concordance between the genotype assay and sequencing was obtained. genotype® mtBdRplus has demonstrated to be easy to implement and to perform in clinical laboratories and useful for a rapid detection of dR M. tuberculosis from decontaminated sputa and clinical isolates. Therefore, this assay could be applied as a rapid tool to predict InH-R and/or RIF-R in dR risk cases.
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April 2013

[Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis].

Rev Argent Microbiol 2012 Jul-Sep;44(3):155-64

Instituto de Biotecnología, CICVyA-INTA Castelar, Dr. Nicolás Repetto y De Los Reseros S/N, (1686) Hurlingham, Buenos Aires, Argentina.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of yIFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.
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January 2013