Suelee Robbe-Austerman

Early Immune Markers Associated with Mycobacterium avium subsp. paratuberculosis Infection in a Neonatal Calf Model

ABSTRACT The objective of this study was to observe early markers of cell-mediated immunity in naïve calves infected with Mycobacterium avium subsp. paratuberculosis and how expression of these markers evolved over the 12-month period of infection. Groups for experimental infection included control (noninfected), oral (infected orally with M. avium subsp. paratuberculosis strain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), intraperitoneal (i.p.) inoculation, and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. One of the earliest markers to emerge was antigen-specific gamma interferon (IFN-γ). Only i.p. inoculated calves had detectable antigen-specific IFN-γ responses at 7 days, with responses of the other infection groups becoming detectable at 90 and 120 days. All infection groups maintained robust IFN-γ responses for the remainder of the study. At 1 month, calves in the oral and oral/M groups had higher antigen-stimulated interleukin-10 (IL-10) levels than calves in the other treatment groups, but IL-10 secretion declined by 12 months for all calves. T-cell activation markers such as CD25, CD26, CD45RO, and CD5 were significantly upregulated in infected calves compared to noninfected controls. Oral inoculation of calves resulted in significantly increased antigen-specific lymphocyte proliferation at 9 and 12 months, as well as inducible nitric oxide synthase (iNOS) secretion at 6 and 12 months. These results demonstrate that infection of naïve calves with M. avium subsp. paratuberculosis invoked early immunologic responses characterized by robust antigen-specific IFN-γ responses and induction of CD25 and CD45RO expression on T-cell subsets. These were followed by antigen-specific lymphocyte proliferation, iNOS secretion, and expression of CD26 and CD5bright markers in the latter part of the 12-month study.

Identification of Mycobacterium spp. of veterinary importance using rpoB gene sequencing

BackgroundStudies conducted on Mycobacterium spp. isolated from human patients indicate that sequencing of a 711 bp portion of the rpoB gene can be useful in assigning a species identity, particularly for members of the Mycobacterium avium complex (MAC). Given that MAC are important pathogens in livestock, companion animals, and zoo/exotic animals, we were interested in evaluating the use of rpoB sequencing for identification of Mycobacterium isolates of veterinary origin.ResultsA total of 386 isolates, collected over 2008 - June 2011 from 378 animals (amphibians, reptiles, birds, and mammals) underwent PCR and sequencing of a ~ 711 bp portion of the rpoB gene; 310 isolates (80%) were identified to the species level based on similarity at ≥ 98% with a reference sequence. The remaining 76 isolates (20%) displayed < 98% similarity with reference sequences and were assigned to a clade based on their location in a neighbor-joining tree containing reference sequences. For a subset of 236 isolates that received both 16S rRNA and rpoB sequencing, 167 (70%) displayed a similar species/clade assignation for both sequencing methods. For the remaining 69 isolates, species/clade identities were different with each sequencing method. Mycobacterium avium subsp. hominissuis was the species most frequently isolated from specimens from pigs, cervids, companion animals, cattle, and exotic/zoo animals.ConclusionsrpoB sequencing proved useful in identifying Mycobacterium isolates of veterinary origin to clade, species, or subspecies levels, particularly for assemblages (such as the MAC) where 16S rRNA sequencing alone is not adequate to demarcate these taxa. rpoB sequencing can represent a cost-effective identification tool suitable for routine use in the veterinary diagnostic laboratory.

Transmission of Brucellosis from Elk to Cattle and Bison, Greater Yellowstone Area, USA, 2002–2012

Bovine brucellosis has been nearly eliminated from livestock in the United States. Bison and elk in the Greater Yellowstone Area remain reservoirs for the disease. During 1990–2002, no known cases occurred in Greater Yellowstone Area livestock. Since then, 17 transmission events from wildlife to livestock have been investigated.

Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock

Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8u2009km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations.

Field Application of Serodiagnostics To Identify Elephants with Tuberculosis prior to Case Confirmation by Culture

ABSTRACT Three serologic methods for antibody detection in elephant tuberculosis (TB), the multiantigen print immunoassay (MAPIA), ElephantTB STAT-PAK kit, and DPP VetTB test, were evaluated using serial serum samples from 14 captive elephants infected with Mycobacterium tuberculosis in 5 countries. In all cases, serological testing was performed prior to the diagnosis of TB by mycobacterial culture of trunk wash or tissue samples collected at necropsy. All elephants produced antibody responses to M. tuberculosis antigens, with 13/14 recognizing ESAT-6 and/or CFP10 proteins. The findings supported the high serodiagnostic test accuracy in detecting infections months to years before M. tuberculosis could be isolated from elephants. The MAPIA and/or DPP VetTB assay demonstrated the potential for monitoring antimycobacterial therapy and predicting TB relapse in treated elephants when continuously used in the posttreatment period. History of exposure to TB and past treatment information should be taken into consideration for proper interpretation of the antibody test results. Data suggest that the more frequent trunk wash culture testing of seropositive elephants may enhance the efficiency of the TB diagnostic algorithm, leading to earlier treatment with improved outcomes.

Persistence of Mycobacterium bovis Bacillus Calmette-Guérin in White-Tailed Deer (Odocoileus Virginianus) after Oral or Parenteral Vaccination

Mycobacterium bovis is the cause of tuberculosis in cattle and a serious zoonotic pathogen, most commonly contracted through consumption of unpasteurized dairy products. To control this zoonosis, many countries have developed bovine tuberculosis eradication programmes. Although relatively successful, efforts are hindered in many regions by spillover from wildlife reservoirs of M.u2003bovis to cattle. Such is the case in the United States where spillover of M.u2003bovis from free‐ranging white‐tailed deer to cattle occurs. One approach to control such inter‐species transmission is vaccination of wildlife. The live, attenuated human vaccine M.u2003bovis Bacillus Calmette‐Guérin (BCG) has been shown to reduce disease severity in white‐tailed deer; however, vaccine persistence within tissues has also been noted. Consumption of venison containing BCG by hunters may present a public health concern as BCG exposure, although unlikely to cause disease, could cause false positive tuberculin skin test results. To examine BCG persistence further, 42 white‐tailed deer were vaccinated orally or subcutaneously (SC) with BCG Danish. Three deer from each group were killed and examined at periods ranging from 2u2003weeks to 11u2003months after vaccination. BCG was recovered from orally vaccinated deer as late as 3u2003months after vaccination, while BCG persisted in SC vaccinated deer for as long as 9u2003months. At no time was BCG isolated from meat; however, prolonged persistence was seen in lymphoid organs. Although vaccine persistence was noted, especially in SC vaccinated deer, the distribution of culture‐positive tissues makes human exposure through consumption unlikely.

Comparison of the MGIT 960, BACTEC 460 TB and solid media for isolation of Mycobacterium bovis in United States veterinary specimens

BackgroundBacteriologic culture remains one of the most important methods to diagnose bovine tuberculosis despite the lengthy incubation time, significant decontamination and media expense, and high biocontainment requirements. Media selection is an important determination of culture sensitivity, and the planned discontinuation of the BACTEC 460xa0TB culture system has challenged veterinary diagnostic laboratories to evaluate alternatives. At the National Veterinary Services Laboratories the BACTEC MGIT 960 and 4 solid media formulations were compared with the BACTEC 460xa0TB system on 6,795 veterinary diagnostic specimens submitted for Mycobacterium bovis culture.ResultsM. bovis was isolated from 2.6% of the samples and atypical mycobacteria from 4.4% of the samples. The BACTEC 12B media isolated significantly more M. bovis (93.1% of positive samples) than MGIT 960 media (81.9%). However, contamination rates were much higher for the MGIT media, 17-24%, compared to 7% for BACTEC, suggesting that contamination was a major cause of MGIT reduced sensitivity. Time to signal positive was 2.37 weeks (95% CI 2.24-2.5) for the MGIT, and 3.2 weeks (95% CI 3.07-3.3) for the BACTEC, both earlier than any solid media. Mycobactosel LJ failed to isolate M. bovis from primary culture. An in-house 7H11 media supplemented with calf sera, hemolyzed blood, malachite green and pyruvate recovered more M. bovis (80.6%) with the least amount of contamination of any other solid media evaluated.ConclusionDecontamination methods may have to be optimized and or MGIT media may have to be altered to reduce contamination in veterinary samples. Despite these issues, the MGIT 960 system is still favored over the use of solid media due to decreased time to recovery and the potential for higher sensitivity.

Isolation of mycobacteria from clinical samples collected in the United States from 2004 to 2011

BackgroundMycobacteria other than M. bovis may interfere with current bovine tuberculosis diagnostic tests resulting in false positive test results. As the prevalence of M. bovis decreases in the United States, interference from other mycobacteria play an increasingly important role in preventing the eradication of M. bovis. To identify mycobacteria other than M. bovis that may be interfering with current diagnostic tests, a retrospective study was performed to identify mycobacteria isolated from clinical tissues at the National Veterinary Services Laboratories between 1 January 2004 and 9 October 2011.ResultsDuring the study period, 2,366 mycobacteria other than M. bovis were isolated from samples submitted for clinical diagnosis of M. bovis. Fifty-five mycobacterial species were isolated during this time period. In cattle, M. avium complex, M. fortuitum/fortuitum complex, M. smegmatis, M. kansasii, and M. terrae complex were the predominate species other than M. bovis isolated from tissues submitted for culture. Mycobacteria other than M. bovis isolated from deer were predominantly M. avium complex, M. terrae/terrae complex, and M. fortuitum/fortuitum complex.ConclusionsThese data provide information characterizing the species and relative prevalence of mycobacteria other than M. bovis that may interfere with current diagnostic tests.

Clinical disease and stage of lactation influence shedding of Mycobacterium avium subspecies paratuberculosis into milk and colostrum of naturally infected dairy cows

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johnes disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n=39) and symptomatic clinical (n=29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrolds egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrolds egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0-60), with negligible positive samples observed in mid (DIM 60-240) and late (DIM 240-305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd.

Descriptive Epidemiology and Whole Genome Sequencing Analysis for an Outbreak of Bovine Tuberculosis in Beef Cattle and White-Tailed Deer in Northwestern Minnesota

Bovine tuberculosis (bTB) was discovered in a Minnesota cow through routine slaughter surveillance in 2005 and the resulting epidemiological investigation led to the discovery of infection in both cattle and white-tailed deer in the state. From 2005 through 2009, a total of 12 beef cattle herds and 27 free-ranging white-tailed deer (Odocoileus virginianus) were found infected in a small geographic region of northwestern Minnesota. Genotyping of isolates determined both cattle and deer shared the same strain of bTB, and it was similar to types found in cattle in the southwestern United States and Mexico. Whole genomic sequencing confirmed the introduction of this infection into Minnesota was recent, with little genetic divergence. Aggressive surveillance and management efforts in both cattle and deer continued from 2010–2012; no additional infections were discovered. Over 10,000 deer were tested and 705 whole herd cattle tests performed in the investigation of this outbreak.