Identification of a predominant genotype of Mycobacterium tuberculosis in Brazilian indigenous population.

After nearly a century of vaccination and six decades of drug therapy, tuberculosis (TB) kills more people annually than any other infectious disease. Substantial challenges to disease eradication remain among vulnerable and underserved populations. The Guarani-Kaiowá people are an indigenous population in Paraguay and the Brazilian state of Mato Grosso do Sul. This community, marginalized in Brazilian society, experiences severe poverty. Like other South American indigenous populations, their TB prevalence is high, but the disease has remained largely unstudied in their communities. Herein, Mycobacterium tuberculosis isolates from local clinics were whole genome sequenced, and a population genetic framework was generated. Phylogenetics show M. tuberculosis isolates in the Guarani-Kaiowá people cluster away from selected reference strains, suggesting divergence. Most cluster in a single group, further characterized as M. tuberculosis sublineage 4.3.3. Closer analysis of SNPs showed numerous variants across the genome, including in drug resistance-associated genes, and with many unique changes fixed in each group. We report that local M. tuberculosis strains have acquired unique polymorphisms in the Guarani-Kaiowá people, and drug resistance characterization is urgently needed to inform public health to ensure proper care and avoid further evolution and spread of drug-resistant TB.

Identification of a predominant genotype of Mycobacterium tuberculosis in Brazilian indigenous population

After nearly a century of vaccination and six decades of drug therapy, tuberculosis (TB) kills more people annually than any other infectious disease. Substantial challenges to disease eradication remain among vulnerable and underserved populations. The Guarani-Kaiowá people are an indigenous population in Paraguay and the Brazilian state of Mato Grosso do Sul. This community, marginalized in Brazilian society, experiences severe poverty. Like other South American indigenous populations, their TB prevalence is high, but the disease has remained largely unstudied in their communities. Herein, Mycobacterium tuberculosis isolates from local clinics were whole genome sequenced, and a population genetic framework was generated. Phylogenetics show M. tuberculosis isolates in the Guarani-Kaiowá people cluster away from selected reference strains, suggesting divergence. Most cluster in a single group, further characterized as M. tuberculosis sublineage 4.3.3. Closer analysis of SNPs showed numerous variants across the genome, including in drug resistance-associated genes, and with many unique changes fixed in each group. We report that local M. tuberculosis strains have acquired unique polymorphisms in the Guarani-Kaiowá people, and drug resistance characterization is urgently needed to inform public health to ensure proper care and avoid further evolution and spread of drug-resistant TB

Rapid baso-apical translocation of Mycobacterium avium ssp. paratuberculosis in mammary epithelial cells in the presence of Escherichia coli.

Infection of mammary gland cells with bacterial pathogens begins with adhesion, invasion, and persistence within the cells or systemic distribution. Some bacteria, such as Escherichia coli, are known to causes bovine mastitis, resulting in acute proinflammatory responses in the mammary tissue. Mycobacterium avium ssp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is able to spread to distant organs after crossing intestinal cells, reaching the mammary gland and potentially being released in milk, infecting calves during suckling. Its exit from systemic sites may be influenced by preexisting inflammation such as that caused by E. coli mastitis. Interactions between E. coli and MAP in mammary epithelial cells have not yet been described. In this study, we posited that E. coli-infected bovine mammary epithelial cells would facilitate baso-apical translocation of MAP in an ex vivo model. We showed that the presence of E. coli in a bovine mammary epithelial cell line (MAC-T) increased baso-apical translocation of MAP to the apical side of the cells. Levels were significantly higher 30 min post-infection and decreased at 120 min post-infection. Cells previously infected with E. coli and MAP or with E. coli alone showed a significant increase in IL1B mRNA expression at 120 min. We detected no significant expression of p38 mitogen-activated protein kinase (mapkp38) or IL10, regardless of treatment. Thereby, the presence of E. coli in MAC-T cells alters the translocation of MAP through epithelial cells, enabling its rapid translocation to the cellular surface. Expression of IL1B was shown to influence the apical-basal translocation of MAP at 120 min. Findings from the current study suggest that MAP translocation into milk is likely enhanced by inflammatory states such as those induced during E. coli mastitis. This is the first report demonstrating the effect of E. coli under MAP coinfection in bovine mammary epithelial cells under experimental conditions.

Effect of feeding heat-treated colostrum on risk for infection with Mycobacterium avium ssp. paratuberculosis, milk production, and longevity in Holstein dairy cows.

In summer 2007, a randomized controlled field trial was initiated on 6 large Midwest commercial dairy farms to investigate the effect of feeding heat-treated (HT) colostrum on transmission of Mycobacterium avium ssp. paratuberculosis (MAP) and on future milk production and longevity within the herd. On each farm, colostrum was collected daily from fresh cows, pooled, divided into 2 aliquots, and then 1 aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60min. A sample from each batch of colostrum was collected for PCR testing (MAP-positive vs. MAP-negative). Newborn heifer calves were removed from the dam within 30 to 60min of birth and systematically assigned to be fed 3.8 L of either fresh (FR; n=434) or heat-treated (HT; n=490) colostrum within 2h of birth. After reaching adulthood (>2 yr old), study animals were tested once annually for 3 yr (2010, 2011, 2012) for infection with MAP using serum ELISA and fecal culture. Lactation records describing milk production data and death or culling events were collected during the 3-yr testing period. Multivariable model logistic and linear regression was used to investigate the effect of feeding HT colostrum on risk for testing positive to MAP during the 3-yr testing period (positive/negative; logistic regression) and on first and second lactation milk yield (kg/cow; linear regression), respectively. Cox proportional hazards regression was used to investigate the effect of feeding HT colostrum on risk and time to removal from the herd. Fifteen percent of all study animals were fed PCR-positive colostrum. By the end of the 3-yr testing period, no difference was noted in the proportion of animals testing positive for MAP, with either serum ELISA or fecal culture, when comparing the HT group (10.5%) versus the FR group (8.1%). There was no effect of treatment on first- (HT=11.797kg; FR=11,671kg) or second-lactation (HT=11,013kg; FR=11,235kg) milk production. The proportion of cows leaving the herd by study conclusion was not different for animals originally fed HT (68.0%) versus FR (71.7%) colostrum. Although a previous study showed that feeding HT colostrum (60°C for 60min) produces short-term benefits, including improved passive transfer of IgG and reduced morbidity in the preweaning period, the current study found no benefit of feeding HT colostrum on long-term outcomes including risk for transmission of Mycobacterium avium ssp. paratuberculosis, milk production in the first and second lactation, and longevity within the herd.

Draft Genome Sequences of Two Pasteurella multocida Strains Isolated from Buffaloes in India with Hemorrhagic Septicemia Disease.

Pasteurella multocida serotype B:2 is the causative agent of hemorrhagic septicemia in cattle and buffaloes in Asia. It is an acute fatal disease and is considered one of the most economically important diseases in this region of the world. We present here the draft genome sequences of strains 2213 and 3213 of P. multocida.

How does a Mycobacterium change its spots? Applying molecular tools to track diverse strains of Mycobacterium avium subspecies paratuberculosis.

Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne's disease research. These data can now be used to define the extent of strain diversity on the farm. However, to perform these important tasks, researchers must have a way to distinguish the many MAP isolates/strains that are present in the environment or host to enable tracking over time. Recent studies have described genetic diversity of the Mycobacterium avium complex (MAC), of which MAP is a member, through pulsed-field gel electrophoresis, single sequence repeats, variable-number tandem repeats, genome rearrangements, single nucleotide polymorphisms and genomewide comparisons to identify insertions and deletions. Combinations of these methods can now provide discrimination sufficient for dependable strain tracking. These molecular epidemiology techniques are being applied to understand transmission of Johne's disease within dairy cattle herds as well as identify which strains predominate in wildlife.

Heat-treated colostrum and reduced morbidity in preweaned dairy calves: results of a randomized trial and examination of mechanisms of effectiveness.

A randomized controlled clinical trial was conducted using 1,071 newborn calves from 6 commercial dairy farms in Minnesota and Wisconsin, with the primary objective being to describe the effects of feeding heat-treated colostrum on serum immunoglobulin G concentration and health in the preweaning period. A secondary objective was to complete a path analysis to identify intermediate factors that may explain how feeding heat-treated colostrum reduced the risk for illness. On each farm, colostrum was collected each day, pooled, and divided into 2 aliquots; then, one aliquot was heat-treated in a commercial batch pasteurizer at 60°C for 60 min. Samples of fresh and heat-treated colostrum were collected for standard microbial culture (total plate count and total coliform count, cfu/mL) and for measurement of immunoglobulin G concentrations (mg/mL). Newborn calves were removed from the dam, generally within 30 to 60 min of birth, and systematically assigned to be fed 3.8L of either fresh (FR, n=518) or heat-treated colostrum (HT, n=553) within 2h of birth. Venous blood samples were collected from calves between 1 and 7d of age for measurement of serum IgG concentrations (mg/mL). All treatment and mortality events were recorded by farm staff between birth and weaning. Regression models found that serum IgG concentrations were significantly higher in calves fed HT colostrum (18.0 ± 1.5 mg/mL) compared with calves fed FR colostrum (15.4 ± 1.5 mg/ml). Survival analysis using Cox proportional hazards regression indicated a significant increase in risk for a treatment event (any cause) in calves fed FR colostrum (36.5%, hazard ratio=1.25) compared with calves fed HT colostrum (30.9%). In addition, we observed a significant increase in risk for treatment for scours in calves fed FR colostrum (20.7%, hazard ratio=1.32) compared with calves fed HT colostrum (16.5%). Path analysis suggested that calves fed HT colostrum were at lower risk for illness because the heat-treatment process caused a significant reduction in colostrum total coliform count, which was associated with a reduced risk for illness as a function of improved serum IgG concentrations.

Heat treatment of colostrum on commercial dairy farms decreases colostrum microbial counts while maintaining colostrum immunoglobulin G concentrations.

This study was conducted on 6 commercial dairy farms in Minnesota and Wisconsin to describe the effect of heat treatment (at 60°C for 60 min) on colostrum, on colostrum bacteria counts, and immunoglobulin G concentrations. First-milking colostrum was collected each day, pooled, divided into 2 aliquots, and then 1 aliquot was heat treated in a commercial batch pasteurizer at 60°C for 60 min. Frozen samples of pre- and post- heat-treated colostrum were submitted for standard microbial culture (total plate count and total coliform count, cfu/mL) and testing for immunoglobulin G concentrations (mg/mL). Data were analyzed from 266 unique batches of colostrum. Linear regression showed that heat treatment decreased colostrum total plate counts (-2.25 log(10)) and coliform counts (-2.49 log(10)), but, overall, did not affect colostrum IgG concentration. Though higher-quality batches of colostrum did experience a greater magnitude of loss of IgG as a result of heat treatment as compared with lower- or intermediate-quality batches of colostrum, the colostral IgG concentrations in these batches remained high overall, and within acceptable limits for feeding. This study demonstrates that batch heat treatment of colostrum at 60°C for 60 min can be successfully conducted on commercial dairy farms by farm staff to decrease colostrum microbial counts while maintaining colostrum IgG concentrations.

Prevalence and characterization of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, isolated from bulk tank milk from Minnesota dairy farms.

Staphylococcus aureus is a common causative agent of bovine mastitis in dairy herds. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals as well as the community is a significant and costly public health concern. S. aureus-related bovine mastitis is a common reason for therapeutic and/or prophylactic use of antibiotics on dairy farms. In this study, herd prevalence of S. aureus, including MRSA, was estimated from bulk tank milk (BTM) from Minnesota farms. A total of 150 pooled BTM samples from 50 farms, collected over 3 seasons (spring, summer, and fall of 2009), were assessed. Herd prevalence of methicillin-susceptible S. aureus (MSSA) was 84%, while MRSA herd prevalence was 4%. A total of 93 MSSA isolates and 2 MRSA isolates were recovered from 150 BTM samples. Antibiotic susceptibility testing of S. aureus isolates showed pansusceptibility in 54 isolates, resistance to a single antibiotic class in 21 isolates, resistance to two antibiotic classes in 13 isolates, and resistance to ≥3 antibiotics classes and thus multidrug resistance in 5 isolates. The two MRSA isolates displayed resistance to ß-lactams, cephalosporins, and lincosamides and were multiresistant. Staphylococcal protein A gene (spa) typing identified spa types t529 and t034 most frequently among methicillin-susceptible isolates, while t121 was observed in MRSA isolates. Seven isolates, including the two MRSA isolates, produced staphylococcal enterotoxins B, C, D, and E on overnight culture. MRSA isolates were further genotyped using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Of the 2 MRSA isolates, one had a composite genotype profile of MLST ST 5-PFGE USA100-unknown spa type, which has been reported among hospital-associated MRSA isolates, while the second isolate carried the MLST ST 8-PFGE USA300-spa type t121 genotype, commonly identified among community-associated MRSA isolates. These results suggest that MRSA genotypes associated with hospitals and community can be isolated from milk at very low rates.

Molecular subtyping of mastitis-associated Klebsiella pneumoniae isolates shows high levels of diversity within and between dairy herds.

Despite advances in controlling mastitis (inflammation of the mammary gland), udder infections caused by Klebsiella pneumoniae continue to affect dairy cattle. Mastitis caused by K. pneumoniae responds poorly to antibiotic treatment, and as a consequence, infections tend to be severe and long lasting. We sought to determine whether a nonrandom distribution of specific genotypes of K. pneumoniae was associated with mastitis from 6 dairy herds located in 4 different states. A total of 635 isolates were obtained and fingerprinted by repetitive DNA sequence PCR. Significant genetic diversity was observed in 4 of the 6 dairy herds analyzed, and a total of 49 genotypic variants were identified. Within a herd, Simpson's diversity indices were 91.0, 94.1, 91.7, 88.6, 53.3, and 64.3% for dairies A, B, C, D, E, and F, respectively. The association between matrices of genetic similarity and matrices of temporal distance was negative in all the dairies analyzed. Four dairies had a high incidence of K. pneumoniae mastitis during the winter. The majority of genotypes were unique to herds of origin, and only 5 genotypes were detected in more than 2 dairies. Genotype 1 (arbitrary designation) occurred most frequently across dairies and was found in 25.2% of all mastitis cases and among 22.8% of reinfected and culled cows in dairy A. Specific genotypes also tended to be associated with a specific bedding type and dairy location. Analysis of molecular variance showed that 18% of the genetic diversity was due to variation among herds within states, and 82% of the genetic diversity was accounted for by variation of genotypes within herds. The data support the idea that mastitis is caused by a diverse group of K. pneumoniae genotypes and thus has major implications for the diagnosis, prevention, and treatment of udder infections in dairy cows.

Influence of type of culture medium on characterization of Mycobacterium avium subsp. paratuberculosis subtypes.

We evaluated the effects of culture and/or enrichment methods on the selection of Mycobacterium avium subsp. paratuberculosis subtypes. M. avium subsp. paratuberculosis isolates from bovine fecal samples processed using a centrifugation protocol were investigated in both liquid (MGIT ParaTb tubes) and solid (Herrold's egg yolk medium) culture media. For this evaluation, M. avium subsp. paratuberculosis subtyping was based on the sequence variation in two of the most discriminatory short sequence repeat loci, i.e., mononucleotide G and trinucleotide GGT, in isolates from liquid and solid cultures. This study identified the existence of one major predominating fingerprint (>13G-5GGT) in bovine fecal samples, regardless of the type of medium used for isolation. Matched-pair analysis of subtypes showed that 69% of samples presented unique subtypes in the two culture media used, while 31% shared the same G-GGT allele. Furthermore, the liquid culture method appeared to select for a more genotypically diverse subtype population than the solid culture method. The variety of subtypes observed between liquid and solid cultures obtained from the same fecal samples suggests that the culture method could provide a "microbiological" bias and lead to a discrepancy in the growth of M. avium subsp. paratuberculosis strains. In conclusion, this study identified that these two types of culture media determined differential growth of M. avium subsp. paratuberculosis strains and that this should be considered in evaluating detection capabilities of diagnostic tests or interpreting data from molecular epidemiological studies performed using conventional solid fecal culture or automated liquid medium culture methods.

Genetic diversity of mastitis-associated Klebsiella pneumoniae in dairy cows.

The objectives of this study were to determine the level of genetic diversity of Klebsiella pneumoniae isolated from clinical mastitis cases and to define genotypes most commonly associated with the disease. Individual quarter milk samples were collected from a single privately owned dairy herd over a 2-yr period and submitted to the Laboratory for Udder Health, Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, for bacteriological culture. Eighty-four K. pneumoniae isolates were obtained and fingerprinted by repetitive DNA sequence PCR, 43 by pulsed-field gel electrophoresis (PFGE), and 29 by multilocus sequence typing (MLST). Significant genetic diversity was observed among the isolates regardless of the fingerprinting method used. Simpson's diversity index was 93.5, 96.1, and 97.0% when analyzed by repetitive DNA sequence PCR (n = 84), pulse field gel electrophoresis (n = 43), and MLST (n = 29), respectively. In some cases more than 1 genotype was obtained from a single milk sample originating from an individual quarter. The majority of infections were observed during the winter and accounted for 69.0% of K. pneumoniae mastitis cases. There was a negative correlation between a matrix of fingerprints similarity and a matrix of temporal distances. The MLST results revealed 5 new and novel allelic types, which have not been previously reported in the MLST database. Three isolates shared MLST types with human clinical isolates, raising the possibility that some K. pneumoniae isolates, of bovine origin, may be capable of causing disease in humans. There were 21 genotypes present within the herd, and there was no evidence for nonrandom distribution of genotypes uniquely associated with mastitis. We have shown, using 3 distinct genotyping methods, that K. pneumoniae isolated from clinical mastitis within a single dairy herd is caused by a genetically diverse population and that multiple genotypes can be isolated from a mastitic quarter. The data suggest that mastitis can be caused by a variety of K. pneumoniae genotypes. Diverse genotypes may have different levels of invasiveness and virulence and may originate from various sources within the dairy.

An overview of transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders of humans and animals associated with an accumulation of abnormal isoforms of prion protein (PrP) in nerve cells. The pathogenesis of TSEs involves conformational conversions of normal cellular PrP (PrP(c)) to abnormal isoforms of PrP (PrP(Sc)). While the protein-only hypothesis has been widely accepted as a causal mechanism of prion diseases, evidence from more recent research suggests a possible involvement of other cellular component(s) or as yet undefined infectious agent(s) in PrP pathogenesis. Although the underlying mechanisms of PrP strain variation and the determinants of interspecies transmissibility have not been fully elucidated, biochemical and molecular findings indicate that bovine spongiform encephalopathy in cattle and new-variant Creutzfeldt-Jakob disease in humans are caused by indistinguishable etiological agent(s). Cumulative evidence suggests that there may be risks of humans acquiring TSEs via a variety of exposures to infected material. The development of highly precise ligands is warranted to detect and differentiate strains, allelic variants and infectious isoforms of these PrPs. This article describes the general features of TSEs and PrP, the current understanding of their pathogenesis, recent advances in prion disease diagnostics, and PrP inactivation.

A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle.

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

Algorithmic approach to high-throughput molecular screening for alpha interferon-resistant genotypes in hepatitis C patients.

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5' untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634-639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.

Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence.

Allelic replacement was used to generate two isogenic lktA deletion mutants of Pasteurella haemolytica serotype 1 that were incapable of synthesizing leukotoxin (Lkt). Southern blot data confirmed that lktA sequences were absent in the two P. haemolytica deletion mutants. Culture supernatants and whole cell lysates from the wild type P. haemolytica, D153 parent strain, but not the lktA deletion mutants, contained immunoreactive and bioactive leukotoxic protein. In addition, only the parent strain was haemolytic when grown on bovine and sheep blood agar plates. Virulence of the lktA deletion mutant, lktA 77, was compared with the parent in an experimentally infected calf model of pneumonic pasteurellosis. Results revealed significant reduction in virulence in the lktA mutant as measured by clinical and lung lesion scores. Notable differences in histological changes such as markedly reduced necrosis and lack of leukocyte degeneration occurred in calves infected with the lktA mutant in comparison with those infected with the parent wild-type strain. Thus, it appears that leukotoxin plays a important role in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Comparative Evaluation of Cleavase Fragment Length Polymorphism With PCR-SSCP and PCR-RFLP to Detect Antimicrobial Agent Resistance in Mycobacterium tuberculosis.

Background: Several molecular methods potentially useful in the detection of Mycobacterium tuberculosis mutations, specifically in rpoB and katG, were compared. Methods and Results: DNA from 24 M. tuberculosis clinical isolates, with mutations associated with resistance to rifampin and/or isoniazid, was analyzed. A 128 bp amplicon, spanning the 81 bp rpoB region containing most mutations leading to rifampin resistance, was analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and a recently introduced mutation scanning method, cleavase fragment length polymorphism (CFLP) analysis. Also, a 350 bp amplicon encompassing that region was analyzed by the CFLP method. CFLP analysis of the 350 bp amplicon (23 isolates) identified 14 of 17 mutants; however, CFLP analysis of the 128 bp amplicon accurately identified all mutants as did PCR-SSCP with interpretative difficulty for two codon 513 mutations. CFLP and PCR-restriction fragment length polymorphism (RFLP) analyses of a 623 bp amplicon encompassing katG codons 315 and 463 showed that the CFLP method identified single and dinucleotide codon 315 substitutions with or without codon 463 (CGG-->CTG) changes, whereas PCR-RFLP (MspI) missed one codon 315 polymorphism (AGC-->ACA) in three isolates. Conclusion: Both PCR-SSCP and CFLP analyses were sensitive in identifying all mutations on short sequences in the rpoB mutants. CFLP appears to be more efficient than SSCP and RFLP for the detection of mutations in large amplicons.

Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.

Animal by-products contaminated with Salmonella in the diets of lactating dairy cows.

As part of a total mixed ration, two rumen-fistulated dairy cows were fed meat and bone meal that had been artificially contaminated with Salmonella spp. Samples from the rumen, feces, and milk were taken 3 d/wk and cultured for salmonella. Rectal temperatures and rumen pH were also measured at the time of sample collection. Over the 2-mo study, salmonella were intermittently recovered from rumen contents, from feces, and from necropsy specimens of rumen contents, cecal contents, and mesenteric lymph nodes. No excretion of salmonella in milk was detected. An elevated rumen pH was associated with increased isolation of salmonella. No clinical illness was observed for either cow. Meat and bone meal that has been contaminated with low concentrations of salmonella is unlikely to result in clinical illness in healthy adult lactating cows. However, dairy producers should continue to be concerned about feed biosecurity and water contamination of animal by-products to prevent and control contamination by salmonella.