Gerald-F. Gerlach

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johnes disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohns disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission.Results164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900 - restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpsons index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property.ConclusionThe results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Methicillin-resistant Staphylococcus pseudintermedius among dogs admitted to a small animal hospital.

The aim of this study was to determine the frequency of carriage of methicillin-resistant Staphylococcus pseudintermedius (MRSP) among dogs admitted to a small animal hospital during a 17-month period, to characterize these isolates and to initially screen for possible factors associated with MRSP carriage. Swabs were taken from the nose/pharynx and the perineum as well as from wounds and skin infections (if present) of 814 dogs before entering the small animal hospital. A questionnaire for background information was completed. The staphylococcal species and methicillin resistance were confirmed pheno- and genotypically. The identified MRSP isolates were characterized by SCCmec typing, testing for susceptibility to 25 antimicrobial agents and SmaI-directed pulsed-field gel electrophoresis. A first screening for possible risk factors for MRSP carriage was performed by means of unifactorial contingency tables and CART analysis. Sixty (7.4%) dogs were positive for MRSP. All MRSP isolates harboured a type II-III SCCmec cassette and showed extended resistance to antimicrobial agents. Fifteen different SmaI patterns were observed. The major factors that clustered with MRSP carriage were former hospitalization and antibiotic treatment within the last six months before sampling. This study showed that only a minor part of the sampled dogs carried multi-resistant MRSP isolates. The facts that prior hospitalization and/or antibiotic therapy are potential associated factors for MRSP carriage underline the necessity of a judicious use of antibiotics in small animal medicine.

Development of a Peptide-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

ABSTRACT Based on phage display technology, a peptide-mediated magnetic separation technique was developed to facilitate selective isolation of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from bulk milk of naturally infected dairy herds. Nine recombinant bacteriophages binding to M. paratuberculosis were isolated from a commercial phage-peptide library encoding random 12-mer peptides. Nucleotide sequencing revealed the deduced sequence of the binding peptides. One peptide with the sequence NYVIHDVPRHPA, designated aMP3, was chemically synthesized with an amino-terminal biotin residue attached via an amino-hexacarbonic acid spacer molecule. Paramagnetic beads coated with the phage or with peptide aMP3 enabled the capture of M. paratuberculosis from milk. Combining this peptide-mediated magnetic separation with an ISMav2-based PCR allowed the detection of M. paratuberculosis in artificially spiked milk down to a concentration of 101 ml−1. Experiments using milk from naturally infected cows and bulk milk samples from infected herds demonstrated that the peptide-mediated capture PCR is sufficiently sensitive to detect single strong shedders in pooled milk samples. The method, for the first time, applies phage display technology to microbial diagnostics and has potential value as a completely standardizable tool for the routine M. paratuberculosis screening of bulk milk samples at acceptable costs.

Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis

ABSTRACT A novel PCR-based restriction fragment length polymorphism analysis of the Brachyspira nox gene was developed. The restriction patterns for Brachyspira hyodysenteriae, B. pilosicoli, B. intermedia, B. murdochii, and B. innocens were highly distinct with two restriction endonucleases only. The assay proved to be user-friendly and robust.

Actinobacillus pleuropneumoniae Iron Transport and Urease Activity: Effects on Bacterial Virulence and Host Immune Response

ABSTRACT Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniaesingle mutants (ΔexbB and ΔureC) and a newly constructed A. pleuropneumoniae double (ΔureC ΔexbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniaeΔexbB mutant nor the double ΔureCΔexbB mutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniae in the porcine respiratory tract. TheA. pleuropneumoniae ΔureC mutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniae ΔureCmutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number ofA. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae ΔureC mutant than in the BALF of those infected with the parent strain. These results imply thatA. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniaeparent strain.

Enzymes involved in anaerobic respiration appear to play a role in Actinobacillus pleuropneumoniae virulence.

ABSTRACT Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to survive on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated sequesters. It was previously demonstrated that a deletion of the anaerobic dimethyl sulfoxide reductase gene (dmsA) results in attenuation in acute disease (N. Baltes, S. Kyaw, I. Hennig-Pauka, and G. F. Gerlach, Infect. Immun. 71:6784-6792, 2003). In the present study, using two-dimensional polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry, we identified an aspartate ammonia-lyase (AspA) which is upregulated upon induction with bronchoalveolar lavage fluid (BALF). This enzyme is involved in the production of fumarate, an alternative electron acceptor under anaerobic conditions. The coding gene (aspA) was cloned and shown to be present in all A. pleuropneumoniae serotype reference strains. The transcriptional start point was identified downstream of a putative FNR binding motif, and BALF-dependent activation of aspA was confirmed by construction of an isogenic A. pleuropneumoniae mutant carrying a chromosomal aspA::luxAB transcriptional fusion. Two aspA deletion mutants, A. pleuropneumoniae ΔaspA and A. pleuropneumoniae ΔaspAΔdmsA, were constructed, both showing reduced growth under anaerobic conditions in vitro. Pigs challenged with either of the two mutants in an aerosol infection model showed a lower lung lesion score than that of the A. pleuropneumoniae wild-type (wt) controls. Pigs challenged with A. pleuropneumoniae ΔaspAΔdmsA had a significantly lower clinical score, and this mutant was rarely reisolated from unaltered lung tissue; in contrast, A. pleuropneumoniae ΔaspA and the A. pleuropneumoniae wt were consistently reisolated in high numbers. These results suggest that enzymes involved in anaerobic respiration are necessary for the pathogens ability to persist on respiratory tract epithelium and play an important role in A. pleuropneumoniae pathogenesis.

Development of an ELISA technique for serodiagnosis of bovine paratuberculosis

A local clinical Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) isolate was cultivated in large-scale culture. A procedure for efficient preparation of a lipoarabinomannan-containing antigen was developed and standardized; 25 mg of purified antigen were obtained per gram of bacterial wet weight. An ELISA based on this antigen was developed. Intra- and interassay variation were determined to be 20% and 27%, respectively. The ELISA was evaluated using the sera of groups of 39 non-randomly selected and 92 randomly selected animals from which ileocaecal lymph nodes were cultured to isolate viable M. paratuberculosis. Combining the results of both groups a positive predictive value of 74% and a negative predictive value of 99% were calculated. The ELISA was licensed by the German regulatory agencies and, in a direct comparison, was found to be superior to both other licensed assays.

Immunization of pigs to prevent disease in humans: construction and protective efficacy of a Salmonella enterica serovar Typhimurium live negative-marker vaccine.

ABSTRACT Zoonotic infections caused by Salmonella enterica serovar Typhimurium pose a constant threat to consumer health, with the pig being a particularly major source of multidrug-resistant isolates. Vaccination, as a promising approach to reduce colonization and shedding, has been scarcely used, as it interferes with current control programs relying on serology as a means of herd classification. In order to overcome this problem, we set out to develop a negative-marker vaccine allowing the differentiation of infected from vaccinated animals (DIVA). Applying an immunoproteomic approach with two-dimensional gel electrophoresis, Western blot, and quadrupole time-of-flight tandem mass spectrometry, we identified the OmpD protein as a suitable negative marker. Using allelic exchange, we generated an isogenic mutant of the licensed live vaccine strain Salmoporc and showed that virulence of Salmoporc and that of the mutant strain, SalmoporcΔompD, were indistinguishable in BALB/c mice. In a pig infection experiment including two oral immunizations with SalmoporcΔompD and challenge with a multiresistant S. enterica serovar Typhimurium DT104 clinical isolate, we confirmed the protective efficacy of SalmoporcΔompD in pigs, showing a significant reduction of both clinical symptoms and colonization of lymph nodes and intestinal tract. OmpD immunogenic epitopes were determined by peptide spot array analyses. Upon testing of several 9-mer peptides, each including an immunogenic epitope, one peptide (positions F100 to Y108) that facilitated the detection of infected animals independent of their vaccination status (DIVA function) was identified. The approach described overcomes the problems currently limiting the use of bacterial live vaccines and holds considerable potential for future developments in the field.

Identification of Dimethyl Sulfoxide Reductase in Actinobacillus pleuropneumoniae and Its Role in Infection

ABSTRACT Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in an A. pleuropneumoniae serotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein of Haemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinished A. pleuropneumoniae genome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of the dmsA ORF. In order to investigate the role of the A. pleuropneumoniae DmsA protein in virulence, an isogenic deletion mutant, A. pleuropneumoniae ΔdmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniae ΔdmsA was attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniae virulence.

Peptide aMptD-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Bulk Milk Samples

ABSTRACT A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 × 102 CFU ml−1 for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 × 102 CFU ml−1) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 × 103 and an association constant of 1.33 × 109. The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.