Preservation of protective capacity of hyperimmune anti-Stx2 bovine colostrum against enterohemorrhagic Escherichia coli O157:H7 pathogenicity after pasteurization and spray-drying processes.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major etiologic agent that causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is the main virulence factor of EHEC responsible for the progression to HUS. Although many laboratories have made efforts to develop an effective treatment for Stx-mediated HUS, a specific therapy has not been found yet. Human consumption of bovine colostrum is known to have therapeutic effects against several gastrointestinal infections because of the peptide and proteins (including antibodies) with direct antimicrobial and endotoxin-neutralizing effects contained in this fluid. We have previously demonstrated that colostrum from Stx type 2 (Stx2)-immunized pregnant cows effectively prevents Stx2 cytotoxicity and EHEC O157:H7 pathogenicity. In this study we evaluated the preservation of the protective properties of hyperimmune colostrum against Stx2 (HIC-Stx2) after pasteurization and spray-drying processes by performing in vitro and in vivo assays. Our results showed that reconstituted HIC-Stx2 colostrum after pasteurization at 60°C for 60 min and spray-dried under optimized conditions preserved specific IgG that successfully neutralized Stx2 cytotoxicity on Vero cells. Furthermore, this pasteurized/dehydrated and reconstituted HIC-Stx2 preserved the protective capacity against EHEC infection in a weaned mice model. The consumption of hyperimmune HIC-Stx2 bovine colostrum could be effective for HUS prevention in humans as well as in EHEC control in calves. However, further studies need to be done to consider its use for controlling EHEC infections.

The genomics of mycobacteria.

The species Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis are the causal agents, respectively, of tuberculosis and paratuberculosis in animals. Both mycobacteria, especially M. bovis, are also important to public health because they can infect humans. In recent years, this and the impact of tuberculosis and paratuberculosis on animal production have led to significant advances in knowledge about both pathogens and their host interactions. This article describes the contribution of genomics and functional genomics to studies of the evolution, virulence, epidemiology and diagnosis of both these pathogenic mycobacteria.

Les mycobactéries Mycobacterium bovis et Mycobacterium avium subsp. paratuberculosis sont les agents étiologiques de la tuberculose et de la paratuberculose, respectivement. En outre, les deux mycobactéries (mais plus particulièrement M. bovis) peuvent infecter l'être humain et jouent donc un rôle en santé publique. En raison de cette importance et des effets de la tuberculose et de la paratuberculose sur la production animale, de grands efforts ont été déployés pour faire avancer nos connaissances sur ces deux agents pathogènes et sur leurs interactions avec leurs hôtes. Les auteurs décrivent la contribution de la génomique et de la génomique fonctionnelle dans les études sur l'évolution, la virulence, l'épidémiologie et le diagnostic de ces deux mycobactéries pathogènes.

Las especies Mycobacterium bovis y Mycobacterium avium subsp. paratuberculosis son los agentes causales de la tuberculosis y la paratuberculosis en animales, respectivamente. Además, ambas micobacterias, pero fundamentalmente M. bovis, son importantes para la salud pública, ya que pueden infectar a los humanos. Debido a esto último y al impacto de la tuberculosis y la paratuberculosis en la producción animal, en los últimos años se ha producido un avance significativo en los conocimientos de ambos agentes patógenos y de la interacción con sus hospedadores. En este artículo describiremos la contribución de la genómica y la genómica funcional a los estudios de evolución, virulencia, epidemiología y diagnóstico de ambas micobacterias patógenas.

Bovine tuberculosis in domestic pigs: Genotyping and distribution of isolates in Argentina.

Bovine tuberculosis is caused by Mycobacterium bovis and affects primarily cattle, among many other mammal species. In this study, 250 isolates of M. bovis collected from pigs slaughtered in Argentina were typed by spoligotyping. Over half of the isolates (66%) grouped into two spoligotypes. Moreover, SB0140 was the most frequent spoligotype detected in the three performed samplings. In addition, 195 isolates were typed through variable number of tandem repeats (VNTR) by selecting 7 loci (MIRU 16­26­ 31 and ETR A­B­C­D). The relationship among the patterns was performed using a goeBURST algorithm and the main clonal complexes grouped 110 isolates (56%). Although pigs shared genotypes with cattle (n = 21), some patterns were detected only in pigs (n=14). These findings suggest the pig as a source ofM. bovis infection to cattle.

Tuberculosis in swine co-infected with Mycobacterium avium subsp. hominissuis and Mycobacterium bovis in a cluster from Argentina.

SUMMARY In Argentina little is known about the epidemiology of tuberculosis (TB) infection in swine. We characterized the epidemiological dynamics of Mycobacterium avium complex (MAC) infection in a swine population of Argentina using molecular tools and spatial analysis techniques. Isolates (n = 196) obtained from TB-like lesions (n = 200) were characterized by polymerase chain reaction. The isolates were positive to either M. bovis (IS6110) (n = 160) or M. avium (IS1245) (n = 16) while the remaining 20 (10.2%) isolates were positive to both M. bovis and M. avium. The detection of both bacteria together suggests co-infection at the animal level. In addition, MAC-positive isolates (n = 36) were classified as M. avium subsp. avium (MAA) (n = 30) and M. avium subsp. hominissuis (MAH) (n = 6), which resulted in five genotypes when they were typed using mycobacterial interspersed repetitive unit, variable number of tandem repeats (MIRU-VNTR). One significant (P = 0.017) spatial clustering of genotypes was detected, in which the proportion of MAH isolates was larger than expected under the null hypothesis of even distribution of genotypes. These results show that in Argentina the proportion of TB cases in pigs caused by M. avium is larger than that reported in earlier studies. The proportion of M. bovis-MAC co-infections was also higher than in previous reports. These results provide valuable information on the epidemiology of MAC infection in swine in Argentina.

Molecular and phenotypic characterisation of Mycobacterium tuberculosis resistant to anti-tuberculosis drugs.

SETTING: Dr Cetrángolo Hospital, Buenos Aires, Argentina. OBJECTIVES: To characterise drug-resistant (DR), multidrug-resistant (MDR-) and extensively drug-resistant (XDR-) Mycobacterium tuberculosis isolates, and identify their genetic profiles, drug resistance levels and resistance-conferring mutations. DESIGN: Phenotypic drug susceptibility testing methods were used to determine drug resistance profiles. Minimal inhibitory concentrations (MICs) of isoniazid (INH), rifampicin (RMP) and levofloxacin (LVX) from 169 DR tuberculosis (TB) isolates, 78 of them monoresistant to INH, 13 to RMP, 7 to LVX, and 71 MDR-TB, were determined. Multiplex allele-specific polymerase chain reaction and DNA sequencing were used to detect mutations in katG, rpoB and gyrA/B genes. Genotyping was performed using spoligotyping and insertion sequence 6110 restriction fragment length polymorphism. RESULTS: In total, 38.9% of the INH-resistant (INH(R)) isolates had an MIC ≥ 32 g/ml; 61.3% of RMP-resistant (RMP(R)) isolates had an MIC ≥ 64 g/ml and 55.6% of the LVX-resistant (LVX(R)) isolates had an MIC 4 ≥ 16 g/ml. The main mutations found in INH(R) isolates were katG315 (53.7%) and inhAP-15 (25.5%), whereas in RMP(R) isolates the main mutations were rpoB531 (61.9%), followed by rpoB526 (16.7%). LVX(R) isolates showed mutations in gyrA94/90. Haarlem, LAM and T were the main spoligotyping families found. katG315 was mainly associated with Haarlem and LAM, whereas inhAP-15 was associated with T. CONCLUSIONS: Several isolates showed an association between high INH(R) levels and katG mutation; others from the Haarlem family were prone to becoming MDR-TB and continue to circulate in the community.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis by multiplex allele-specific polymerase chain reaction.

SETTING: Dr Cetrangolo Hospital, Buenos Aires Province, Argentina. OBJECTIVE: To evaluate a multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis (MDR-TB) clinical isolates and to describe the main mutations conferring resistance to isoniazid (INH) and rifampicin (RMP). DESIGN: Drug-resistant Mycobacterium tuberculosis clinical isolates were tested to detect mutations using MAS-PCR. The genes involved were katG, inhA promoter and rpoB. RESULTS: Among 193 clinical isolates included in the study, 52.6% of the INH-resistant isolates presented a mutation in the katG (315) gene, 28.1% in the inhAP (-15) and 3.0% in both. For the rpoB gene, 60% of the RMP-resistant isolates showed a mutation in codon 531, 17.5% in 526 and 2.5% in 516. Results were compared with those obtained by sequencing, and 100% concordance was obtained for the detection of the mutation in katG (315), 94.1% for inhAP (-15), and 97.8% for rpoB. The global concordance between both methods was 98%. CONCLUSIONS: The MAS-PCR system allowed the simultaneous and rapid detection of approximately 80.0% of the drug-resistant clinical isolates. This method could be used as a rapid and simple screening tool to detect drug-resistant TB in clinical practice.

Evaluation of an immunomagnetic capture method followed by PCR to detect Mycobacterium bovis in tissue samples from cattle / Evaluación de un método de captura inmunomagnética seguida de PCR para la detección de Mycobacterium bovis en tejidos de ganado

Tuberculosis is one of the most important infectious diseases worldwide. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), an important animal pathogen with public health implications as it is a zoonosis. Currently, the diagnosis of BTB is based on the caudal fold test of the Tuberculin Skin Test (TST). Post-mortem bacterial culture is carried out to confirm the diagnosis, and then specific biochemical tests are performed for the characterization of the etiologic agent. Culture takes at least 4 to 8 weeks to develop. The diagnosis by molecular tests such as PCR can provide fast and reliable results, significantly decreasing the time of confirmation (from two months to two days), thus allowing the possibility of taking control actions to prevent the spread of the disease in herds. In this work the use of an immunomagnetic separation capture followed by PCR (IMS-PCR) based on the IS6110 element showed a detection threshold corresponding to 10 CFU in M. bovis-spiked PBS. In the case of infected bovine fresh tissues, after five replicates, the minimum value of detection was 1000 CFU in 100% of the trials (5/5). This paper attempts to provide a sensitive, rapid and specific technique for the diagnosis of bovine tuberculosis, and opens up the possibility of a direct application in the control and eradication of this cattle disease.

La tuberculosis es una de las enfermedades infecciosas más importantes. Mycobacterium bovis es el agente causal de la tuberculosis bovina (TBB), un patógeno animal y zoonótico. En la actualidad, el diagnóstico de TBB se basa en la prueba intradérmica de la tuberculina. El cultivo bacteriano post mortem se lleva a cabo para confirmar el diagnóstico y a continuación se realizan pruebas bioquímicas específicas para la caracterización del agente etiológico. El cultivo bacteriano toma por lo menos 4 a 8 semanas para su desarrollo. El diagnóstico mediante pruebas moleculares como PCR puede proporcionar resultados rápidos y robustos, con un considerable acortamiento hasta la confirmación del diagnóstico (de 2 meses a 2 días). En este trabajo, el uso de captura inmunomagnética seguida de PCR (IMS-PCR) dirigida al elemento IS6110 mostró un umbral de detección correspondiente a 10 UFC en M. bovis diluido en PBS. En el caso de tejidos bovinos inoculados experimentalmente después de 5 réplicas, el valor mínimo de detección fue de 1000 UFC en el 100% de los ensayos. Este artículo aspira a proporcionar una técnica sensible, rápida y específica para el diagnóstico de la tuberculosis bovina, con el fin de abrir la posibilidad de una aplicación directa en el control y la erradicación de esta enfermedad en el ganado.

Evaluation of an immunomagnetic capture method followed by PCR to detect Mycobacterium bovis in tissue samples from cattle.

Tuberculosis is one of the most important infectious diseases worldwide. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), an important animal pathogen with public health implications as it is a zoonosis. Currently, the diagnosis of BTB is based on the caudal fold test of the Tuberculin Skin Test (TST). Post-mortem bacterial culture is carried out to confirm the diagnosis, and then specific biochemical tests are performed for the characterization of the etiologic agent. Culture takes at least 4 to 8 weeks to develop. The diagnosis by molecular tests such as PCR can provide fast and reliable results, significantly decreasing the time of confirmation (from two months to two days), thus allowing the possibility of taking control actions to prevent the spread of the disease in herds. In this work the use of an immunomagnetic separation capture followed by PCR (IMS-PCR) based on the IS6110 element showed a detection threshold corresponding to 10 CFU in M. bovis-spiked PBS. In the case of infected bovine fresh tissues, after five replicates, the minimum value of detection was 1000 CFU in 100% of the trials (5/5). This paper attempts to provide a sensitive, rapid and specific technique for the diagnosis of bovine tuberculosis, and opens up the possibility of a direct application in the control and eradication of this cattle disease.

Mycobacterium bovis in Argentina: isolates from cats typified by spoligotyping / Mycobacterium bovis en Argentina: aislamientos de gatos tipificados por spoligotyping

In the present work, 19 Mycobacterium bovis isolates from different cats were typified by spoligotyping. We detected nine spoligotypes. There was only one cluster, which grouped 11 of the isolates (57.9%), showing the main spoligotype from cattle from Argentina. The rest of the spoligotypes presented only one isolate each. Five of them were not found in cattle, and were unique and exclusive of cats. The isolates studied show that tuberculosis of bovine origin in cats constitutes a potential public health problem in Buenos Aires region. The identification of genotypes from non-natural hosts could contribute to understand the spread of bovine tuberculosis. This is the first report showing genetic profiles of M. bovis isolates in felines from Argentina.

En el presente trabajo se tipificaron por spoligotyping 19 aislamientos de M. bovis de diferentes gatos. Se detectaron 9 espoligotipos y un único agrupamiento o cluster integrado por 11 aislamientos (57,9%) y relacionado con el principal espoligotipo de bovinos de Argentina. El resto de los espoligotipos detectados presentaron solamente un aislamiento cada uno; 5 de ellos no se encontraron en bovinos y fueron únicos y exclusivos de gatos. La presencia de estos aislamientos indica que la tuberculosis bovina en los gatos constituye un potencial problema de salud pública en la ciudad de Buenos Aires. La identificación de genotipos de aislamientos de M. bovis de hospedadores no convencionales podría contribuir a la mejor comprensión de la diseminación de la tuberculosis bovina. Este es el primer informe en el que se muestran los perfiles genotípicos de aislamientos de M. bovis obtenidos de felinos de Argentina.

Mycobacterium bovis with different genotypes and from different hosts induce dissimilar immunopathological lesions in a mouse model of tuberculosis.

With the hypothesis that genetic variability of Mycobacterium bovis could influence virulence and immunopathology, five M. bovis strains were selected from an epidemiological study in Argentina on the basis of their prevalence in cattle and occurrence in other species. We then determined the virulence and the immunopathology evoked by these strains in a well-characterized mouse model of progressive pulmonary tuberculosis. The reference strain AN5 was used as a control. BALB/c mice infected with this M. bovis reference strain showed 50% survival after 4 months of infection, with moderate bacillary counts in the lung. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In contrast, strain 04-303, isolated from a wild boar, was the most lethal and its most striking feature was sudden pneumonia with extensive necrosis. Strain 04-302, also isolated from wild boar but with a different spoligotype, induced similar pathology but to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02-2B (from human) were less virulent, permitting higher survival after 4 months of infection and limited tissue damage. Strain AN5 and the cattle and human isolates induced rapid, high and stable expression of interferon (IFN)-gamma and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN-gamma, tumour necrosis factor-alpha and iNOS. Interestingly, these more virulent strains induced very low expression of murine beta defensin 4 (mBD-4); whereas, the control strain AN5 induced progressive expression of this anti-microbial peptide, peaking at day 120. The less virulent strains induced high mBD-4 expression during early infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also showed variable virulence. This variability can be attributed to the induction of a different pattern of immune response.

Mycobacterium bovis in Argentina: isolates from cats typified by spoligotyping.

In the present work, 19 Mycobacterium bovis isolates from different cats were typified by spoligotyping. We detected nine spoligotypes. There was only one cluster, which grouped 11 of the isolates (57.9%), showing the main spoligotype from cattle from Argentina. The rest of the spoligotypes presented only one isolate each. Five of them were not found in cattle, and were unique and exclusive of cats. The isolates studied show that tuberculosis of bovine origin in cats constitutes a potential public health problem in Buenos Aires region. The identification of genotypes from non-natural hosts could contribute to understand the spread of bovine tuberculosis. This is the first report showing genetic profiles of M. bovis isolates in felines from Argentina.

[The genotype of the principal Mycobacterium bovis in Argentina is also that of the British Isles: did bovine tuberculosis come from Great Britain?]. / El genotipo de Mycobacterium bovis mayoritario en la Argentina lo es también en las islas Británicas: la tuberculosis bovina provino de Gran Bretaña?

Bovine tuberculosis is a highly prevalent animal disease in Argentina. In this work evidence was obtained showing that a major Mycobacterium bovis group in Argentina had been introduced with the bovine bulls imported from the United Kingdom at the end of the XIX century. This evidence came from two sources: historical, obtained by bibliographical references, and from laboratory results, using a molecular typing method called spoligotyping. These strains are also present in other countries that introduced cattle from the same origin.

El genotipo de Mycobacterium bovis mayoritario en la Argentina lo es también en las islas Británicas: la tuberculosis bovina provino de Gran Bretaña? / [The genotype of the principal Mycobacterium bovis in Argentina is also that of the British Isles: did bovine tuberculosis come from Great Britain?]

Bovine tuberculosis is a highly prevalent animal disease in Argentina. In this work evidence was obtained showing that a major Mycobacterium bovis group in Argentina had been introduced with the bovine bulls imported from the United Kingdom at the end of the XIX century. This evidence came from two sources: historical, obtained by bibliographical references, and from laboratory results, using a molecular typing method called spoligotyping. These strains are also present in other countries that introduced cattle from the same origin.

El genotipo de Mycobacterium bovis mayoritario en la Argentina lo es también en las islas Británicas: la tuberculosis bovina provino de Gran Bretaña? / [The genotype of the principal Mycobacterium bovis in Argentina is also that of the British Isles: did bovine tuberculosis come from Great Britain?]

Bovine tuberculosis is a highly prevalent animal disease in Argentina. In this work evidence was obtained showing that a major Mycobacterium bovis group in Argentina had been introduced with the bovine bulls imported from the United Kingdom at the end of the XIX century. This evidence came from two sources: historical, obtained by bibliographical references, and from laboratory results, using a molecular typing method called spoligotyping. These strains are also present in other countries that introduced cattle from the same origin.

PCR-restriction fragment length polymorphism analysis (PRA) of Mycobacterium leprae from human lepromas and from a natural case of an armadillo of Corrientes, Argentina.

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that allows identification of the species without the need for specialized equipment. Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in internal organs such as lymph nodes, spleen and liver, as well as in nerves and skin. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans. Mycobacterium leprae were recognized in those naturally infected animals through different techniques. Three samples of extracted DNA of the mycobacteria present in the spleen, liver and popliteal lymph node of a naturally infected animal during the Experimental Program in Armadillo (PEA) and three samples of human lepromas were processed by PRA. The patterns of the six samples analyzed were identical and were characteristic of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of armadillos naturally infected with the Hansen bacillus.

Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendations for standardised nomenclature.

Spacer oligonucleotide typing (spoligotyping) is widely used for differentiation of bacteria of the Mycobacterium tuberculosis complex. However, the absence of any standardised method for concise description of spoligotypes makes it difficult to compare the results from different laboratories. This paper describes unambiguous, interconvertible systems for the designation of spoligotype patterns, the adoption of which will be beneficial to mycobacterial research.

The sae locus of Staphylococcus aureus encodes a two-component regulatory system.

Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system.

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents

Polyamines and regulation of ornithine biosynthesis in Escherichia coli.

The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.