Characterization of an aerosol generation system to assess inhalation risks of aerosolized nano-enabled consumer products.

Objective: The aerosolization of common nano-enabled consumer products such as cosmetics has significantly increased engineered nanoparticle inhalation risks. While several studies have investigated the impact of cosmetic dermal exposures, inhalation hazards of aerosolized cosmetics are much less known but could pose considerable harm to users due to potential co-exposure of nanoparticles and other product components.Materials and Methods: In this study, we developed a fully automated aerosol generation system to examine the aerosol properties of four aerosolized nano-enabled cosmetics using real-time monitoring and sampling instrumentation. Physicochemical characterization of aerosols was conducted using scanning electron microscopy coupled with energy dispersive x-ray spectroscopy (SEM-EDX). Characterization and calibration of animal exposure pods coupled to the system were also performed by measuring and comparing particle concentrations between pods.Results and Discussion: Results show peak emissions are shade dependent and varied between 12,000-22,000 particles/cm3 with modal diameters ranging from 36 nm-1.3 µm. SEM-EDX analysis determined that the original products and collected aerosols have similar morphological features consisting of micron-sized particles decorated with nanoparticles and crystalline structures. Mean total particle concentration in pods at 5 and 10 mg/m3 target levels were 2.22E + 05 #/cm3 and 4.33E + 05 #/cm3, respectively, with <10% variability between pods.Conclusions: The fully automated exposure platform described herein provides reproducible aerosol generation, conforms to recommended guidelines on chemical testing, and therefore is suitable for future in vivo toxicological assessments to examine potential respiratory hazards of aerosolized nano-enabled consumer products.

Developing Multipollutant Exposure Indicators of Traffic Pollution: The Dorm Room Inhalation to Vehicle Emissions (DRIVE) Study.

Introduction: The Dorm Room Inhalation to Vehicle Emissions (DRIVE2) study was conducted to measure traditional single-pollutant and novel multipollutant traffic indicators along a complete emission-to-exposure pathway. The overarching goal of the study was to evaluate the suitability of these indicators for use as primary traffic exposure metrics in panel-based and small-cohort epidemiological studies. Methods: Intensive field sampling was conducted on the campus of the Georgia Institute of Technology (GIT) between September 2014 and January 2015 at 8 monitoring sites (2 indoors and 6 outdoors) ranging from 5 m to 2.3 km from the busiest and most congested highway artery in Atlanta. In addition, 54 GIT students living in one of two dormitories either near (20 m) or far (1.4 km) from the highway were recruited to conduct personal exposure sampling and weekly biomonitoring. The pollutants measured were selected to provide information about the heterogeneous particulate and gaseous composition of primary traffic emissions, including the traditional traffic-related species (e.g., carbon monoxide [CO], nitrogen dioxide [NO2], nitric oxide [NO], fine particulate matter [PM2.5], and black carbon [BC]), and of secondary species (e.g., ozone [O3] and sulfate as well as organic carbon [OC], which is both primary and secondary) from traffic and other sources. Along with these pollutants, we also measured two multipollutant traffic indicators: integrated mobile source indicators (IMSIs) and fine particulate matter oxidative potential (FPMOP). IMSIs are derived from elemental carbon (EC), CO, and nitrogen oxide (NOx) concentrations, along with the fractions of these species emitted by gasoline and diesel vehicles, to construct integrated estimates of gasoline and diesel vehicle impacts. Our FPMOP indicator was based on an acellular assay involving the depletion of dithiothreitol (DTT), considering both water-soluble and insoluble components (referred to as FPMOPtotal-DTT). In addition, a limited assessment of 18 low-cost sensors was added to the study to supplement the four original aims. Results: Pollutant levels measured during the study showed a low impact by this highway hotspot source on its surrounding vicinity. These findings are broadly consistent with results from other studies throughout North America showing decreased relative contributions to urban air pollution from primary traffic emissions. We view these reductions as an indication of a changing near-road environment, facilitated by the effectiveness of mobile source emission controls. Many of the primary pollutant species, including NO, CO, and BC, decreased to near background levels by 20 to 30 m from the highway source. Patterns of correlation among the sites also varied by pollutant and time of day. NO2 exhibited spatial trends that differed from those of the other single-pollutant primary traffic indicators. We believe this was caused by kinetic limitations in the photochemical chemistry, associated with primary emission reductions, required to convert the NO-dominant primary NOx, emitted from automobiles, to NO2. This finding provides some indication of limitations in the use of NO2 as a primary traffic exposure indicator in panel-based health effect studies. Roadside monitoring of NO, CO, and BC tended to be more strongly correlated with sites, both near and far from the road, during morning rush hour periods and often weakly to moderately correlated during other time periods of the day. This pattern was likely associated with diurnal changes in mixing and chemistry and their impact on spatial heterogeneity across the campus. Among our candidate multipollutant primary traffic indicators, we report several key findings related to the use of oxidative potential (OP)-based indicators. Although earlier studies have reported elevated levels of FPMOP in direct exhaust emissions, we found that atmospheric processing further enhanced FPMOPtotal-DTT, likely associated with the oxidation of primary polycyclic aromatic hydrocarbons (PAHs) to quinones and hydroxyquinones and with the oxidization and water solubility of metals. This has important implications in terms both of the utility of FPMOPtotal-DTT as a marker for exhaust emissions and of the importance of atmospheric processing of particulate matter (PM) being tied to potential health outcomes. The results from the personal exposure monitoring also point to the complexity and diversity of the spatiotemporal variability patterns among the study monitoring sites and the importance of accounting for location and spatial mobility when estimating exposures in panel-based and small-cohort studies. This was most clearly demonstrated with the personal BC measurements, where ambient roadside monitoring was shown to be a poor surrogate for exposures to BC. Alternative surrogates, including ambient and indoor BC at the participants' respective dorms, were more strongly associated with personal BC, and knowledge of the participants' mean proximity to the highway was also shown to explain a substantial level of the variability in corresponding personal exposures to both BC and NO2. In addition, untargeted metabolomic indicators measured in plasma and saliva, which represent emerging methods for measuring exposure, were used to extract approximately 20,000 and 30,000 features from plasma and saliva, respectively. Using hydrophilic interaction liquid chromatography (HILIC) in the positive ion mode, we identified 221 plasma features that differed significantly between the two dorm cohorts. The bimodal distribution of these features in the HILIC column was highly idiosyncratic; one peak consisted of features with elevated intensities for participants living in the near dorm; the other consisted of features with elevated intensities for participants in the far dorm. Both peaks were characterized by relatively short retention times, indicative of the hydrophobicity of the identified features. The results from the metabolomics analyses provide a strong basis for continuing this work toward specific chemical validation of putative biomarkers of traffic-related pollution. Finally, the study had a supplemental aim of examining the performance of 18 low-cost CO, NO, NO2, O3, and PM2.5 pollutant sensors. These were colocated alongside the other study monitors and assessed for their ability to capture temporal trends observed by the reference monitoring instrumentation. Generally, we found the performance of the low-cost gas-phase sensors to be promising after extensive calibration; the uncalibrated measurements alone, however, would likely not have led to reliable results. The low-cost PM sensors we evaluated had poor accuracy, although PM sensor technology is evolving quickly and warrants future attention. Conclusions: An immediate implication of the changing near-road environment is that future studies aimed at characterizing hotspots related to mobile sources and their impacts on health will need to consider multiple approaches for characterizing spatial gradients and exposures. Specifically and most directly, the mobile source contributions to ambient concentrations of single-pollutant indicators of traffic exposure are not as distinguishable to the degree that they have been in the past. Collectively, the study suggests that characterizing exposures to traffic-related pollutants, which is already difficult, will become more difficult because of the reduction in traffic-related emissions. Additional multi-tiered approaches should be considered along with traditional measurements, including the use of alternative OP measures beyond those based on DTT assays, metabolomics, low-cost sensors, and air quality modeling.

Virulence of two strains of mycobacterium bovis in cattle following aerosol infection.

Over the past two decades, highly virulent strains of Mycobacterium tuberculosis have emerged and spread rapidly in man, suggesting a selective advantage based on virulence. A similar scenario has not been described for Mycobacterium bovis infection in cattle (i.e. bovine tuberculosis). An epidemiological investigation of a recent outbreak of bovine tuberculosis in a USA dairy indicated that the causative strain of M. bovis (strain 10-7428) was particularly virulent, with rapid spread within the herd. In the present study, the virulence of this strain (10-7428) was directly compared in the target host with a well-characterized strain (95-1315) of relevance to the USA bovine tuberculosis eradication programme. Aerosol inoculation of 10(4) colony forming units of M. bovis 95-1315 (n = 8) or 10-7428 (n = 8) resulted in a similar distribution and severity of gross and microscopical lesions of tuberculosis as well as mycobacterial colonization, primarily affecting the lungs and lung-associated lymph nodes. Specific cell-mediated and antibody responses, including kinetics of the response, as well as antigen recognition profiles, were also comparable between the two treatment groups. Present findings demonstrate that M. bovis strains 95-1315 and 10-7428 have similar virulence when administered to cattle via aerosol inoculation. Other factors such as livestock management practices likely affected the severity of the outbreak in the dairy.

Discriminant construct validity of ImPACT™: a companion study.

In a previous analysis of ImPACT scores relative to traditional neuropsychological tests (NP) and experimental tasks (Maerlender et al., 2010 ) we demonstrated convergent construct validity for the primary ImPACT test-score composites. A complete analysis of discriminant validity was not undertaken at that time. Here, test scores from the 54 collegiate football and hockey players were re-analyzed to specifically address the discriminant validity of the ImPACT™ composite scores using a multiply operationalized correlation matrix of multi-trait multi-method data. In the method used here, discriminant validity is determined by obtaining non-significant correlations between a target score when correlated with the average of the other trait measurements (multiply-operationalized multi-trait-mono-method analysis). Results showed that the ImPACT™ Verbal Memory (p = .044), Visual Memory (p = .006), and Visual Motor Speed (p = .000) scores were highly correlated with composites of the other scores, while the Reaction Time composite demonstrated adequate discriminant validity (p = .145). In comparison all of the NP composites showed good discrimination (all p-values >.05, except for Reaction Time p = .05). Thus the apparent lack of discriminability between three of four composite scores in this sample raises questions about using ImPACT™ composite scores to support specific construct-oriented interpretations. Taken together, the discriminant and convergent construct validity properties of ImPACT™ indicate construct sensitivity, but limited construct specificity.

Cognitive effects of one season of head impacts in a cohort of collegiate contact sport athletes.

OBJECTIVE: To determine whether exposure to repetitive head impacts over a single season negatively affects cognitive performance in collegiate contact sport athletes. METHODS: This is a prospective cohort study at 3 Division I National Collegiate Athletic Association athletic programs. Participants were 214 Division I college varsity football and ice hockey players who wore instrumented helmets that recorded the acceleration-time history of the head following impact, and 45 noncontact sport athletes. All athletes were assessed prior to and shortly after the season with a cognitive screening battery (ImPACT) and a subgroup of athletes also were assessed with 7 measures from a neuropsychological test battery. RESULTS: Few cognitive differences were found between the athlete groups at the preseason or postseason assessments. However, a higher percentage of the contact sport athletes performed more poorly than predicted postseason on a measure of new learning (California Verbal Learning Test) compared to the noncontact athletes (24% vs 3.6%; p < 0.006). On 2 postseason cognitive measures (ImPACT Reaction Time and Trails 4/B), poorer performance was significantly associated with higher scores on several head impact exposure metrics. CONCLUSION: Repetitive head impacts over the course of a single season may negatively impact learning in some collegiate athletes. Further work is needed to assess whether such effects are short term or persistent.

Performance of immunochromatographic and ELISA tests for detecting fallow deer infected with Mycobacterium bovis.

Fallow deer (Dama dama) are widely distributed as natural or naturalised populations, as well as in game parks and deer farms. We used 157 fallow deer sampled in populations considered to be Mycobacterium tuberculosis complex (MTC) free and 73 Mycobacterium bovis-infected fallow deer confirmed postmortem by culture to evaluate the diagnostic performance of two tests for the detection of anti-mycobacterial antibodies: the dual path platform (DPP) VetTB assay and the bovine purified protein derivative (bPPD) ELISA. We also compared their sensitivity with that of the skin test, analyzed the effect of haemolysis degree on the antibody detection and described the relationship between the test readings and presence/absence of gross tuberculosis (TB) compatible lesions. Sensitivity of bPPD ELISA was 51% at a specificity of 96%. Depending on the cut-off value selected, the sensitivity of DPP VetTB ranged from 62 to 71%, while its specificity was 88-95%. In the subgroup of M. bovis-infected deer for which the skin test data were available (33 of 73); this method detected 76% of culture-positive animals, although the specificity of the intradermal test was not determined in this study. When the DPP VetTB and skin test data were combined, the resulting sensitivity obtained in this sub-group of M. bovis-infected deer increased to 97%. Gross pathology identified TB compatible lesions (TBL) in 89% culture-confirmed fallow deer. The infected animals with visible lesions had significantly higher readings in the DPP VetTB, but not in the bPPD ELISA. Only high levels of haemolysis decreased antibody test sensitivity and this effect was more evident for the bPPD ELISA. The results allowed inferring a number of management recommendations for rapid detection of MTC infection in live fallow deer and in surveys on hunter-harvested cervids.

Computed tomographic examination of South American sea lions (Otaria flavescens) with suspected Mycobacterium pinnipedii infection.

Ten South American sea lions (Otaria flavescens) were presented for clinical evaluation and diagnosis of tuberculosis following known exposure to Mycobacterium pinnipedii. CT was used to determine whether foci of calcification in mediastinal lymph nodes, typically associated with pinniped tuberculosis, could be detected and whether CT was a useful diagnostic modality, in conjunction with other tests, for the diagnosis of tuberculosis in this species. Blood was collected from the caudal gluteal vein of each animal for serological testing using commercially available serological tests (ElephantTB STAT-PAK and DPP Vet; Chembio Diagnostic Systems) and a multiantigen print immunoassay (MAPIA), carried out at Chembio to verify the in-house kits. In four of nine animals that underwent CT scanning, lesions consistent with pinniped tuberculosis were apparent and these were confirmed at subsequent postmortem examination. The five remaining animals did not show any abnormalities on CT, with three being negative on serological tests, which were considered to be normal and potentially used as reference images for healthy sea lions. One animal could not be CT scanned due to its large size and weight (510 kg).

Proceedings from the Ice Hockey Summit on concussion: a call to action.

The objective of this proceedings is to integrate the concussion in sport literature and sport science research on safety in ice hockey to develop an action plan to reduce the risk, incidence, severity, and consequences of concussion in ice hockey. A rationale paper outlining a collaborative action plan to address concussions in hockey was posted for review two months prior to the Ice Hockey Summit: Action on Concussion. Focused presentations devoted specifically to concussion in ice hockey were presented during the Summit and breakout sessions were used to develop strategies to reduce concussion in the sport. This proceedings and a detailed scientific review (a matrix of solutions) were written to disseminate the evidence-based information and resulting concussion reduction strategies. The manuscripts were reviewed by the authors, advisors and contributors to ensure that the opinions and recommendations reflect the current level of knowledge on concussion in hockey. Six components of a potential solution were articulated in the Rationale paper and became the topics for breakout groups that followed the professional, scientific lectures. Topics that formed the core of the action plan were: metrics and databases; recognizing, managing and return to play; hockey equipment and ice arenas; prevention and education; rules and regulations; and expedient communication of the outcomes. The attendees in breakout sessions identified action items for each section. The most highly ranked action items were brought to a vote in the open assembly, using an Audience Response System (ARS). The strategic planning process was conducted to assess: Where are we at?; Where must we get to?; and What strategies are necessary to make progress on the prioritized action items? Three prioritized action items for each component of the solution and the percentage of the votes received are listed in the body of this proceedings.

Examination of the construct validity of ImPACT™ computerized test, traditional, and experimental neuropsychological measures.

Although computerized neuropsychological screening is becoming a standard for sports concussion identification and management, convergent validity studies are limited. Such studies are important for several reasons: reference to established measures is needed to establish validity; examination of the computerized battery relative to a more traditional comprehensive battery will help understand the strengths and limitations of the computer battery; and such an examination will help inform the output of the computerized battery. We compared scores on the ImPACT™ battery to a comprehensive battery of traditional neuropsychological measures and several experimental measures used in the assessment of sports-related concussion in 54 healthy male athletes. Convergent validity was demonstrated for four of the five ImPACT™ domain scores. Two cognitive domains often compromised as a result of mild TBI were not directly identified by the ImPACT™ battery: sustained attention and auditory working memory. Affective symptoms correlated with performance on measures of attention and working memory. In this healthy sample the correlations between the domains covered by ImPACT™ and the neuropsychological battery supports ImPACT™ as a useful screening tool for assessing many of the cognitive factors related to mTBI. However, the data suggest other sources of data need to be considered when identifying and managing concussions.

Ante-mortem testing wild fallow deer for bovine tuberculosis.

This study aimed to maximize the sensitivity of bovine tuberculosis detection in living wild fallow deer (Dama dama) under field conditions. We evaluated the rapid test (RT; CervidTB STAT-PAK Assay, Chembio Diagnostic Systems, Inc., USA) in comparison with the comparative cervical skin test (CCT). A total of 134 fallow deer were captured between January and March 2008. At time 0, 0.1 ml of avian purified protein derivative (avian PPD; Cooper-Zeltia, Spain), 0.1 ml bovine PPD (Cooper-Zeltia, Spain), 0.1 ml negative control PBS and 0.1 ml of a positive control (the mitogen phytohaemagglutinin, PHA; containing 250 mg PHA, diluted in PBS) were injected intradermally at four shaved sites in the neck. The skin fold thickness at each injection site was measured at time 0 and 72 h (3 repeats each time). Animals with a skin test response of 2mm or more at the bovine PPD injection site and animals with any visible reactivity in the RT were necropsied and tissues submitted for culture and for histopathology. A total of 36 fallow deer were considered reactors to bovine PPD or to the RT (apparent prevalence 27%). Regarding both bovine PPD reactivity and the skin fold increase at the PHA injection site, we found significant effects of age and sex by age interaction. Adult males had the largest responses. Mycobacterium bovis was isolated from lymphoid tissues of 21 fallow deer. Skin test sensitivity, as compared to M. bovis culture confirmed deer, was 80.1% (17/21). But, the CCT alone would have missed 4 of 21 culture confirmed animals. RT sensitivity, based on culture confirmed deer, was also 80.1% (17/21). Similarly, the RT alone would have missed another 4 of 21 culture confirmed deer. However, combining the CCT and the RT allowed for detecting all 21 culture positive fallow deer. We conclude that the combined application of the RT and the skin testing can maximize the sensitivity of bTB detection in living fallow deer, thus facilitating control programs for wildlife disease surveillance.

Immune responses in cattle inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii.

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.

Tuberculosis caused by Mycobacterium microti in South American camelids.

BACKGROUND: Infection with Mycobacterium microti can cause chronic disease in animals and threaten human health through its zoonotic potential. OBJECTIVE: To describe clinical findings, diagnostic investigations, necropsy, and epidemiology results in South American camelids (SAC) infected with M. microti, member of the Mycobacterium tuberculosis complex. ANIMALS: Eleven SAC with tuberculous lesions. METHODS: Description of 10 llamas and 1 alpaca, aged 4-18 years, from 6 herds with a history of wasting and weakness admitted to the Vetsuisse-Faculty of Berne over 8 years. RESULTS: Clinical signs included weight loss, recumbency, and anorexia in late stages of the disease. Respiratory problems were seen in 6 animals of 11. No consistent hematologic abnormalities were identified. Suspect animals were examined in detail by abdominal ultrasonography and thoracic radiology. Abnormal findings such as enlarged mediastinal, mesenteric, or hepatic lymph nodes were seen only in animals with advanced disease. Single comparative intradermal tuberculin test with bovine protein purified derivate (PPD) and avian PPD was negative in all animals. At necropsy, typical tuberculous lesions were found, and confirmed by bacteriological smear and culture, molecular methods, or both. CONCLUSIONS AND CLINICAL IMPORTANCE: Infection caused by M. microti should be considered a differential diagnosis in chronic debilitating disease with or without respiratory signs in SAC. Antemortem confirmation of the diagnosis remains challenging at any stage of infection. Because cases of M. microti infection have been reported in immunocompromized human patients, the zoonotic potential of the organism should be kept in mind when dealing with this disease in SAC.

Humoral immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination and experimental challenge with M. bovis.

Monitoring of the kinetics of production of serum antibodies to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and the efficacy of intervention strategies in several species. The humoral immune responses to multiple M. bovis antigens by white-tailed deer vaccinated with BCG orally via a lipid-formulated bait (n=5), orally in liquid form (n=5), and subcutaneously (n=6) were evaluated over time after vaccination and after experimental challenge with virulent M. bovis and were compared to the responses by unvaccinated deer (n=6). Antibody responses were evaluated by using a rapid test (RT), a multiantigen print immunoassay (MAPIA), a lipoarabinomannan enzyme-linked immunosorbent assay (LAM-ELISA), and immunoblotting to whole-cell sonicate and recombinant antigen MPB83. MAPIA and RT detected minimal to no antibody responses over those at the baseline to multiple M. bovis antigens in vaccinated white-tailed deer after challenge. This was in contrast to the presence of more readily detectable antibody responses in nonvaccinated deer with more advanced disease. The LAM-ELISA results indicated an overall decrease in the level of production of detectable antibodies against lipoarabinomannan-enriched mycobacterial antigen in vaccinated animals compared to that in nonvaccinated animals after challenge. Immunoblot data were inconsistent but did suggest the occurrence of unique antibody responses by certain vaccinated groups to Ag85 and HSP70. These findings support further research toward the improvement and potential use of antibody-based assays, such as MAPIA, RT, and LAM-ELISA, as tools for the antemortem assessment of disease progression in white-tailed deer in both experimental and field vaccine trials.

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife.

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.

Mycobacterium pinnipedii: transmission from South American sea lion (Otaria byronia) to Bactrian camel (Camelus bactrianus bactrianus) and Malayan tapirs (Tapirus indicus).

Tuberculosis infections caused by Mycobacterium (M.) pinnipedii in a South American sea lion, Bactrian camel, and Malayan tapirs kept in two zoological gardens spanning a time period of 5 years are reported. The zoos were linked by the transfer of one tapir. Conventional bacteriological and molecular methods were applied to detect the pathogen. Spoligotyping and MIRU/VNTR-typing performed to assess the genetic similarity revealed identical molecular characteristics of the isolates from all animals involved. Anti-tuberculosis antibodies were detected using ELISA and a recently developed serological rapid test. The study shows that: (i) using molecular methods, the assessment of the genetic relationship of infectious agents helps to confirm the routes of infection, and that (ii) immunological tests may help to detect tuberculosis infections ante mortem more reliably and early. This would prevent the transfer of tuberculosis by asymptomatic animals.

Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti.

Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.

Tuberculosis outbreak in a dromedary racing herd and rapid serological detection of infected camels.

A recent outbreak of tuberculosis (TB) in a dromedary racing herd of 58 animals involved 3 infected animals. Disease was confirmed at necropsy by finding gross lesions from which Mycobacterium bovis (antelope type) was isolated. Sera collected from the camels in this herd were used to evaluate two new serological methods, Multiantigen Print Immunoassay (MAPIA) and rapid test (RT) developed using the lateral-flow technology, in comparison with the intradermal tuberculin tests. Antibodies were found in all three infected dromedaries by both RT and MAPIA, but not in the remaining 55 animals in the herd. With the limited number of animals tested in this study, the serological assays showed the potential for convenient, rapid, and accurate diagnosis of TB in live camels.

Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii.

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

Early antibody responses to experimental Mycobacterium bovis infection of cattle.

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.