Tod Stuber

Recovery of Mycobacterium bovis from soft fresh cheese originating in Mexico

ABSTRACT 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.

Molecular epidemiology of Mycobacterium bovis : Usefulness in international trade

Tuberculosis (TB) represents a barrier for free trade of livestock between Mexico and the United States of America (US). In spite of efforts from Mexico to export TB-free animals, some of those found with TB lesions in slaughterhouses in the US are traced back to that country. Therefore, the purpose of this study was to determine, through molecular epidemiology, the most probable source of infection for cattle found with TB lesions in the US. Ninety M. bovis isolates, 50 from Mexico obtained from cattle in 8 different states, and 40 from the US from cattle, deer, elk and feral pigs from 7 different states were included in the study. All samples were analyzed in both laboratories, Mexico and the US, following the same protocol for molecular analysis by spoligotyping. Twenty-seven clusters, ranging from 1 to 18 genetically similar strains were found. Some clustering by country was observed, strains from cattle and deer in Michigan in the US fell into the same cluster, suggesting transmission between species. These results, combined with epidemiological information suggest that despite of the possibility that some animals with lesions in the US come from Mexico as false negatives, the US has its own source of infection, must probably in dairy cattle and wildlife. Genetic diversity of isolates from Mexico was larger than that in the US, which could be a consequence of the endemic status of the disease and the indiscriminate movement of animals between regions.

Molecular Epidemiology of Brucella abortus Isolates from Cattle, Elk, and Bison in the United States, 1998 to 2011

ABSTRACT A variable-number tandem repeat (VNTR) protocol targeting 10 loci in the Brucella abortus genome was used to assess genetic diversity among 366 field isolates recovered from cattle, bison, and elk in the Greater Yellowstone Area (GYA) and Texas during 1998 to 2011. Minimum spanning tree (MST) and unweighted-pair group method with arithmetic mean (UPGMA) analyses of VNTR data identified 237 different VNTR types, among which 14 prominent clusters of isolates could be identified. Cattle isolates from Texas segregated into three clusters: one comprised of field isolates from 1998 to 2005, one comprised of vaccination-associated infections, and one associated with an outbreak in Starr County in January 2011. An isolate obtained from a feral sow trapped on property adjacent to the Starr County herd in May 2011 clustered with the cattle isolates, suggesting a role for feral swine as B. abortus reservoirs in Starr County. Isolates from a 2005 cattle outbreak in Wyoming displayed VNTR-10 profiles matching those of strains recovered from Wyoming and Idaho elk. Additionally, isolates associated with cattle outbreaks in Idaho in 2002, Montana in 2008 and 2011, and Wyoming in 2010 primarily clustered with isolates recovered from GYA elk. This study indicates that elk play a predominant role in the transmission of B. abortus to cattle located in the GYA.

Molecular Relationship between Strains of M. bovis from Mexico and Those from Countries with Free Trade of Cattle with Mexico

The purpose of this study was to identify relationships between spoligotypes of M. bovis from cattle in Mexico and those reported in countries with free trade of cattle with Mexico: Australia, Canada, New Zealand and the United States of America. Mexican spoligotypes were obtained from isolates collected from cattle in different parts of the country. Spoligotypes from Canada and New Zealand were obtained from different reports in the literature. Those from the United States were obtained from the database of the National Veterinary Services Laboratory in APHIS-USDA. In order to perform the analysis in a single data set, spoligotypes were all converted to binary data and classified according to www.mbovis.org or www.pasteur-guadeloupe.fr:8081. Epidemiologic information included country and species infected. From 3,198 isolates, 174 different spoligotypes were obtained, 95 were orphans. Ninety one percent of the isolates came from the Unites States (n = 1,609) and Mexico (n = 1,323). Spoligotype SB0265 is shared between Canada and the United States in cattle and wildlife. Six spoligotypes, SB0673, SB0121, SB0145, SB0971, SB0140 and SB1165, were frequent in cattle and wildlife in the United States and cattle in Mexico, suggesting wide exchange of strains. Spoligotype SB0669 was found only in Mexico. Spoligotype SB0140 was the most common in Australia and the sixth in the United States and Mexico. In a phylogenetic analysis, spoligotype SB0140 appears as the oldest spoligotype in the data set, suggesting this as the ancestral spoligotype for all spoligotypes in the five countries. Some spoligotypes are shared by animals and humans, corroborating the zoonotic importance of M. bovis.

Anatomical distribution of Mycobacterium bovis genotypes in experimentally infected white-tailed deer.

Mycobacterium bovis (M. bovis) causes tuberculosis in white-tailed deer (WTD). Natural infection of WTD with M. bovis is most closely mimicked by instilling inoculum into palatine tonsillar crypts. One hundred fifty days after intratonsillar inoculation, M. bovis was cultured from 30 tissues originating from 14 deer. Whole-genome sequencing (WGS) was performed on the original inoculum, single colonies subcultured from the original inoculum, and M. bovis isolated from each culture positive tissue. Single nucleotide polymorphisms (SNP) were identified by comparing the derived sequences to the reference strain AF2122/97. Results indicate that the majority of the SNPs that were identified were homogeneous between the inoculum and the isolates from the tissues. The majority of individual tissues had different WGS genotypes from each other, suggesting that dissemination of M. bovis beyond the initial site of infection may require few mycobacteria representing a bottleneck.

Use of whole-genome sequencing and evaluation of the apparent sensitivity and specificity of antemortem tuberculosis tests in the investigation of an unusual outbreak of Mycobacterium bovis infection in a Michigan dairy herd

OBJECTIVE To describe use of whole-genome sequencing (WGS) and evaluate the apparent sensitivity and specificity of antemortem tuberculosis tests during investigation of an unusual outbreak of Mycobacterium bovis infection in a Michigan dairy herd. DESIGN Bovine tuberculosis (bTB) outbreak investigation. ANIMALS Cattle, cats, dog, and wildlife. PROCEDURES All cattle in the index dairy herd were screened for bTB with the caudal fold test (CFT), and cattle ≥ 6 months old were also screened with a γ-interferon (γIFN) assay. The index herd was depopulated along with all barn cats and a dog that were fed unpasteurized milk from the herd. Select isolates from M bovis-infected animals from the index herd and other bTB-affected herds underwent WGS. Wildlife around all affected premises was examined for bTB. RESULTS No evidence of bTB was found in any wildlife examined. Within the index herd, 53 of 451 (11.8%) cattle and 12 of 21 (57%) cats were confirmed to be infected with M bovis. Prevalence of M bovis-infected cattle was greatest among 4- to 7-month-old calves (16/49 [33%]) followed by adult cows (36/203 [18%]). The apparent sensitivity and specificity were 86.8% and 92.7% for the CFT and 80.4% and 96.5% for the γIFN assay when results for those tests were interpreted separately and 96.1% and 91.7% when results were interpreted in parallel. Results of WGS revealed that M bovis-infected barn cats and cattle from the index herd and 6 beef operations were infected with the same strain of M bovis. Of the 6 bTB-affected beef operations identified during the investigation, 3 were linked to the index herd only by WGS results; there was no record of movement of livestock or waste milk from the index herd to those operations. CONCLUSIONS AND CLINICAL RELEVANCE Whole-genome sequencing enhanced the epidemiological investigation and should be used in all disease investigations. Performing the CFT and γIFN assay in parallel improved the antemortem ability to detect M bovis-infected animals. Contact with M bovis-infected cattle and contaminated milk were major risk factors for transmission of bTB within and between herds of this outbreak.

Whole genome sequencing of Mycobacterium bovis to obtain molecular fingerprints in human and cattle isolates from Baja California, Mexico

OBJECTIVES To determine genetic diversity by comparing the whole genome sequences of cattle and human Mycobacterium bovis isolates from Baja California. METHODS A whole genome sequencing strategy was used to obtain the molecular fingerprints of 172 isolates of M. bovis obtained from Baja California, Mexico; 155 isolates were from cattle and 17 isolates were from humans. Spoligotypes were characterized in silico and single nucleotide polymorphism (SNP) differences between the isolates were evaluated. RESULTS A total of 12 M. bovis spoligotype patterns were identified in cattle and humans. Two predominant spoligotypes patterns were seen in both cattle and humans: SB0145 and SB1040. The SB0145 spoligotype represented 59% of cattle isolates (n=91) and 65% of human isolates (n=11), while the SB1040 spoligotype represented 30% of cattle isolates (n=47) and 30% of human isolates (n=5). When evaluating SNP differences, the human isolates were intimately intertwined with the cattle isolates. CONCLUSIONS All isolates from humans had spoligotype patterns that matched those observed in the cattle isolates, and all human isolates shared common ancestors with cattle in Baja California based on SNP analysis. This suggests that most human tuberculosis caused by M. bovis in Baja California is derived from M. bovis circulating in Baja California cattle. These results reinforce the importance of bovine tuberculosis surveillance and control in this region.

Whole genome sequencing of Mycobacterium bovis isolated from livestock in the United States, 1989-2018

The United States official bovine tuberculosis (bTB) eradication program has utilized genotyping for Mycobacterium bovis isolates since 2000 and whole genome sequencing was implemented in 2013. The program has been highly successful, yet as bTB prevalence has reached historic lows, a small number of new bTB-affected cattle herds occur annually. Therefore, understanding the epidemiology of bTB transmission is critically important, in order to target limited resources for surveillance and achieve eradication. This evaluation described the diversity and epidemiology of M. bovis isolates identified in the USA livestock. Isolates from animals within the bTB endemic area of Michigan were excluded. Broad diversity was found among 1,248 isolates, collected from affected cattle and farmed cervids herds and fed cattle during 1989–2018. Nearly 70% of isolates from 109 herds/cases during 1999–2018 were European clonal complex 1 and 30% were European clonal complex 2. The sources of infection based on the herd investigation were known for 41% of herds/cases and 59% were not epidemiologically linked to another USA origin herd. Whole genome sequencing results were consistent with the investigation findings and previously unrecognized links between herds and cases were disclosed. For herds/cases with an unknown source of infection, WGS results suggested several possible sources, including undocumented cattle movement, imported cattle and humans. The use of WGS in new cases has reduced the time and costs associated with epidemiological investigations. Within herd SNP diversity was evaluated by examining 18 herds with 10 or more isolates sequenced. Forty percent of isolates had not diverged or accumulated any SNPs, and 86% of the isolates had accumulated 3 or fewer SNPs. The results of WGS does not support a bTB reservoir in USA cattle. The bTB eradication program appears to be highly effective as the majority of herds/cases in the USA are unique strains with limited herd to herd transmission.

Whole-Genome Sequencing of Porcine Epidemic Diarrhea Virus by Illumina MiSeq Platform

Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the family Coronaviridae. PEDV was identified as an emerging pathogen in US pig populations in 2013. Since then, this virus has been detected in at least 31 states in the USA and has caused significant economic loss to the swine industry. Active surveillance and characterization of PEDV are essential for monitoring the virus. Obtaining comprehensive information about the PEDV genome can improve our understanding of the evolution of PEDV viruses, and the emergence of new strains, and can enhance vaccine designs. In this chapter, both a targeted amplification method and a random-priming method are described to amplify the complete genome of PEDV for sequencing using the MiSeq platform. Overall, this protocol provides a useful two-pronged approach to complete whole-genome sequences of PEDV depending on the amount of virus in the clinical samples.