Field Trial with Vaccine Candidates Against Bovine Tuberculosis Among Likely Infected Cattle in a Natural Transmission Setting.

Background/Objectives: Vaccines may improve the control and eradication of bovine tuberculosis. However, the evaluation of experimental candidates requires the assessment of the protection, excretion, transmission and biosafety. A natural transmission trial among likely infected animals was conducted. Methods: Seventy-four male heifers were randomly distributed (five groups) and vaccinated subcutaneously with attenuated strains (M. bovis Δmce2 or M. bovis Δmce2-phoP), a recombinant M. bovis BCG Pasteur (BCGr) or M. bovis BCG Pasteur. Then, they cohoused with a naturally infected bTB cohort under field conditions exposed to the infection. Results: A 23% of transmission of wild-type strains was confirmed (non-vaccinated group). Strikingly, first vaccination did not induce immune response (caudal fold test and IFN-gamma release assay). However, after 74 days of exposure to bTB, animals were re-vaccinated. Although their sensitization increased throughout the trial, the vaccines did not confer significant protection, when compared to the non-vaccinated group, as demonstrated by pathology progression of lesions and confirmatory tools. Besides, the likelihood of acquiring the infection was similar in all groups compared to the non-vaccinated group (p > 0.076). Respiratory and digestive excretion of viable vaccine candidates was undetectable. To note, the group vaccinated with M. bovis Δmce2-phoP exhibited the highest proportion of animals without macroscopic lesions, compared to the one vaccinated with BCG, although this was not statistically supported. Conclusions: This highlights that further evaluation of these vaccines would not guarantee better protection. The limitations detected during the trial are discussed regarding the transmission rate of M. bovis wild-type, the imperfect test for studying sensitization, the need for a DIVA diagnosis and management conditions of the trials performed under routine husbandry conditions. Re-vaccination of likely infected bovines did not highlight a conclusive result, even suggesting a detrimental effect on those vaccinated with M. bovis BCG.

Enterohemorrhagic Escherichia coli O157:H7 antigens produced in transgenic lettuce effective as an oral vaccine in mice.

KEY MESSAGE: Transgene with recombination sites to address biosafety concerns engineered into lettuce to produce EspB and γ-intimin C280 for oral vaccination against EHEC O157:H7. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen where ruminant farm animals, mainly bovine, serve as reservoirs. Bovine vaccination has been used to prevent disease outbreaks, and the current method relies on vaccines subcutaneously injected three times per year. Since EHEC O157:H7 colonizes mucosal surfaces, an oral vaccine that produces an IgA response could be more convenient. Here, we report on oral vaccination against EHEC O157:H7 in mice orally gavaged with transgenic lettuce that produces EHEC O157:H7 antigens EspB and γ-intimin C280. Younger leaves accumulated a higher concentration of antigens; and in unexpanded leaves of 30-day-old T2 plants, EspB and γ-intimin C280 were up to 32 and 51 µg/g fresh weight, respectively. Mice orally gavaged with lettuce powders containing < 3 µg antigens for 6 days showed a mucosal immune response with reduced colonization of EHEC O157:H7. This suggests that the transgenic lettuce has potential to be used for bovine vaccination. To promote the biosafety of crop plants producing medically relevant proteins, recombination sites were built into our transgenic lines that would permit optional marker removal by Cre-lox recombination, as well as transgene deletion in pollen by CinH-RS2 recombination. The ability to upgrade the transgenic lettuce by stacking additional antigen genes or replacing older genes with newer versions would also be possible through the combined use of Bxb-att and Cre-lox recombination systems.

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

Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis, primarily affecting the lungs. The M. tuberculosis 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.

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

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, fliCH7, efa, iha, and toxB genes, with stx2a/stx2c 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.

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

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.

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

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.

Vaccination with a BCG strain overexpressing Ag85B protects cattle against Mycobacterium bovis challenge.

Mycobacterium bovis is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with leuD. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with M. bovis, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur.

Assessment of the immune responses induced in cattle after inoculation of a Mycobacterium bovis strain deleted in two mce2 genes.

The generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. Rationally attenuated Mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. However, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental M. bovis strains. Therefore, the aim of this study was to obtain an M. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. We showed that the activation of CD4+ T cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. Moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mRNA Th-1 cytokines than the parental strain. Therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis.

Detection of Mycobacterium bovis-infected dairy herds using PCR in bulk tank milk samples.

Bovine tuberculosis (bTB) is a chronic and zoonotic disease due to Mycobacterium bovis. The tuberculosis eradication campaign carried out in Argentina has considerably improved the health situation of the herds. Here we evaluated a strategy to detect M. bovis-infected herds by Touch-Down IS6110 polymerase chain reaction (PCR) in bulk tank raw milk from dairy farms. We evaluated 177 samples from herds with the official tuberculosis free certificate (TFC) and 80 from herds without the certificate, non-tuberculosis-free certificate (NTFC), from 10 departments of Santa Fe province, Argentina. To avoid the effect of Taq polymerase inhibitors, a dilution of DNA template was performed. Positive PCR results were obtained in 102 (40%) of the samples, whereas negative ones were obtained in 155 (60%) of the samples. Importantly, 44% of NTFC and 38% of TFC samples were positive. All samples were subjected to culture in Löwenstein Jensen and Stonebrink media with no positive isolation. The negative predictive value (NPV) of PCR in the TFC group was 95%, while the positive predictive value (PPV) of PCR in the NTFC group was 51%. Based on these results, this work proposes a method that should be applied regularly to detect M. bovis--infected dairy herds, complementary to the official test of tuberculin, or purifed protein derivative (PPD), to control dairy herds, especially those free of tuberculosis.

Knockout mutation of p27-p55 operon severely reduces replication of Mycobacterium bovis in a macrophagic cell line and survival in a mouse model of infection.

Integrity of p27-p55 operon has been demonstrated to be crucial for replication of Mycobacterium tuberculosis, the main agent of human tuberculosis, in the mouse model of infection. However, the individual contribution of each gene of the operon for the virulence of pathogenic Mycobacterium spp. still remains unclear. The operon is formed by two genes, p27 and p55. p27 gene encodes a lipoprotein that binds triacylated glycolipids and modulates the host immune responses by inhibiting the MHC-II Ag processing. Besides, p55 encodes an efflux pump that, together with P27, is involved in resistance to drugs. In this study, we evaluated the individual contribution of P27 and P55 to the virulence of Mycobacterium bovis, the etiological agent for bovine tuberculosis. Knockout mutation of p27-p55 operon in M. bovis severely decreased the virulence of the bacteria when assessed in a progressive model of pulmonary tuberculosis in Balb/c mice. In addition, the mutant strain showed poor replication in a murine macrophagic cell line. Virulence and intracellular replication were only restored when the mutant strain was complemented with a copy of the whole operon. The reintroduction of p55 into the mutant strain partially restored the virulence of the bacteria while no complementation was achieved with p27 individual gene. 

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Mycobacterium bovis in Swine: Spoligotyping of Isolates from Argentina.

A total of 143 Mycobacterium bovis isolates of pigs, from the most productive swine area in Argentina, were typed by spoligotyping. Twenty-two different spoligotypes were identified, and 133 (93%) isolates were grouped into 12 clusters. One of them, designed SB0140, was the most frequent because it held 83 (58%) isolates. This spoligotype also grouped 362 (43%) out of 841 isolates from previously typed cattle and, thus, constitutes the most frequent in our country. In addition, 135 (94%) isolates revealed spoligotypes identical to those of cattle, showing an epidemiological link. On the other hand, there were seven novel spoligotypes, six of which were also unique since they had only one isolate each. This study aimed to identify the spoligotypes of M. bovis isolated from pigs to contribute to a better understanding of the distribution of bovine tuberculosis in the main productive area of Argentina.

Increased IL-17 expression is associated with pathology in a bovine model of tuberculosis.

The identification of bovine tuberculosis (bTB) biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and to improve the disease diagnosis and prognosis. The aim of this study was to understand the changing profile of the immune responses during the course of infection and to identify biomarkers associated with pathology. Here we describe the immune response developed in experimentally infected cattle with field Mycobacterium bovis strains. Blood samples were taken from each animal at different time points after M. bovis intratracheal infection and lymphocyte subset activation and cytokine mRNA expression were determined from peripheral blood mononuclear cells in response to purified protein derivative (PPDB). We found that CD4 and CD8 activation during the early stages of infection, together with IL-17 gene expression, were positively associated with pathology. The results of this study provide evidences of the role of IL-17 in the immunopathology of tuberculosis and support the use of IL-17 as a potential biomarker with predictive value of prognosis in bTB.

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.