Suelee Robbe-Austerman

Pathogenesis of Mycobacterium avium subsp. paratuberculosis in neonatal calves after oral or intraperitoneal experimental infection.

Understanding the host response to Mycobacterium avium subsp. paratuberculosis is critical to the development of effective vaccines and therapeutics for the control of this disease in the field. The current study compared the effectiveness of oral and intraperitoneal (IP) methods of experimental inoculation and two strains of M. avium subsp. paratuberculosis (strain K-10 and clinical isolate 509) on the level of infection and lesion development. Calves were inoculated with 4x10(11) to 8x10(12)cfu live bacteria, depending upon treatment group. Fecal shedding of M. avium subsp. paratuberculosis was minimal and infrequent over the course of the study for calves that received strain K-10 (oral and IP), however, calves orally inoculated with the clinical isolate shed high numbers of bacteria in their feces up to 4 months post-inoculation. Colonization was present in a number of intestinal tissues and lymph nodes with the lowest number of affected tissues in the IP calves and the highest for calves receiving the clinical isolate via oral inoculation. Microscopic lesions were predominantly found in the ileal and jejunal sections of small intestine and their associated lymph nodes, as well as the ileocecal valve and node. These data suggest that a variety of experimental infection regimes can be effective but oral inoculation with a clinical isolate may result in greater colonization of tissues and fecal shedding of M. avium subsp. paratuberculosis.

Comparative genomic analysis of Mycobacterium avium subspecies obtained from multiple host species

BackgroundMycobacterium avium (M. avium) subspecies vary widely in both pathogenicity and host specificity, but the genetic features contributing to this diversity remain unclear.ResultsA comparative genomic approach was used to identify large sequence polymorphisms among M. avium subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate M. avium subsp. paratuberculosis (MAP) K-10. Open reading frames (ORFs) were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to MAP K-10 DNA. Multiple large polymorphic regions were found in the genomes of MAP isolates obtained from sheep. One of these clusters encodes glycopeptidolipid biosynthesis enzymes which have not previously been identified in MAP. M. avium subsp. silvaticum isolates were observed to have a hybridization profile very similar to yet distinguishable from M. avium subsp. avium. Isolates obtained from cattle (n = 5), birds (n = 4), goats (n = 3), bison (n = 3), and humans (n = 9) were indistinguishable from cattle isolate MAP K-10.ConclusionGenome diversity in M. avium subspecies appears to be mediated by large sequence polymorphisms that are commonly associated with mobile genetic elements. Subspecies and host adapted isolates of M. avium were distinguishable by the presence or absence of specific polymorphisms.

Bovine Tuberculosis in a Nebraska Herd of Farmed Elk and Fallow Deer: A Failure of the Tuberculin Skin Test and Opportunities for Serodiagnosis

In 2009, Mycobacterium bovis infection was detected in a herd of 60 elk (Cervus elaphus) and 50 fallow deer (Dama dama) in Nebraska, USA. Upon depopulation of the herd, the prevalence of bovine tuberculosis (TB) was estimated at ∼71–75%, based upon histopathology and culture results. Particularly with elk, gross lesions were often severe and extensive. One year ago, the majority of the elk had been tested for TB by single cervical test (SCT), and all were negative. After initial detection of a tuberculous elk in this herd, 42 of the 59 elk were tested by SCT. Of the 42 SCT-tested elk, 28 were TB-infected with only 3/28 reacting upon SCT. After SCT, serum samples were collected from the infected elk and fallow deer from this herd at necropsy and tested by three antibody detection methods including multiantigen print immunoassay, cervidTB STAT-PAK, and dual path platform VetTB (DPP). Serologic test sensitivity ranged from 79 to 97% depending on the test format and host species. Together, these findings demonstrate the opportunities for use of serodiagnosis in the rapid detection of TB in elk and fallow deer.

Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction

Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from feces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct fecal polymerase chain reaction (PCR) is becoming more widely used, demonstrating similar sensitivity and specificity to culture. To ensure efficient and reproducible PCR results from a difficult sample matrix such as feces, there are many obstacles that a DNA extraction method must overcome, including the presence of inhibitors and the thick waxy cell wall of MAP. In the current study, 6 commercial DNA extraction kits were evaluated using fecal samples from naturally infected cattle shedding various amounts of MAP. Upon extraction, DNA purity and yield were measured, and real-time PCR was performed for detection of the insertion sequence (IS)900 and ISMAP02 targets. The kits evaluated showed significant differences in the purity and yield of DNA obtained. The best results were observed with kits E and A, having identified 94% (16/17) and 76% (13/17) of the positive samples by IS900 PCR, respectively. Both of these kits utilized bead beating in a lysis solution for cell disruption, followed by spin column technology (kit E) or magnetic bead–based technology (kit A) for nucleic acid isolation and purification. Two kits (A and F) demonstrated improved performance when used in conjunction with the respective manufacturer’s PCR test. The present study demonstrates the importance of choosing the correct methodology for the most accurate diagnosis of paratuberculosis through fecal PCR.

Augmentation of secreted and intracellular gamma interferon following johnin purified protein derivative sensitization of cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Measurement of secreted interferon (IFN)-γ has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-γ assay. In the present study, healthy noninfected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative (JPPD) and effects on secreted and intracellular IFN-γ were observed. Intradermal injection resulted in significant increases in secreted IFN-γ for subclinically infected cows after stimulation of peripheral blood mononuclear cells (PBMC) with concanavalin A or M. paratuberculosis antigen preparations (whole-cell sonicate and JPPD) on days 7 and 10 postinjection. Intracellular IFN-γ was increased after intradermal injection in total PBMC for all treatment groups and was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T-cell populations were further defined by CD45RO expression, intracellular IFN-γ was higher within CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells for subclinically and clinically infected cows but similar within these subpopulations for healthy controls. These results indicate that intradermal sensitization of cows in the subclinical stage of infection will upregulate expression of IFN-γ, enhancing the sensitivity of this assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.

Evaluation of the Gamma Interferon ELISA in Sheep Subclinically Infected with Mycobacterium Avium Subspecies Paratuberculosis Using a Whole-Cell Sonicate or a Johnin Purified-Protein Derivative

The aim of the study reported here was to estimate the sensitivity and specificity of the gamma interferon (IFN-γ) ELISA for paratuberculosis in sheep using receiver-operating characteristic analysis. Bacteriologic culture of tissues was used to define the reference positive population (n = 33). Two reference negative populations were used: culture-negative sheep from infected flocks (n = 77), and sheep from noninfected flocks (n = 358). We also evaluated the accuracy of 2 Mycobacterium avium subspecies paratuberculosis (MAP) antigen preparations, a whole-cell sonicate (MpS) and a johnin purified-protein derivative (PPDj). The source of the reference negative sheep used in the analysis affected overall accuracy of the IFN-γ ELISA. The area under the curve was 0.683 (95% confidence interval 0.574–0.787), using culture-negative sheep from infected flocks, was 0.831 (0.764–0.889), using sheep from noninfected flocks for the MpS, and was 0.809 (0.726–0.881) and 0.897 (0.862–0.925) for the PPDj, respectively. Using the MpS, the cut point that classified the most sheep correctly was an optical density reading of 0.20, for sensitivity of 40.7% (19.4–57.6) and specificity of 88.7% (77.0–95.7) or 97.6% (93.04–99.5), depending on the reference negative population used. Using the PPDj, the cut point that classified the most sheep correctly was 0.25 for sensitivity of 66.7% (47.2–82.7) and specificity of 93.5% (85.5–97.9) or 98.3% (96.4–99.4), respectively. The PPDj was more accurate at identifying MAP-infected sheep than was the MpS (P = 0.034).

IDENTIFICATION OF BRUCELLA SUIS FROM FERAL SWINE IN SELECTED STATES IN THE USA

Abstract Serologic tests currently available for brucellosis diagnosis detect antibodies to Brucella but do not distinguish between species of Brucella. Although Brucella suis is known to circulate within various feral swine (Sus scrofa) populations, our objective was to determine the primary species of Brucella circulating in feral swine populations in areas of the US with high brucellosis prevalence. We cultured lymph nodes from 183 feral swine. We identified 22 isolates from 21 animals, and all isolates were genotyped as B. suis. Most isolates were B. suis biovar 1, with the exception of two genetically distinct isolates from one feral swine in Hawaii, which were identified as B. suis biovar 3. Serum from each feral swine was also tested by the fluorescence polarization assay when possible, but only 52% (95% CL = 29.8–74.3) of culture-positive animals were antibody positive. Our results indicate that brucellosis infections in feral swine within the US are typically caused by B. suis. However, improved serologic tests are needed to more accurately determine exposure to Brucella spp. and to monitor disease trends in feral swine populations.

Investigations on Deer to Deer and Deer to Cattle Transmission of the Vaccine Mycobacterium bovis Bacillus Calmette-Guerin (BCG)

Mycobacterium bovis is the causative agent of tuberculosis in animals and can cause tuberculosis in humans clinically indistinguishable from that caused by Mycobacterium tuberculosis. Efforts to eradicate bovine tuberculosis have significantly decreased prevalence in developed countries. However, some countries have found it impossible to eradicate bovine tuberculosis due to the presence of a wildlife reservoir of M. bovis. In Michigan, USA there exists a reservoir of M. bovis in free-ranging white-tailed deer. Vaccination with M. bovis BCG is one approach to controlling tuberculosis in wildlife. Nevertheless, use of a live vaccine generates concerns about exposure risk to non-target species, including domestic livestock. Unintentional exposure of cattle to BCG may result in increased numbers of false positive tuberculin skin test reactions. Twenty-nine white-tailed deer received 1 SC dose of 107 colony-forming units of M. bovis BCG Danish 1331 (n=19) or no vaccination (n=10). Vaccinated and non-vaccinated deer were comingled with opportunity for direct and indirect contact. Twelve unvaccinated Holstein calves were housed in a separate paddock with no means of direct contact with deer; however, indirect contact through the sharing of feed and water was permitted. After 180 days, 11 out of 15 vaccinated deer and 4 out of 8 non-vaccinated deer were classified as reactors using the tuberculin skin test. All 12 calves were categorized as non-reactors, by both the tuberculin skin test and BOVIGAM™ assay. Vaccination of free-ranging white-tailed deer with BCG Danish is unlikely to have a deleterious effect on tuberculosis surveillance measures in cattle.

Emerging Tuberculosis Pathogen Hijacks Social Communication Behavior in the Group-Living Banded Mongoose (Mungos mungo)

ABSTRACT An emerging Mycobacterium tuberculosis complex (MTC) pathogen, M. mungi, infects wild banded mongooses (Mungos mungo) in Northern Botswana, causing significant mortality. This MTC pathogen did not appear to be transmitted through a primary aerosol or oral route. We utilized histopathology, spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), quantitative PCR (qPCR), and molecular markers (regions of difference [RDs] from various MTC members, including region of difference 1 [RD1] from M. bovis BCG [RD1BCG], M. microti [RD1mic], and M. pinnipedii [RD1seal], genes Rv1510 [RD4], Rv1970 [RD7], Rv3877/8 [RD1], and Rv3120 [RD12], insertion element IS1561, the 16S RNA gene, and gene Rv0577 [cfp32]), including the newly characterized mongoose-specific deletion in RD1 (RD1mon), in order to demonstrate the presence of M. mungi DNA in infected mongooses and investigate pathogen invasion and exposure mechanisms. M. mungi DNA was identified in 29% of nasal planum samples (n = 52), 56% of nasal rinses and swabs (n = 9), 53% of oral swabs (n = 19), 22% of urine samples (n = 23), 33% of anal gland tissue (n = 18), and 39% of anal gland secretions (n = 44). The occurrence of extremely low cycle threshold values obtained with qPCR in anal gland and nasal planum samples indicates that high levels of M. mungi can be found in these tissue types. Histological data were consistent with these results, suggesting that pathogen invasion occurs through breaks in the nasal planum and/or skin of the mongoose host, which are in frequent contact with anal gland secretions and urine during olfactory communication behavior. Lesions in the lung, when present, occurred only with disseminated disease. No environmental sources of M. mungi DNA could be found. We report primary environmental transmission of an MTC pathogen that occurs in association with social communication behavior. IMPORTANCE Organisms causing infectious disease evolve modes of transmission that exploit environmental and host conditions favoring pathogen spread and persistence. We report a novel mode of environmental infectious disease transmission that occurs in association with olfactory secretions (e.g., urine and anal gland secretions), allowing pathogen exposure to occur within and between social groups through intricate social communication behaviors of the banded mongoose host. The presence of M. mungi in these environmentally deposited secretions would effectively circumvent natural social barriers (e.g., territoriality), facilitating between-group pathogen transmission in the absence of direct physical contact, a rare occurrence in this highly territorial species. This work identifies an important potential mechanism of pathogen transmission of epidemiological significance in social species. We also provide evidence of a novel mechanism of pathogen transmission for the MTC complex, where pathogen movement in the environment and host exposure dynamics are driven by social behavior. Organisms causing infectious disease evolve modes of transmission that exploit environmental and host conditions favoring pathogen spread and persistence. We report a novel mode of environmental infectious disease transmission that occurs in association with olfactory secretions (e.g., urine and anal gland secretions), allowing pathogen exposure to occur within and between social groups through intricate social communication behaviors of the banded mongoose host. The presence of M. mungi in these environmentally deposited secretions would effectively circumvent natural social barriers (e.g., territoriality), facilitating between-group pathogen transmission in the absence of direct physical contact, a rare occurrence in this highly territorial species. This work identifies an important potential mechanism of pathogen transmission of epidemiological significance in social species. We also provide evidence of a novel mechanism of pathogen transmission for the MTC complex, where pathogen movement in the environment and host exposure dynamics are driven by social behavior.

Identification of Source of Brucella suis Infection in Human by Using Whole-Genome Sequencing, United States and Tonga.

Brucella suis infection was diagnosed in a man from Tonga, Polynesia, who had butchered swine in Oregon, USA. Although the US commercial swine herd is designated brucellosis-free, exposure history suggested infection from commercial pigs. We used whole-genome sequencing to determine that the man was infected in Tonga, averting a field investigation.