Evaluation of a New Strategy for Control of Bovine Tuberculosis in Michigan White-Tailed Deer: Year 1 Progress Report

The State of Michigan is striving to eliminate bovine tuberculosis (Tb) infection among free-ranging white-tailed deer in the northeastern Lower Peninsula of the state. Aggressive reduction in the overall deer population abundance may help to further reduce TB prevalence, but this course of action is unacceptable to many hunters and landowners. Targeted culling of sick deer would likely be far more acceptable to these stakeholders, so in the winter of 2003 the Michigan Department of Natural Resources pilot-trialed a new strategy based on live-trapping and Tb-testing of wild deer. The field study was conducted in a township with relatively high TB prevalence within Deer Management Unit 452 in the northeastern Lower Peninsula. Over a 2-month trapping period, 119 individual deer were live-trapped, blood sampled, fitted with a radio-collar, and released. A total of 31 of these deer were subsequently classified as Tb-suspect by at least one of five blood tests employed (however there was a low level of agreement among tests). A delay in testing meant that only six of these suspect deer were culled by sharpshooters before pre-programmed release of their radio-collars, after which they could no longer be located. Mycobacterium bovis was cultured from one of these six suspect deer; the other five were negative on culture. All target deer were located to within shooting range with 1 – 2 days of effort, and all the radio-collars on the apparently-healthy deer dropped off after the intended 90-day interval, and were thereafter recovered for re-use. There was considerable support for this pilot project among hunters, farmers, state and federal agriculture agencies, the media and the general public, and so we recommend that further field trials be undertaken using this technique. The initial focus of these trials should be on improving the efficacy and reliability of the blood testing procedure.

Enhanced phagocytosis of Corynebacterium pseudotuberculosis by monocyte-macrophage cells from goats naturally infected with caprine arthritis encephalitis virus

Sanches B.G.S., Souza F.N., Azedo M.R., Batista C.F., Bertagnon H.G., Blagitz M.G. & Della Libera A.M.M.P. 2012. [Enhanced phagocytosis of Corynebacterium pseudotuberculosis by monocyte-macrophage cells from goats naturally infected with caprine arthritis encephalitis virus.] Fagocitose intensificada de Corynebacterium pseudotuberculosis por celulas da serie monocito-macrofago de caprinos naturalmente infectados pelo virus da artrite encefalite. Pesquisa Veterinaria Brasileira 32(12):1225-1229. Departamento de Clinica Medica, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Avenida Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitaria, Sao Paulo, SP 05508-270, Brazil. E-mail: camilafb@usp.br Caprine arthritis encephalitis (CAE) and caseous lymphadenitis (CL) have high incidence and transmissibility in small ruminants. Since both virus have tropism for macrophages and monocytes and affect the innate immune response, it is believed that CAE can predispose the animal to infection by Corynebacteruim pseudotuberculosis, the etiological agent of CL. To confirm this hypothesis, we evaluated phagocytosis from the monocyte-macrophage cells from 30 Saanen goats. Goats were uniformly divided in two groups according to results of agar gel immunodiffusion test for CAE virus (CAEV). Peripheral blood mononuclear cells were isolated by density gradient centrifugation and the monocyte-macrophage cells were isolated from the mononuclear cells by their adhesion properties in plaques. Afterwards, phagocytosis of C. psudotuberculosis was performed for two hours at 37 degrees C, 5% of CO2, and assessed by microscopic visualization. There was no difference in the percentage of monocyte-macrophage cells that phagocytozed C. bovis between groups (P = 0.41). However, when phagocytosis rates were classified according to the number of C. pseudotuberculosis phagocyted, the percentage of monocyte-macrophage cells that internalized more than 12 bacteria were higher in serologically CAEV positive animals compared to the serologically negative ones (P < 0.001). Furthermore, a positive and significant correlation (r = 0.488; P = 0.006) between the percentage of monocyte-macrophage cells that internalized more than 12 bacteria and the percentage of monocyte that were carrying out phagocytosis was also encountered in serologically CAEV positive goats, however the same were not observed in serologically negative ones. These results demonstrated an alteration in the intensity of C. pseudotuberculosis phagocytosis by monocytes-macrophages from goats infected by CAEV. Thus, these results indicated that goats infected with CAEV may be more susceptible to CL.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Cryptosporidium parvum infection is very important with respect to public health, owing to foodborne and waterborne outbreaks and gastrointestinal illness in immunocompetent and immunocompromised persons. In cattle, infection with this species manifests either as a subclinical disease or with diarrheal illness, which occurs more often in the presence of other infectious agents than when alone. The aim of this study was to develop a real-time polymerase chain reaction (PCR) assay for the detection of C. parvum in calf fecal samples and to compare the results of this assay with those of the method routinely used for the diagnosis of Cryptosporidium spp., nested PCR targeting the 18S rRNA gene. Two hundred and nine fecal samples from calves ranging in age from 1 day to 6 months were examined using real-time PCR specific for the actin gene of C. parvum and by a nested PCR targeting the 18S rRNA gene of Cryptosporidium spp. Using real-time PCR detection, 73.2% (153 out of 209) of the samples were positive for C. parvum, while 56.5% (118 out of 209) of the samples were positive for Cryptosporidium spp. when the nested PCR amplification method was used for the detection. The analytical sensitivity of the real-time PCR was approximately one C. parvum oocyst. There was no significant nonspecific DNA amplification of any of the following species and genotype: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium galli, Cryptosporidium ryanae, Cryptosporidium serpentis, or avian genotype II. Thus, we conclude that real-time PCR targeting the actin gene is a sensitive and specific method for the detection of C. parvum in calf fecal samples.

Isolierung von Essigsäure-, Propionsäure- und Buttersäure-bildenden Bakterien aus Biogasanlagen

In der vorliegenden Arbeit wurden Essigsäure-, Propionsäure und Buttersäure-bildende Bakterien aus einer thermophilen und drei mesophilen Biogasanlagen sowie aus zwei Hochdruck-Biogas-Laborfermentern isoliert. Die Fermenter waren mit dem nachwachsenden Rohstoff Maissilage, teilweise mit Rinder- oder Schweinegülle und weiteren festen Inputstoffen gefüttert. Für die Isolierung von Säure-bildenden Bakterien wurde ein Mineralsalzmedium verwendet, welchem als Kohlenstoffquelle Na-DL-Laktat, Succinat, Ethanol, Glycerin, Glucose oder eine Aminosäuremischung (Alanin, Serin, Threonin, Glutaminsäure, Methionin und Cystein) hinzugefügt wurde. Hierbei handelt es sich um Substrate, welche beim anaeroben Abbau während der Hydrolyse oder der primären Gärung entstehen können. Die erhaltenen Isolate waren in der Lage, aus diesen Substraten Essigsäure, Propionsäure oder Buttersäure zu bilden. Insgesamt wurden aus den beprobten Anlagen 49 Isolate gewonnen, welche zu den Phyla Firmicutes, Tenericutes oder Thermotogae gehörten. Mit Hilfe von 16S rDNA-Sequenzen konnten die meisten Isolate als Clostridium sporosphaeroides, Defluviitoga tunisiensis und Dendrosporobacter sp. identifiziert werden. Die Bildung von Essigsäure, Propionsäure oder Buttersäure wurde in Kulturen von Isolaten festgestellt, welche als folgende Arten identifiziert wurden: Bacillus thermoamylovorans, Clostridium aminovalericum, Clostridium cochlearium/Clostridium tetani, Clostridium sporosphaeroides, Dendrosporobacter sp., Proteiniborus sp., Selenomonas bovis und Tepidanaerobacter sp. Zwei Isolate, verwandt mit Thermoanaerobacterium thermosaccharolyticum, konnten Buttersäure und Milchsäure bilden. In Kulturen von Defluviitoga tunisiensis wurde Essigsäurebildung festgestellt. Ein Vergleich der 16S rDNA-Sequenzen mit Datenbanken und die Ergebnisse der PCR-Amplifikationen mit Isolat-spezifischen Primerpaaren ergaben zusätzlich Hinweise, dass es sich bei einigen Isolaten um neue Arten handeln könnte (z. B. Stamm Tepidanaerobacter sp. AS34, Stamm Proteiniborus sp. ASG1.4, Stamm Dendrosporobacter sp. LG2.4, Stamm Desulfotomaculum sp. EG2.4, Stamm Gallicola sp. SG1.4B und Stamm Acholeplasma sp. ASSH51). Durch die Entwicklung Isolat-spezifischer Primerpaare, abgeleitet von 16S rDNA-Sequenzen der Isolate oder Referenzstämmen, konnten die Isolate in Biogasanlagen detektiert und mittels qPCR quantifiziert werden (hauptsächlich im Bereich zwischen 1000 bis 100000000 Kopien der 16S rDNA/g BGA-Probe). Weiterhin konnten die Isolate mit Hilfe physiologischer Versuche charakterisiert und deren Rolle in der anaeroben Abbaukette diskutiert werden. Die Art Defluviitoga tunisiensis scheint eine große Bedeutung in Biogasanlagen zu spielen. Defluviitoga tunisiensis wurde am häufigsten in Untersuchungen im Rahmen der vorliegenden Arbeit isoliert und konnte auch mit Hilfe des entwickelten Primerpaares in hohen Abundanzen in den beprobten Biogasanlagen detektiert werden (10000 - 100000000 Kopien der 16S rDNA/g BGA-Probe). Die manuelle Annotation des Gesamtgenoms sowie die Substratverwertungsversuche haben gezeigt, dass Defluviitoga tunisiensis ein sehr breites Substratspektrum in der Verwertung von Kohlenhydraten besitzt und dadurch möglicherweise eine wichtige Rolle bei der Verwertung von Biomasse in Biogasanlagen einnimmt. Mit Hilfe der Ergebnisse der vorliegenden Arbeit konnten somit neue Einblicke in die zweite Stufe des anaeroben Abbaus, die Acidogenese, in Biogasanlagen gegeben werden. rn

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

Partial overlap of anti-mycobacterial, and anti-Saccharomyces cerevisiae mannan antibodies in Crohn's disease

AIM: To test whether humoral immune reaction against mycobacteria may play a role in anti-Saccharomyces cerevisiae antibodies (ASCA) generation in Crohn's disease (CD) and/or whether it correlates with clinical subtypes. METHODS: The dominant ASCA epitope was detected by Galanthus nivalis lectin (GNL)-binding assay. ASCA and IgG against mycobacterial lysates (M avium, M smegmatis, M chelonae, M bovis BCG, M avium ssp. paratuberculosis (MAP)] or purified lipoarabinomannans (LAM) were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice. RESULTS: GNL bound to different extents to mycobacterial lysates, abundantly to purified mannose-capped (Man) LAM from M tuberculosis, but not to uncapped LAM from M smegmatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA (r = 0.37-0.64; P = 0.003-P < 0.001). ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent cross-reactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP (P = 0.024, 0.004 and 0.045, respectively). CONCLUSION: Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.

Molecular and serological diagnosis of Bartonella infection in 61 dogs from the United States

Molecular diagnosis of canine bartonellosis can be extremely challenging and often requires the use of an enrichment culture approach followed by PCR amplification of bacterial DNA. HYPOTHESES: (1) The use of enrichment culture with PCR will increase molecular detection of bacteremia and will expand the diversity of Bartonella species detected. (2) Serological testing for Bartonella henselae and Bartonella vinsonii subsp. berkhoffii does not correlate with documentation of bacteremia. ANIMALS: Between 2003 and 2009, 924 samples from 663 dogs were submitted to the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory for diagnostic testing with the Bartonella α-Proteobacteria growth medium (BAPGM) platform. Test results and medical records of those dogs were retrospectively reviewed. METHODS: PCR amplification of Bartonella sp. DNA after extraction from patient samples was compared with PCR after BAPGM enrichment culture. Indirect immunofluorescent antibody assays, used to detect B. henselae and B. vinsonii subsp. berkhoffii antibodies, were compared with PCR. RESULTS: Sixty-one of 663 dogs were culture positive or had Bartonella DNA detected by PCR, including B. henselae (30/61), B. vinsonii subsp. berkhoffii (17/61), Bartonella koehlerae (7/61), Bartonella volans-like (2/61), and Bartonella bovis (2/61). Coinfection with more than 1 Bartonella sp. was documented in 9/61 dogs. BAPGM culture was required for PCR detection in 32/61 cases. Only 7/19 and 4/10 infected dogs tested by IFA were B. henselae and B. vinsonii subsp. berkhoffii seroreactive, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs were most often infected with B. henselae or B. vinsonii subsp. berkhoffii based on PCR and enrichment culture, coinfection was documented, and various Bartonella species were identified. Most infected dogs did not have detectable Bartonella antibodies.

An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection

We compared clinicopathologic findings in dogs with Bartonella infection to Bartonella spp. negative dogs suspected of a vector-borne disease. Cases (n=47) and controls (n=93) were selected on the basis of positive or negative enrichment culture PCR results, respectively. Signalment, clinicopathologic findings and treatments were extracted from medical records. DNA sequencing identified Bartonella henselae (n=28, 59.6%), Bartonella vinsonii subsp. berkhoffii (n=20, 42.6%), Bartonella koehlerae (n=3, 6.4%), Bartonella volans-like (n=3, 6.4%) and Bartonella bovis (n=1, 2.1%). There were no significant differences in age, breed, size, sex or neuter status between cases and controls. Dogs infected with Bartonella sp. often had a history of weight loss [OR=2.82; 95% CI: 1.08-7.56] and were hypoglobulinemic [OR=4.26; 95% CI: 1.31-14.41]. With the exception of weight loss and hypoglobulinemia, clinicopathologic abnormalities in Bartonella-infected dogs in this study were similar to dogs suspected of other vector-borne infections.

Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4-7.8) but none of the 269 red deer (0%; 0-1.4). M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.

Differences in primary sites of infection between zoonotic and human tuberculosis: results from a worldwide systematic review.

Tuberculosis (TB) is one of the most devastating infectious diseases worldwide. Whilst global burden estimates for M. tuberculosis infection (MtTB) are well established, accurate data on the contribution of zoonotic TB (zTB) caused by M. bovis or M. caprae to human TB are scarce. The association of M. bovis infection with extrapulmonary tuberculosis has been suggested repeatedly, though there is little scientific evidence available to support this relationship. The present study aimed to determine globally the occurrence of extrapulmonary TB and the primary site (i.e. primary body location affected) of zTB in comparison with MtTB, based on previously published reports. A systematic literature review was conducted in 32 different bibliographic databases, selecting reports on zTB written in English, French, German, Spanish or Portuguese. Data from 27 reports from Africa, America, Europe and the Western Pacific Region were extracted for analyses. Low income countries, in Africa and South-East Asia, were highly underrepresented in the dataset. The median proportion of extrapulmonary TB cases was significantly increased among zTB in comparison with data from registries of Europe and USA, reporting mainly MtTB cases (47% versus 22% in Europe, 73% versus 30% in the USA). These findings were confirmed by analyses of eight studies reporting on the proportions of extrapulmonary TB in comparable populations of zTB and MtTB cases (median 63% versus 22%). Also, disparities of primary sites of extrapulmonary TB between zTB and MtTB were detected. Our findings, based on global data, confirm the widely suggested association between zTB and extrapulmonary disease. Different disability weights for zTB and MtTB should be considered and we recommend separate burden estimates for the two diseases.

[Mastitis management in Swiss dairy farms with udder health problems]

The objective of this study was to describe the udder health management in Swiss dairy herds with udder health problems. One hundred dairy herds with a yield-corrected somatic cell count of 200'000 to 300'000 cells/ml during 2010 were selected. Data concerning farm structure, housing system, milking technique, milking procedures, dry-cow and mastitis management were collected during farm visits between September and December 2011. In addition, quarter milk samples were collected for bacteriological culturing from cows with a composite somatic cell count ≥ 150'000 cells/ml. The highest quarter level prevalence was 12.3 % for C. bovis. Eighty-two percent of the pipeline milking machines in tie-stalls and 88 % of the milking parlours fulfilled the criteria for the vacuum drop, and only 74 % of the pipeline milking machines met the criteria of the 10-l-water test. Eighty-five percent of the farms changed their milk liners too late. The correct order of teat preparation before cluster attachment was carried out by 37 % of the farmers only. With these results, Swiss dairy farmers and herd health veterinarians can be directed to common mistakes in mastitis management. The data will be used for future information campaigns to improve udder health in Swiss dairy farms.

Bovine Tuberculosis Risk Factors for British Herds Before and After the 2001 Foot-and-Mouth Epidemic: What have we Learned from the TB99 and CCS2005 Studies?

Over the last couple of decades, the UK experienced a substantial increase in the incidence and geographical spread of bovine tuberculosis (TB), in particular since the epidemic of foot-and-mouth disease (FMD) in 2001. The initiation of the Randomized Badger Culling Trial (RBCT) in 1998 in south-west England provided an opportunity for an in-depth collection of questionnaire data (covering farming practices, herd management and husbandry, trading and wildlife activity) from herds having experienced a TB breakdown between 1998 and early 2006 and randomly selected control herds, both within and outside the RBCT (the so-called TB99 and CCS2005 case-control studies). The data collated were split into four separate and comparable substudies related to either the pre-FMD or post-FMD period, which are brought together and discussed here for the first time. The findings suggest that the risk factors associated with TB breakdowns may have changed. Higher Mycobacterium bovis prevalence in badgers following the FMD epidemic may have contributed to the identification of the presence of badgers on a farm as a prominent TB risk factor only post-FMD. The strong emergence of contact/trading TB risk factors post-FMD suggests that the purchasing and movement of cattle, which took place to restock FMD-affected areas after 2001, may have exacerbated the TB problem. Post-FMD analyses also highlighted the potential impact of environmental factors on TB risk. Although no unique and universal solution exists to reduce the transmission of TB to and among British cattle, there is an evidence to suggest that applying the broad principles of biosecurity on farms reduces the risk of infection. However, with trading remaining as an important route of local and long-distance TB transmission, improvements in the detection of infected animals during pre- and post-movement testing should further reduce the geographical spread of the disease.

Molecular detection of Bartonella spp. in deer ked pupae, adult keds and moose blood in Finland

SUMMARY The deer ked (Lipoptena cervi) is a haematophagous ectoparasite of cervids that harbours haemotrophic Bartonella. A prerequisite for the vector competence of the deer ked is the vertical transmission of the pathogen from the mother to its progeny and transstadial transmission from pupa to winged adult. We screened 1154 pupae and 59 pools of winged adult deer keds from different areas in Finland for Bartonella DNA using PCR. Altogether 13 pupa samples and one winged adult deer ked were positive for the presence of Bartonella DNA. The amplified sequences were closely related to either B. schoenbuchensis or B. bovis. The same lineages were identified in eight blood samples collected from free-ranging moose. This is the first demonstration of Bartonella spp. DNA in a winged adult deer ked and, thus, evidence for potential transstadial transmission of Bartonella spp. in the species.

Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.