Estimating Diagnostic Test Accuracies for Brachyspira hyodysenteriae Accounting for the Complexities of Population Structure in Food Animals

For swine dysentery, which is caused by Brachyspira hyodysenteriae infection and is an economically important disease in intensive pig production systems worldwide, a perfect or error-free diagnostic test ("gold standard") is not available. In the absence of a gold standard, Bayesian latent class modelling is a well-established methodology for robust diagnostic test evaluation. In contrast to risk factor studies in food animals, where adjustment for within group correlations is both usual and required for good statistical practice, diagnostic test evaluation studies rarely take such clustering aspects into account, which can result in misleading results. The aim of the present study was to estimate test accuracies of a PCR originally designed for use as a confirmatory test, displaying a high diagnostic specificity, and cultural examination for B. hyodysenteriae. This estimation was conducted based on results of 239 samples from 103 herds originating from routine diagnostic sampling. Using Bayesian latent class modelling comprising of a hierarchical beta-binomial approach (which allowed prevalence across individual herds to vary as herd level random effect), robust estimates for the sensitivities of PCR and culture, as well as for the specificity of PCR, were obtained. The estimated diagnostic sensitivity of PCR (95% CI) and culture were 73.2% (62.3; 82.9) and 88.6% (74.9; 99.3), respectively. The estimated specificity of the PCR was 96.2% (90.9; 99.8). For test evaluation studies, a Bayesian latent class approach is well suited for addressing the considerable complexities of population structure in food animals.

Ein nicht alltägliches türkisches Souvenir

Schlussfolgerungen für die Praxis Bei entsprechender Exposition in einem Endemiegebiet und typischer Klinik mit schmerzlosem, zentral nekrotischem, kutanen Ulkus sollte an einen kutanen Anthrax gedacht werden. Bei Einsendung einer Probe in das Primärlabor sollte unbedingt über die Verdachtsdiagnose informiert werden, damit die entsprechenden Biosicherheitsvorgaben eingehalten und eine Gefährdung von Personal und Umwelt ausgeschlossen werden können. Nach Abnahme eines Wundabstrichs bzw. einer Biopsie sollte unverzüglich eine antibiotische Therapie mit Ciprofloxacin oder Clindamycin für sieben Tage eingeleitet werden. Schon der Verdachtsfall ist in der Schweiz innerhalb von zwei Stunden meldepflichtig.

Diagnosis of diabetes insipidus observed in Swiss Duroc boars

Background Diabetes insipidus (DI) is a rare disease in humans and animals, which is caused by the lack of production, malfunction or dysfunction of the distal nephron to the antidiuretic effect of the antidiuretic hormone (ADH). Diagnosis requires a thorough medical history, clinical examination and further laboratory confirmation. This case report describes the appearance of DI in five Duroc boars in Switzerland. Case presentation Two purebred intact Duroc boars at the age of 8 months and 1.5 years, respectively, with a history of polyuric and polydipsic symptoms had been referred to the Swine Clinic in Berne. Based on the case history, the results of clinical examination and the analysis of blood and urine, a tentative diagnosis of DI was concluded. Finally, the diagnosis was confirmed by findings from a modified water deprivation test, macroscopic examinations and histopathology. Following the diagnosis, three genes known to be involved in inherited DI in humans were analyzed in order to explore a possible genetic background of the affected boars. Conclusion The etiology of DI in pigs is supposed to be the same as in humans, although this disease has never been described in pigs before. Thus, although occurring only on rare occasions, DI should be considered as a differential diagnosis in pigs with polyuria and polydipsia. It seems that a modified water deprivation test may be a helpful tool for confirming a diagnosis in pigs. Since hereditary forms of DI have been described in humans, the occurrence of DI in pigs should be considered in breeding programs although we were not able to identify a disease associated mutation.

Bacillus anthracis Diversity and Geographic Potential across Nigeria, Cameroon and Chad: Further Support of a Novel West African Lineage.

Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning.

Evaluation of a Commercial ELISA for the Detection of Antibodies to Sarcoptes scabiei in Wild Boar (Sus scrofa).

Sarcoptic mange occurs in free-ranging wild boar (Sus scrofa) but has been poorly described in this species. We evaluated the performance of a commercial indirect enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of sarcoptic mange in domestic swine when applied to wild boar sera. We tested 96 sera from wild boar in populations without mange history ("truly noninfected") collected in Switzerland between December 2012 and February 2014, and 141 sera from free-ranging wild boar presenting mange-like lesions, including 50 live animals captured and sampled multiple times in France between May and August 2006 and three cases submitted to necropsy in Switzerland between April 2010 and February 2014. Mite infestation was confirmed by skin scraping in 20 of them ("truly infected"). We defined sensitivity of the test as the proportion of truly infected that were found ELISA-positive, and specificity as the proportion of truly noninfected that were found negative. Sensitivity and specificity were 75% and 80%, respectively. Success of antibody detection increased with the chronicity of lesions, and seroconversion was documented in 19 of 27 wild boar sampled multiple times that were initially negative or doubtful. In conclusion, the evaluated ELISA has been successfully applied to wild boar sera. It appears to be unreliable for early detection in individual animals but may represent a useful tool for population surveys.

Capsule gene regulation in Bacillus anthracis and implications for virulence

The poly-D-glutamic acid capsule of Bacillus anthracis is considered essential for lethal anthrax disease. Yet investigations of capsule function have been limited primarily to attenuated B. anthracis strains lacking certain genetic elements. In work presented in this thesis, I constructed and characterized a genetically complete (pXO1 + pXO2+) B. anthracis strain (UT500) and isogenic mutants deleted for two previously identified capsule gene regulators, atxA and acpA, and a newly-identified regulator, acpB. Results of transcriptional analysis and microscopy revealed that atxA controls expression of the first gene of the capsule biosynthesis operon, capB, via positive transcriptional regulation of acpA and acpB. acpA and acpB appear to be partial functional homologs. Deletion of either gene alone has little effect on capsule synthesis. However, a mutant deleted for both acpA and acpB is noncapsulated. Thus, in contrast to previously published models, my results suggest that atxA is the master regulator of cap gene expression in a genetically complete strain. A detailed transcriptional analysis of capB and the regulatory genes was performed to establish the effects of the regulators and CO2/bicarbonate on specific mRNAs of target genes. CO2/bicarbonate is a well-established signal for B. anthracis capsule synthesis in culture. Taqman RT-PCR results indicated that growth in the presence of elevated CO2 greatly increased expression of acpA, acpB and capB but not atxA. 5′ end mapping of capB and acpA revealed atxA-regulated and atxA-independent transcriptional start sites for both genes. All atxA-regulated start sites were also CO2-regulated. A single atxA-independent start site was identified 5 ′ of acpB. However, RT-PCR analysis indicated that capD and acpB are co-transcribed. Thus, it is likely that atxA-mediated control of acpB expression occurs via transcriptional activation of the atxA-regulated start sites of capB. Finally, I examined the contribution of the B. anthracis capsule to virulence. The virulence of the parent strain, mutants deleted for the capsule biosynthesis genes ( capBCAD), and mutants missing the capsule regulator genes was compared using a mouse model for inhalation anthrax. The data indicate that in this model, capsule is essential for virulence. Mice survived infection with the noncapsulated capBCAD and acpA acpB mutants. These mutants initiated germination in the lung, but did not disseminate to the spleen. The acpA mutant had an LD50 value similar to the parent strain and was able to disseminate and cause lethal infection. Unexpectedly, the acpB mutant had a higher LD 50 and a reduced ability to disseminate. During in vitro culture, the acpB single mutant produces capsule and toxin similar to the parent strain. It is likely that acpB regulates the expression of downstream genes that contribute to the virulence of B. anthracis. ^

Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Introduction to public health preparedness for disaster and emergency response

The unprecedented attacks of September 11, 2001, and the subsequent anthrax-related events thrust our nation's often forgotten public health system into the forefront of public attention. A strong public health system with a well-prepared workforce plays a critical role in preparing for and responding to the threat of bioterrorism and other disasters and emergencies. Technical expertise is critical as is a basic awareness and understanding of core public health competencies especially as they relate to disaster and emergency response is also imperative for a public health agency to function as a vital Emergency Response team member. Ideally this training should begin at the Public Health graduate level so as to provide the baseline core tools to be able to function as a vital team member when they are practicing out in the real world. Online learning is an efficient and effective method for providing public health education to in a flexible format to meet the needs of busy student-professions. This Public Health Disaster Preparedness online course developed during an Emergency Response state program practicum is a practical and proficient approach to accomplish this endeavor. ^

Functional Characterization of AtxA, the Bacillus anthracis Virulence Regulator

Coordinated expression of virulence genes in Bacillus anthracis occurs via a multi-faceted signal transduction pathway that is dependent upon the AtxA protein. Intricate control of atxA gene transcription and AtxA protein function have become apparent from studies of AtxA-induced synthesis of the anthrax toxin proteins and the poly-D-glutamic acid capsule, two factors with important roles in B. anthracis pathogenesis. The amino-terminal region of the AtxA protein contains winged-helix (WH) and helix-turn-helix (HTH) motifs, structural features associated with DNA-binding. Using filter binding assays, I determined that AtxA interacted non-specifically at a low nanomolar affinity with a target promoter (Plef) and AtxA-independent promoters. AtxA also contains motifs associated with phosphoenolpyruvate: sugar phosphotransferase system (PTS) regulation. These PTS-regulated domains, PRD1 and PRD2, are within the central amino acid sequence. Specific histidines in the PRDs serve as sites of phosphorylation (H199 and H379). Phosphorylation of H199 increases AtxA activity; whereas, H379 phosphorylation decreases AtxA function. For my dissertation, I hypothesized that AtxA binds target promoters to activate transcription and that DNA-binding activity is regulated via structural changes within the PRDs and a carboxy-terminal EIIB-like motif that are induced by phosphorylation and ligand binding. I determined that AtxA has one large protease-inaccessible domain containing the PRDs and the carboxy-terminal end of the protein. These results suggest that AtxA has a domain that is distinct from the putative DNA-binding region of the protein. My data indicate that AtxA activity is associated with AtxA multimerization. Oligomeric AtxA was detected when co-affinity purification, non-denaturing gel electrophoresis, and bis(maleimido)hexane (BMH) cross-linking techniques were employed. I exploited the specificity of BMH for cysteine residues to show that AtxA was cross-linked at C402, implicating the carboxy-terminal EIIB-like region in protein-protein interactions. In addition, higher amounts of the cross-linked dimeric form of AtxA were observed when cells were cultured in conditions that promote toxin gene expression. Based on the results, I propose that AtxA multimerization requires the EIIB-like motif and multimerization of AtxA positively impacts function. I investigated the role of the PTS in the function of AtxA and the impact of phosphomimetic residues on AtxA multimerization. B. anthracis Enzyme I (EI) and HPr did not facilitate phosphorylation of AtxA in vitro. Moreover, markerless deletion of ptsHI in B. anthracis did not perturb AtxA function. Taken together, these results suggest that proteins other than the PTS phosphorylate AtxA. Point mutations mimicking phosphohistidine (H to D) and non-phosphorylated histidine (H to A) were tested for an impact on AtxA activity and multimerization. AtxA H199D, AtxA H199A, and AtxA H379A displayed multimerization phenotypes similar to that of the native protein, whereas AtxA H379D was not susceptible to BMH cross-linking or co-affinity purification with AtxA-His. These data suggest that phosphorylation of H379 may decrease AtxA activity by preventing AtxA multimerization. Overall, my data support the following model of AtxA function. AtxA binds to target gene promoters in an oligomeric state. AtxA activity is increased in response to the host-related signal bicarbonate/CO2 because this signal enhances AtxA multimerization. In contrast, AtxA activity is decreased by phosphorylation at H379 because multimerization is inhibited. Future studies will address the interplay between bicarbonate/CO2 signaling and phosphorylation on AtxA function.

Control of the master virulence regulatory gene atxA in Bacillus anthracis

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact atxA transcript levels, and the timing and steady state level of atxA expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the atxA promoter. The AbrB-binding site has been described, but additional cis-acting control sequences have not been defined. Using transcriptional lacZ fusions, electrophoretic mobility shift assays, and Western blot analysis, the cis-acting elements and trans-acting factors involved in regulation of atxA in B. anthracis strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a trans-acting protein(s) other than AbrB negatively impacts atxA transcription when it binds specifically to a 9-bp palindrome within atxA promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the trans-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner. The identity of the trans-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the trans-acting repressor protein(s) indirectly controls B. anthracis development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both atxA- and pXO2-61-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper B. anthracis development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on atxA expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of atxA. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an abrB-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an abrB-null mutant. In addition, the abrB-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the atxA promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the abrB-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an abrB-null mutant. Overall, this work highlights the complex regulatory network that governs expression of atxA and provides an additional role for AtxA in B. anthracis development.

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

Circadian rhythm profiles in porcine ICU subjects

Purpose: To explore the natural trajectory of circadian rhythms of sedation requirement, core body temperature (CBT), pulmonary mechanics (PM), and gas exchange (GE) in mechanically ventilated swine, as these variables affect the duration of mechanical ventilation. ^ Design: A secondary analysis to describe and compare circadian rhythms of study variables in swine mechanically ventilated for ≤ 7 days. ^ Setting: Porcine Intensive Care Unit (ICU).^ Sample: Six male swine. ^ Methods: Sedation requirements were recorded hourly and the CBT, PM and GE variables were sampled every 1 s – 1 min for ≤ 7 days. The data sets for each pig with > 5 days ICU length of stay were divided into one section representing the first 3 days and one section representing subsequent days. The Lomb periodogram was used to estimate the circadian time period for each variable, and cosinor analysis with the estimated time period to obtain amplitude and mesor. Circadian to ultradian bandpower ratio to assess rhythm quality and stability over time and goodness-of-fit index to describe biological significance of a rhythm were used. Together, these two parameters were used to define rhythm robustness over time. The masking effect of sedation as a potential confounder of the circadian rhythms of CBT, PM, and GE was explored, and circadian rhythm profiles of CBT of pigs in the ICU setting were compared with those of the same pigs in the ambulatory setting. ^ Results: All pigs had significant rhythms in CBT, respiratory rate, and peripheral oxygen saturation across ICU data sets. Healthier pigs had more robust rhythms of study variables over time. Sedation did not appear to mask the circadian rhythms of CBT, PM, and GE. The circadian rhythm of CBT was less robust in the ICU setting than in the ambulatory setting. ^ Conclusions: Individual subject observations provided preliminary evidence that robustness of rhythms varies with subject acuity. Comparison of profiles of circadian rhythms among ICU subjects with similar acuity and disease processes is warranted to determine if the profiles in the present study are reproducible. Identification of consistent patterns may provide insight into subject morbidity and timing of such therapeutic interventions as weaning from mechanical ventilation. ^

ATXA -dependent gene expression in Bacillus anthracis

The Bacillus anthracis toxin genes, cya, lef , and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. I determined that several phenotypes are associated with the atxA gene. In addition to being toxin-deficient, an atxA -null mutant grows poorly on minimal media and sporulates early compared to the parent strain. Furthermore, an atxA-null mutant has an altered 2-D gel protein profile. I used a genetic approach to find additional atxA-regulated genes. Random transcriptional lacZ fusions were generated in B. anthracis using transposon Tn 917-LTV3. Transposon-insertion libraries were screened for mutants expressing increased β-galactosidase activity in 5% CO2. Introduction of an atxA-null mutation in these mutants revealed that 79% of the CO2-regulated fusions were also atxA-dependent. DNA sequence analysis of transposon insertion sites in mutants carrying CO 2/atxA-regulated fusions revealed ten mutants harboring transposon insertions in loci distinct from the toxin genes. The majority of the tcr (toxin co-regulated) loci mapped within the pXO1 pathogenicity island. These results indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins. ^ Characterization of one tcr locus revealed a new regulatory gene, pagR. The pagR gene (300 nt) is located downstream of pag. pagR is cotranscribed with pag and is responsible for autogenous control of the operon. pagR also represses expression of cya and lef. Repression of toxin gene expression by pagR may be mediated by atxA. The steady state level of atxA mRNA is increased in a pagR mutant. Recombinant PagR protein purified from Escherichia coli did not specifically bind the promoter regions of pagA or atxA. An unidentified factor in B. anthracis crude extracts, however, was able to bind the atxA promoter in the absence of PagR or AtxA. These investigations increase our knowledge of virulence regulation in B. anthracis and ultimately will lead to a better understanding of anthrax disease. ^

Effects of Bedding on Pig Performance

More pigs are being produced in bedded systems such as hoop barns due to the increase in demand for niche pork. Most of the current swine nutrition information has been generated in confinement settings without bedding. As omnivores, pigs ingest some bedding. There are questions about the effects of the ingested bedding on growth and feed utilization by the modern market pig. More knowledge in this area will allow for more accurate feed formulation in bedded systems.