Administration of non-pathogenic isolates of Escherichia coli and Clostridium perfringens type A to piglets in a herd affected with a high incidence of neonatal diarrhoea.

A bacterial cocktail of living strains of Clostridium perfringens type A (CPA) without ß2-toxin gene and non-pathogenic Escherichia coli was administered orally to newborn piglets before first colostrum intake and on 2 consecutive days on a farm with a high incidence of diarrhoea and antibiotic treatment in suckling piglets associated with E. coli and CPA. This clinical field study was driven by the hypothetic principle of competitive exclusion of pathogenic bacteria due to prior colonization of the gut mucosal surface by non-pathogenic strains of the same bacterial species with the aim of preventing disease. Although CPA strains used in this study did not produce toxins in vitro, their lack of pathogenicity cannot be conclusively confirmed. The health status of the herd was impaired by a high incidence of postpartum dysgalactia syndrome in sows (70%) and a high incidence of neonatal diarrhoea caused by enterotoxigenic E. coli and CPA during the study. No obvious adverse effect of the bacterial treatment occurred. On average, more piglets were weaned in litters treated (P=0.009). Visual pathological alterations in the small intestinal wall were more frequent in dead piglets of the control group (P=0.004) and necrotizing enteritis was only found in that group. A higher average daily weight gain of piglets in the control group (P<0.001) may be due to an increased milk uptake due to less competition in the smaller litters. The bacterial cocktail was tested under field conditions for its potential to stabilize gut health status in suckling piglets before disease development due to colibacillosis and clostridial infections; however, the gut flora stabilizing effect of the bacterial cocktail was not clearly discernible in this study. Further basic research is needed to confirm the positive effects of the bacterial treatment used and to identify additional potential bacterial candidates for competitive exclusion.

[Computed tomography of the pig lung. An innovative approach to the definition of the pulmonary health status]. / Computertomographie der Lunge des Schweines. Ein innovativer Ansatz zur Definition des Lungengesundheitsstatus.

OBJECTIVE: In veterinary medicine computed tomography (CT) imaging has gained importance in recent years, especially for diagnostics in pets, but also during the course of experimental studies in animal models for human medicine. In this study the applicability of CT as an imaging method for the depiction of the porcine thorax and in particular of the pig lung was evaluated. MATERIAL AND METHODS: CT examinations were performed with 11 healthy pigs of two age groups. For evaluation, CT findings were related to clinical, radiological, macroscopical, microscopical, and microbiological findings. RESULTS: Clinically relevant anatomical structures were determined and recorded using transverse slices. In ventral recumbency, lung parenchyma density measurements at the levels of the second, fourth and seventh thoracic vertebrae resulted in significantly higher densities of the ventral in comparison to those of the dorsal lung quadrants. CONCLUSION AND CLINICAL RELEVANCE: Computed tomography is a valuable tool for the high-contrast depiction of the porcine lung without superposition. In future studies this CT reference base for unaltered pig lungs may facilitate the identification of anatomical structures within the porcine lung as well as the assessment of pathological lung alterations.

Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide-spot array.

Enzootic pneumonia (EP) in pigs caused by Mycoplasma hyopneumoniae is a highly prevalent, chronic respiratory disease, which causes considerable economic losses in the swine industry. Most herds are vaccinated, but classical bacterin vaccines do not prevent colonization and it is not possible to detect flourishing M. hyopneumoniae infections in vaccinated herds since commonly used commercial ELISAs cannot differentiate infected from vaccinated animals. To solve this problem, new immunogenic proteins, up-regulated or solely expressed during infection, need to be identified. For this purpose a peptide-spot array was constructed which presents 105 potential linear B-cell epitopes identified by in silico analysis in 35 putative lipoproteins encoded on the genome of M. hyopneumoniae type strain 232. Subjecting this array to immunoblotting using porcine convalescent serum revealed a single strongly immunoreactive epitope on the Mhp366 protein which did not react with serum from bacterin-immunized pigs. In addition, it was not possible to detect Mhp366 in total cell lysates of in vitro grown M. hyopneumoniae strains, using a polyclonal rabbit serum raised against a recombinant GST-Mhp366 fusion protein. To investigate the possibility of using an Mhp366-based ELISA in the field for differentiating vaccinated herds with and without a flourishing infection it was shown that (i) homologues of the corresponding mhp366 gene were present in all 17 M. hyopneumoniae strains and porcine lung samples tested from different geographic origins and (ii) an ELISA based on epitope-specific synthetic peptides as solid phase antigen allowed a classification of field samples. Therefore, Mhp366 might be the first antigen identified which facilitates the detection of flourishing M. hyopneumoniae infections even in vaccinated herds.

Characterization of the porcine transferrin gene (TF) and its association with disease severity following an experimental Actinobacillus pleuropneumoniae infection.

Transferrin (TF)-mediated provision of iron is essential for a productive infection by many bacterial pathogens, and iron-depletion of TF is a first line defence against bacterial infections. Therefore, the transferrin (TF) gene can be considered a candidate gene for disease resistance. We obtained the complete DNA sequence of the porcine TF gene, which spans 40 kb and contains 17 exons. We identified polymorphisms on a panel of 10 different pig breeds. Comparative intra- and interbreed sequence analysis revealed 62 polymorphisms in the TF gene including one microsatellite. Ten polymorphisms were located in the coding sequence of the TF gene. Four SNPs (c.902A>T, c.980G>A, c.1417A>G, c.1810A>C) were predicted to cause amino acid exchanges (p.Lys301Ile, p.Arg327Lys, p.Lys473Glu, p.Asn604His). We performed association analyses using six selected TF markers and 116 pigs experimentally infected with Actinobacillus pleuropneumoniae serotype 7. The analysis showed breed-specific TF allele frequencies. In German Landrace, we found evidence for a possible association of the severity of A. pleuropneumoniae infection with TF genotypes.

[Comparison of the diagnostic significance of clinical, radiographic and ultrasonographic results after an experimental aerosol infection of pigs with Actinobacillus pleuropneumoniae]. / Vergleich der diagnostischen Aussagekraft klinischer, röntgenologischer und sonographischer Befunde bei der experimentellen Infektion des Schweines mit Actinobacillus pleuropneumoniae.

Scoring schemes for clinical, ultrasonographic and radiographic findings in pigs were developed based upon a standardized animal model for Actinobacillus pleuropneumoniae infection.The results of these methods were compared to each other as well as with the corresponding pathomorphological findings during necropsy. Altogether 69 pigs of different breeding lines (Hampshire, Pietrain and German Landrace were examined. Positive correlations were found between the results of all three methods as well as with the necropsy scores (p <0.0001). Different pathomorphological findings were detected either by radiographic or by ultrasonographic examination dependent upon the type of lung tissue alterations: Alterations of the pleura as well as sequestration of lung tissue on the lung surface could be clearly identified during the ultrasonographic examination while deep tissue alterations with no contact to the lung surface could be detected reliably by radiographic examination. Both methods complement each other, and the application of a combined ultrasonographic and radiographic examination of the thorax allows a comprehensive inspection of the lung condition. Particularly during the acute phase of the disease the extent of lung tissue damage can be estimated more precisely than by clinical examination alone.

Agreement between ELISA and complement fixation test used for diagnosing of paratuberculosis in goats.

An ELISA with a lipoarabinomannan as an antigen, developed for diagnosis of bovine paratuberculosis, has been adapted for use in goats, and compared with complement fixation test. Kappa value of 0.62 indicated good agreement between CFT and the adapted ELISA and proved that the investigated ELISA may be helpful in diagnosis of Mycobacterium avium subsp. paratuberculosis infection in goats. The ELISA has been used to screen a randomly selected representative sample of Polish breeding goat population (21.78% of herds, 21.33% of goats). It has been demonstrated that only 2.42% of animals coming from 15.79% of herds were seropositive. Within-herd seroprevalence varied from 1.69% to 38.10%. Most of the infected animals (67.07%) were 3- 4-years-old. No seropositive cases were found in group up to 1-year-old animals.

[Functional genome analysis investigating resistance to respiratory tract disease in a porcine Actinobacillus pleuropneumoniae infection model]. / Funktionelle Genomanalyse zur Resistenz gegen Atemwegsinfektionen am Beispiel der Actinobacillus-Pleuropneumonie des Schweins.

Here we present the work of the multidisciplinary consortium IRAS (Development of Genetic Markers for Immune Defence and Resistance in the Porcine Respiratory Tract) which includes different commercial and research institutions and was formed as a response to the call "Functional Genome Analysis in the Animal Organism (FUGATO)" by the German Ministry of Education and Research. IRAS started work in the fall of 2005 and--using the experimental infection of pigs with Actinobacillus pleuropneumoniae as model pathogen--aims at i) characterizing the course of infection by clinical as well as advanced laboratory tools (phenotypic-genetic approach) and ii) defining the diversity and distribution of allels known to be associated with immune defence in mouse and man (homolog-genetic approach). The intention is to identify genetic markers for increased resistance to infection thereby providing additional tools for the estimation of breeding values to the pig industry.

[Composting of poultry carcasses as an alternative method for disposal in case of an outbreak of an epizootic disease: first results]. / Kompostierung von Geflügeltierkörpern als alternative Beseitigungsmöglichkeit im Tierseuchenkrisenfall: erste Ergebnisse.

Composting of poultry carcasses represents an alternative method for disposal in case of an outbreak of an epizootic disease. Two composting experiments, each with a different construction of the compost pile, were carried out in a stable. In the first experiment two layers of turkey carcasses were formed. This compost pile covered with straw was directly built on the ground. In the second experiment no layers of carcasses were formed, and it was assembled on straw bales covered with plastic foil. One part of this compost pile was covered with straw, the other one was additionally covered with plastic foil. In the first experiment in the upper layers of the compost pile temperatures of up to 54.9 degrees C were reached and the decomposition of carcasses was very advanced with no soft tissues remaining after 30 days. In contrast temperatures of only 45.2 degrees C were reached in the lower layers and decomposition was far less advanced. This difference in decomposition was most likely caused by the temperature difference observed. In the second experiment the near complete decomposition seen in the upper layers of the compost pile at the first trial, was not achieved. Decomposition was more advanced in the straw covered part of this compost pile than in the part covered with straw and plastic foil. On the other hand, higher temperatures of up to 48.4 degrees C were measured in the lower layers of this compost pile most likely as a result of the increased heat insulation in particular to the ground.

Comparison of three common methods of lung lavage in healthy pigs.

Bronchoscopic, endotracheal and transtracheal lung lavage were evaluated in 38 healthy pigs taken from a nucleus herd in a good state of health with respect to their applicability in practice and the traceability of bacteria, cellular parameters and the antimicrobial peptide PR-39 in the respective lavage fluid samples. The total cell count, qualitative morphological cellular characteristics as well as PR-39 could be determined in all lavage fluid samples, while quantitative cell differentiation was not possible in endotracheal lavage samples. The comparison of the three methods resulted in a higher proportion of polymorphonuclear neutrophil granulocytes (PMNs) and higher concentrations of PR-39 in transtracheal samples. For this reason different valuation standards with respect to PMNs and PR-39 concentrations are presupposed for transtracheal lavage samples. The occurrence of pavement epithelial cells as well as the number of contaminating bacterial species per sample was the lowest in transtracheal lavage. Mycoplasma hyopneumoniae polymerase chain reaction appeared to have the highest diagnostic sensitivity in combination with bronchoscopic lavage. In conclusion, bronchoscopic and transtracheal lavage were considered to be more appropriate for bacteriological and cytological diagnostics than endotracheal lavage.

Respiratory disease markers in porcine bronchoalveolar lavage fluid.

In bronchoalveolar lavage fluid (BALF) of pigs originating from different herds bacteria, cells and the antibacterial peptide PR-39 were examined to gain information about the lung health status. In a high health nucleus herd 56% and in low health herds 20-100% of the examined pigs were found positive for potentially pathogenic bacteria. Based on these findings, a novel definition for bacterial respiratory tract disease was established using an 8% cut-off for the relative number of neutrophils in bronchoscopic and a 40% cut-off in transtracheal BALF in combination with the occurrence of potentially pathogenic microorganisms. The antibacterial peptide PR-39 was highly correlated to this definition of respiratory disease. An assessment of the bacteriological respiratory health status appears to be possibly based on the determination of PR-39 concentrations in BALF using different cut-off values according to the lavage method (2.5 nM for bronchoscopic and 5 nM for transtracheal BALF).

[Observations on the control and eradication of paratuberculosis in dairy herds]. / Uberlegungen zur Kontrolle und Bekämpfung der Paratuberkulose in Milchviehbeständen.

Johne's disease is widely seen in dairy herds in Germany. Estimates based primarily on epidemiological surveys in neighbouring states assume that 5 to 15 % of German herds are infected. In the past three years several authors have reported that the causative agent of Johne's disease, Mycobacterium avium subspecies paratuberculosis (MAP), is found ubiquitously in the environment and can be isolated from a number of different animals, including non ruminants. These results imply that MAP should be considered an environmental pathogen. Based on this assumption a concept for control and eradication of Johne's disease is presented aiming at minimizing the future spread of disease and reducing environmental contamination with the pathogen at low costs. The concept includes the classification of herds based on an bulk milk ELISA followed by a robot-compatible bulk milk PCR in ELISA-positive herds only. Due to the comparatively low costs combined with the high specificity of the approach a detection of heavily infected herds ("tip of the iceberg") all over the country would be possible; based on the eradication of strong shedders in these herds the input of MAP into the environment would be reduced considerably.

[Considerations concerning diagnostic certainties and cut-off values for a bulk milk ELISA for Mycobacterium avium ssp. paratuberculosis]. / Uberlegungen zu diagnostischen Sicherheiten und Cut-Off-Werten für einen Sammelmilch-ELISA auf Paratuberkulose.

Serological tests for the examination of individual samples from single animals are evaluated based on their ability to detect true positives above a defined threshold value. If results are obtained not from an individual but from a bulk sample this concept usually is adopted such that the threshold is set to allow the detection of a single positive sample within the pool. In conjunction with the development of a diagnostic paratuberculosis ELISA for the examination of bulk milk samples it is discussed which interpolations of this concept are justified when defining the true status of a herd based on the test parameters and the seroprevalence within the herd. Here, bulk milk from up to 50 animals each and the corresponding individual samples of 4241 dairy cows from 28 herds in the state of Brandenburg are investigated, and results are subjected to different evaluation approaches. Based on epidemiological considerations and test parameters a "critical prevalence" is defined which then serves as basis for the deduction of a cut-off value to be used for bulk milk samples. Finally, the practical relevance of this approach is demonstrated by suggesting an initial scheme for paratuberculosis classification of dairy herds with respect to possible control measures.

[Establishment and evaluation of an ELISA for the detection of antibodies in milk against Mycobacterium avium subspecies paratuberculosis]. / Etablierung und Evaluation eines ELISA zum Nachweis von Antikörpern gegen Mycobacterium avium subspecies paratuberculosis in Milch.

Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) is the etiological agent of paratuberculosis (Johne's Disease), a chronic granulomatous enteritis of ruminants occurring worldwide with increasing frequency and leading to growing economic losses. Continuous surveillance of dairy farms would be advisable, particularly with respect to the increasing economic importance of paratuberculosis and the high tenacity of the pathogen, which can persist in the environment for many months. So far, such measures have not been taken as the cost-intensive collection of serum samples would have been required. Based on these considerations, it was the aim of this study to evaluate an economically viable diagnostic method for antibody detection using milk samples. This objective was reached by establishing a milk-ELISA. A commercially available test (Svanovir-ELISA by Svanova, Sweden) was chosen, because this ELISA has an excellent specificity with respect to cultural examination of the ileocaecal lymph node ("Gold-Standard"). The Svanovir-ELISA could be successfully adapted for testing milk for antibodies against M. paratuberculosis. The milk is skimmed by centrifugation and is diluted 1:10 for testing. The inter-assay-variation was 17%. A comparative antibody analysis done in parallel with milk and serum samples from 601 dairy cows using the Svanovir-ELISA showed a significant correlation between the results obtained with both methods. The optimal "cut-off" for the milk-ELISA of 46 EUMS (> 46 EUMS = positive) resulting in a specificity of 94.6% and a sensitivity of 60.9% was confirmed by receiver-operator characteristics (ROC) analysis. In the meantime the Svanovir-ELISA has been licensed for use with milk samples in Germany.

[Paratuberculosis: the pathogen and routes of infection]. / Paratuberkulose: Erreger und Ubertragungswege.

Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is a chronic and incurable enteritis of ruminants with economic importance worldwide. The infectious agent is an acid-fast rod defined solely based on its mycobactin-dependent growth in vitro and the presence of insertion element IS900. The bacterium, which is difficult to culture primarily due to its extremely slow growth, occurs not only in cattle but also in other ruminant. In addition, it has been isolated from non-ruminant species. Despite its wide spectrum of potential hosts the contact between adult cattle and calves is the predominant route of infection within a herd as well as among herds. To interrupt this route of infection hygienic measures, primarily for the housing and feeding of calves, as well as diagnostic measures prior to trading of cattle are urgently required.

[Modelling of prevalence development in a paratuberculosis control program in a dairy herd]. / Modellierung der Prävalenzentwicklung bei einer Paratuberkulosesanierung in einem Milchviehbestand.

A modelling approach to calculate the success of a paratuberculosis control programme in dairy herds is presented. The essential parameters of the model are the prevalence at the beginning of the programme, diagnostic sensitivity and specificity of the tests used, discipline in culling test-positive animals, turnover in the herd, percentage of replacement with own stock and paratuberculosis prevalence in animals bought into the herd from outside, and a general hygiene-based factor. Diagnostic measures and time schedule used in the modelling approach are given by the paratuberculosis-control-programme of the local board for infectious disease control in food animals in the state of Lower Saxony. It was found by the model-calculations that in case of a high initial prevalence the anticipated six-year duration of the control programme is justified in order to ensure a lasting improvement of herd health. If hygienic measures are strictly obeyed and all test positive animals are culled a clear reduction on paratuberculosis prevalence can be achieved within the first year. According to the model in the second and third year the prevalence will increase again despite ongoing diagnostic measures in order to decrease again continuously with the beginning of the fourth year. Given an initial prevalence of 10%, 20% or 30% the prevalence after six years is calculated to be at 3%, 5% or 8% when all measures are followed as given in the control programme. The presented programme seems to be appropriate to predict prevalence development in paratuberculosis infected dairy herds if the herds are managed according to the guidelines of the "Tierseuchenkasse Niedersachsen", the local board for infectious disease control in food animals in the state of Lower Saxony, Germany. It becomes apparent that within six years a high decrease of the prevalence in the herds, but not a complete eradication of disease can be achieved by consistently complying with the rules given in these guidelines.

[Eradication of paratuberculosis in dairy herds: determination of the initial herd prevalence and modelling of prevalence development]. / Sanierung von Milchviehherden mit Paratuberkulose: Bestimmung der Anfangsprävalenz und Modellierung der Prävalenzentwicklung.

A prerequisite for the success of any eradication programme is the accurate determination of the initial herd prevalence as well as a herd-specific prediction of prevalence development. This prerequisite is not currently given for the eradication of paratuberculosis in infected herds. In the work presented a method to predict the initial paratuberculosis prevalence in infected herds is presented; it is based on the formation of two groups (ELISA-positive and negative) and the determination of generally applicable factors (positive predictive value [ppvn] of the ELISA and sensitivity of fecal culture in the ELISA-negative group [senF]). The ppvn of the ELISA was determined to be 0.6 based on the cultural examination of the ileocaecal lymph node of 64 ELISA-positive animals; the value for senF was set to be 0.64 based on the cultural examination of feces and ileocaecal lymph nodes of 40 ELISA-negative animals. To calculate the initial herd prevalence the number of animals in each of the groups was multiplied with the ppvn of the ELISA or with the reciprocal value of senF (1.5). The values were added and divided by the size of the herd. The practicability of this model was examined on nine herds with a total of 708 animals. The development of herd prevalence was modelled based on the examination scheme given in the paratuberculosis control programme of the "Niedersächsische Tierseuchenkasse" (local board for infectious disease control in food animals in the state of Lower Saxony, Germany). For the calculation a yearly turnover-rate of 33% with restocking from within the herd and a possibility of paratuberculosis diagnosis only in animals two years and older were assumed. The development of herd prevalence is exemplarily presented for four herds with different initial prevalences.

Isolation and diagnostic potential of ISMav2, a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis.

A novel Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) specific insertion sequence has been identified by representational difference analysis and designated as ISMav2. ISMav2 has no similarity to known mycobacterial IS elements but shows more than 50% identity to a non-composite transposon of Streptomyces coelicolor at the DNA and protein level. ISMav2 is present in at least three copies on the genome as assessed by Southern blot analysis and its potential value as a diagnostic tool was confirmed by PCR analyses on 79 M. paratuberculosis field isolates, nine M. avium ssp. avium isolates, and the reference strains of nine other mycobacterial species.

Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infections in goats.

An ELISA was developed for the diagnosis of Corynebacterium pseudotuberculosis infections in goats. A bacterial whole cell extract was used as solid-phase antigen, and serum from a culture-positive animal served as the internal reference standard. The well-to-well and assay-to-assay variations were determined to be 12.7 and 33.0%, respectively. A cut off value was determined by parallel testing of 142 sera (112 ELISA-positive, 30 ELISA-negative) in a Western blot, and the correlation between both tests was highly significant (K=0, 93). In addition, the reliability of the ELISA for the detection of infected herds was proven in a double blind study testing 910 sera from 74 goat herds.

Actinobacillus pleuropneumoniae iron transport and urease activity: effects on bacterial virulence and host immune response.

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. pleuropneumoniae single mutants (DeltaexbB and DeltaureC) and a newly constructed A. pleuropneumoniae double (DeltaureC DeltaexbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniae DeltaexbB mutant nor the double DeltaureC DeltaexbB 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. The A. pleuropneumoniae DeltaureC 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 DeltaureC mutant, 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 of A. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae DeltaureC mutant than in the BALF of those infected with the parent strain. These results imply that A. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniae parent strain.

Cloning and characterization of the gene coding for NADPH-sulfite reductase hemoprotein from Actinobacillus pleuropneumoniae and use of the protein product as a vaccine.

An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate and screened with serum produced in pigs that had been vaccinated with the anionic fraction of a sodium chloride extract. One E. coli transformant was isolated that produced a large amount of a protein with an electrophoretic mobility of about 67,000 molecular mass. The A. pleuropneumoniae-derived DNA encoding the protein was localized and characterized by restriction enzyme digestion and nucleotide sequence analysis which showed strong homology with the cysI gene of E. coli. One open reading frame of 1764 bases in length was detected which encoded a cysI protein from serotype 1, with a calculated molecular mass of 66,678. The DNA encoding the protein was labeled with radio-isotope and the homologous gene was isolated from an A. pleuropneumoniae serotype 5a library. The serotype 5a gene was the same length, but the cysI protein from serotype 5a was slightly larger (66,849) due to 8 substitutions in the amino acid sequence. Expression plasmids containing cysI from either serotype of A. pleuropneumoniae complemented an E. coli cysI mutant. Pigs vaccinated with the recombinant cysI were protected from challenge with A. pleuropneumoniae of the homologous serotype.