Recovery of Mycobacterium bovis from Soft Fresh Cheese Originating in Mexico

Recent outbreaks of human tuberculosis in the United States caused by Mycobacterium bovis have implicated cheese originating in Mexico as a source of these infections. A total of 203 samples of cheese originating in Mexico were cultured, and M. bovis was recovered from one specimen. Therefore, M. bovis can be recovered from cheese and may be a source of human infections.     

Pulmonary mycoplasmosis in farmed white-tailed deer (Odocoileus virginianus)

An outbreak of respiratory disease at a farmed cervid facility resulted in isolation and identification of Mycoplasma boris in four affected white-tailed deer (Odocoileus virginianus) fawns. Microscopically, pulmonary lesions similar to those associated with M. bovis infections in calves, inclulding lymphoplasmacytic peribronchiolar cuffing and caseonecrotic bronchiectasis, were present. Arcanobacterium pyogenes was recovered from lung tissue as well. This report indicates that M. bovis can be associated with respiratory disease in white-tailed deer.     

Increasing Prevalence of Mycoplasma bovis in Danish Cattle

A study on the prevalence of mycoplasmas in pneumonic bovine lungs was performed on material submitted for diagnostic purposes at the Danish Veterinary Laboratory, Copenhagen. Among the 50 examined cases 43 (86.0%) were found to be infected with mycoplasmas. The predominant mycoplasmas were Ureaplasma spp. (72.0%), M. dispar (48.0%) and M. bovis (24.0%). Other mycoplasmas were M. bovirhinis (20.0%) and M. bovigenitalium (6.0%). Among the infected lungs multiple species infections were predominant (76.7%) over single species infections (23.3%) with M. dispar-Ureaplasma (25.6%), M. bovis-Ureaplasma (18.6%) and M. dispar-M. bovirhinis-Ureaplasma (11.6%) infections being the most frequently encountered combinations. There appears to be an increasing prevalence of M. bovis (24.0%)) as compared to earlier reports (0.6–2.0%), thus calling for special attention upon this mycoplasma. Pulsed field gel electrophoresis (PFGE) analysis of 11 field isolates of M. bovis from 9 different farms revealed different profiles except for 2 isolates which were recovered from the same farm. Because mycoplasmas belonging to the ‘M. mycoides cluster’ were not encountered during this study; it appears that the Danish cattle population is still free from this group of mycoplasma in spite of their presence in some other European countries.     

Spoligotype analysis of Mycobacterium bovis isolates from Northern México

Bovine tuberculosis is still rife in Latin America, producing huge economic losses. There are very few studies of the way this disease is spread through this geographical region, particularly in countries that border those that are almost free of Mycobacterium bovis. In this work, we have analyzed the spacer oligonucleotide typing (spoligotype) patterns of M. bovis isolates from cattle at Ciudad Juárez, a Mexican city close to El Paso, Texas. Fifty-eight M. bovis isolates collected from a herd in Northern Mexico were studied by spoligotyping. Nine spoligotype patterns were observed in total. Two were predominant (SB0121 and SB0140) and accounted for 50% and 14% of the isolates, respectively. Six patterns were found to be already described in an international M. bovis spoligotype database, while the other three (SB0985, SB0986, and SB0987) were novel. Interestingly, none of the isolates corresponded to any other Mexican pattern previously reported. This is the first spoligotype analysis of M. bovis strains from a border city between Mexico and the United States. The necessity for further studies to formulate a better identification of M. bovis strains within, and its dissemination between, the two countries is discussed.     

A common high molecular weight antigen of Babesia bovis isolates from Mexico

Cattle from an area of Mexico endemic with Babesia bovis infections have a dominant antibody response to a 152kDa antigen of the Tamaulipas strain of B. bovis. A mAb termed PB/5, showing a specific reactivity to this 152kDa antigen in Western blots, was identified. The mAb which reacted with the blunt end of B. bovis in an indirect fluorescent antibody test also reacted to a 152kDa antigen in two other isolates (Nuevo Leon and Yucatan), and a 175kDa antigen in the Huasteca B. bovis isolate from Mexico. Polyclonal monospecific sera from a calf inoculated with mAb-affinity purified 152kDa antigen (Tamaulipas strain) identified B. bovis by the indirect fluorescent antibody test and two antigens of B. bovis (65kDa and 152kDa) in Western blot. Since the epitope reacting to the mAb PB/5 is conserved, this antigen provides a basis for developing a diagnostic test or an immunogen.     

On human tuberculosis due to M. bovis. A review

The present survey of literature is devoted to the problem of tuberculosis due to M. bovis in man. On the basis of an analysis of literary data the author summarizes materials characterizing the state of the problem in the USSR in recent years. It has been shown that in spite of general low incidence rate of M. bovis infections in human population there are regions with high level of cattle raising (Kazakh SSR, Novosibirsk Region of the RSFSR) where both the infection rate and morbidity rate due to this agent are much higher. The role of consumption of milk infected with M. bovis in causing the disease especially among children is demonstrated. The author underlines the fact that transmission of M. bovis from man to man is practically very rare and therefore this type of tuberculosis in human population is not autonomous and disappears as soon as the tuberculosis of cattle is irradicated. Data on sources and chains of infection of man with M. bovis as well as some clinical aspects of the infection are presented.     

Source of enterococci in a farmhouse raw-milk cheese

Enterococci are widely distributed in raw-milk cheeses and are generally thought to positively affect flavor development. Their natural habitats are the human and animal intestinal tracts, but they are also found in soil, on plants, and in the intestines of insects and birds. The source of enterococci in raw-milk cheese is unknown. In the present study, an epidemiological approach with pulsed-field gel electrophoresis (PFGE) was used to type 646 Enterococcus strains which were isolated from a Cheddar-type cheese, the milk it was made from, the feces of cows and humans associated with the cheese-making unit, and the environment, including the milking equipment, the water used on the farm, and the cows' teats. Nine different PFGE patterns, three of Enterococcus casseliflavus, five of Enterococcus faecalis, and one of Enterococcus durans, were found. The same three clones, one of E. faecalis and two of E. casseliflavus, dominated almost all of the milk, cheese, and human fecal samples. The two E. casseliflavus clones were also found in the bulk tank and the milking machine even after chlorination, suggesting that a niche where enterococci could grow was present and that contamination with enterococci begins with the milking equipment. It is likely but unproven that the enterococci present in the human feces are due to consumption of the cheese. Cow feces were not considered the source of enterococci in the cheese, as Enterococcus faecium and Streptococcus bovis, which largely dominated the cows' intestinal tracts, were not found in either the milk or the cheese.     

Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review

Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.     

Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.     

Alteration of raw-milk cheese by Pseudomonas spp.: monitoring the sources of contamination using fluorescence spectroscopy and metabolic profiling

Thirty Pseudomonas spp. strains isolated from milk, water, cheese centre and cheese surface in two traditional workshops manufacturing raw milk St. Nectaire cheese were characterised by fluorescence spectroscopy and Biolog metabolic profiling. Factorial discriminant analysis (FDA) of the two data sets revealed clear linkages between groups of isolates. In the first workshop, milk could be incriminated as the sole source of cheese contamination. In the second one, milk and cheese centre isolates were found similar but surface cheese contaminants could be linked to a secondary contamination originating from water. Thus, it is possible to characterise, differentiate and trace Pseudomonas spp. strains using the fluorescence and metabolic profiling techniques. In addition, the two data sets were found highly correlated by canonical correlation analysis (CCA). Fluorescence spectroscopy however showed several advantages because of its low cost and processing speed.     

Consumption of Camembert cheese stimulates commensal enterococci in healthy human intestinal microbiota

Enterococci are natural inhabitants of the human gastrointestinal tract and the main Gram-positive and facultative anaerobic cocci recovered in human faeces. They are also present in a variety of fermented dairy and meat products, and some rare isolates are responsible for severe infections such as endocarditis and meningitis. The aim of the present study was to evaluate the effect of Camembert cheese consumption by healthy human volunteers on the faecal enterococcal population. A highly specific real-time quantitative PCR approach was designed and used to type enterococcal species in human faeces. Two species were found, Enterococcus faecalis and Enterococcus faecium, and only the Enterococcus faecalis population was significantly enhanced after Camembert cheese consumption, whereas Escherichia coli population and the dominant microbiota remained unaffected throughout the trial.     

Is Mycobacterium bovis in the environment important for the persistence of bovine tuberculosis?

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in cattle and wildlife. Direct aerosol contact is thought to be the primary route of infection between conspecifics, whereas indirect transmission via an environmental reservoir of M. bovis is generally perceived not to be a significant source for infection. Here, we report on the application of molecular technology (PCR) to quantify the prevalence of M. bovis in the environment and to explore its epidemiological significance. We show that the detectability of viable M. bovis at badger setts and latrines is strongly linked to the frequency of M. bovis excretion by infected badgers, and that putative M. bovis in the environment is prevalent on a large proportion of endemic cattle farms in Britain. These results raise important questions about the role of an environmental reservoir in bTB persistence.     

Survival of Mycobacterium bovis on feedstuffs commonly used as supplemental feed for white-tailed deer (Odocoileus virginianus)

Mycobacterium bovis, the causative agent of bovine tuberculosis, has become established in free-ranging white-tailed deer Odocoileus virginianus in northeastern Michigan. The practice of supplemental feeding of white-tailed deer during the winter is believed to contribute to transmission of M. bovis between deer. The current study was conducted to determine the ability of M. bovis to survive on various feedstuffs commonly used as supplemental feed for deer in northeast Michigan (i.e., apples, corn, carrots, sugar beets, potatoes, and hay) and the effect of maintenance at -20 C, 8 C, and 23 C on survival. Mycobacterium bovis survived on all feedstuffs at all temperatures tested for at least 7 days. At 23 C, M. bovis could still be isolated from samples of apples, corn and potatoes at 112 days. This study suggests that contamination of feedstuffs by M. bovis-infected deer could act as a source of indirect transmission between deer because M. bovis is able to survive in temperatures similar to those recorded during winter months in northeastern Michigan. Current efforts to ban or control supplemental feeding of deer should have a positive effect on decreasing transmission of M. bovis among deer.     

Polymorphism in the rpoT gene in Mycobacterium leprae isolates obtained from Latin American countries and its possible correlation with the spread of leprosy

The genotypes of Mycobacterium leprae isolates originating from Mexico, Peru and Paraguay were analysed for the polymorphism of short tandem repeats in the rpoT gene. The genotype with four copies of the six-base tandem repeats in the rpoT gene was prominently predominant in Mexico, but the genotype of all isolates from Peru and Paraguay contained three copies of the six-base tandem repeats. These obvious different distributions might reflect the spread of leprosy by the different strains of M. leprae harboured by the various human races that moved to the American continent, as has been demonstrated in other infectious diseases.     

The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth

Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis--its requirement for pyruvate in glycerol medium--we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guérin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.     

Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species -- M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti" -- were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, Sao Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.     

The complete genome sequence of Mycoplasma bovis strain Hubei-1

Infection by Mycoplasma bovis (M. bovis) can induce diseases, such as pneumonia and otitis media in young calves and mastitis and arthritis in older animals. Here, we report the finished and annotated genome sequence of M. bovis strain Hubei-1, a strain isolated in 2008 that caused calf pneumonia on a Chinese farm. The genome of M. bovis strain Hubei-1 contains a single circular chromosome of 953,114 bp with a 29.37% GC content. We identified 803 open reading frames (ORFs) that occupy 89.5% of the genome. While 34 ORFs were Hubei-1 specific, 662 ORFs had orthologs in the M. bovis type strain PG45 genome. Genome analysis validated lateral gene transfer between M. bovis and the Mycoplasma mycoides subspecies mycoides, while phylogenetic analysis found that the closest M. bovis neighbor is Mycoplasma agalactiae. Glycerol may be the main carbon and energy source of M. bovis, and most of the biosynthesis pathways were incomplete. We report that 47 lipoproteins, 12 extracellular proteins and 18 transmembrane proteins are phase-variable and may help M. bovis escape the immune response. Besides lipoproteins and phase-variable proteins, genomic analysis found two possible pathogenicity islands, which consist of four genes and 11 genes each, and several other virulence factors including hemolysin, lipoate protein ligase, dihydrolipoamide dehydrogenase, extracellular cysteine protease and 5'-nucleotidase.     

Rapid selection of complement-inhibiting protein variants in group A Streptococcus epidemic waves.

Serotype M1 group A Streptococcus strains cause epidemic waves of human infections long thought to be mono- or pauciclonal. The gene encoding an extracellular group A Streptococcus protein (streptococcal inhibitor of complement) that inhibits human complement was sequenced in 1,132 M1 strains recovered from population-based surveillance of infections in Canada, Finland and the United States. Epidemic waves are composed of strains expressing a remarkably heterogeneous array of variants of streptococcal inhibitor of complement that arise very rapidly by natural selection on mucosal surfaces. Thus, our results enhance the understanding of pathogen population dynamics in epidemic waves and infectious disease reemergence.     

Genetic diversity among Mycobacterium tuberculosis isolates from Mexican patients

Forty-six isolates of the Mycobacterium tuberculosis complex were typified by PCR of the IS6110 region and by Mycobacterium bovis specific primers JB21/JB22. Isolate MVG01 was typified as M. bovis, being the first record of a case of human tuberculosis caused by this species in Mexico. RAPD-PCR was used to describe the genetic diversity of the remaining 45 M. tuberculosis complex isolates. The corrected genotypic diversity value calculated for the analyzed population was 0.96, the estimated mean gene diversity was 0.235, and the corrected Shannon-Weiner index was 2.15. All allele-loci combinations generated showed significant linkage disequilibria. The distribution of genetic variation was analyzed both by the unweighted pair group method with arithmetic averages clustering and by principal coordinates analysis. Unweighted pair group method with arithmetic averages clustering resulted in a tree with four main clusters and one unclustered strain (MVG20), the principal coordinates analysis strain distribution pattern being consistent with this grouping. The obtained results suggest that the studied isolates belong to a clonal population having significant genetic diversity. Our genetic diversity results are comparable with those reported for other populations of M. tuberculosis, although only three RAPD primers were used.