Competitive exclusion: A tool to combat extended-spectrum ß-lactamase-producing Escherichia coli strains in chickens.

Apart from effective hygiene regimes in poultry houses, measures to increase the resistance of birds against intestinal colonisation with EEC (= extended-spectrum ß-lactamases [ESBL] and AmpC-type [AmpC] beta-lactamses producing Escherichia coli) might be one tool to reduce the risk of transferring these organisms from broilers via poultry meat to humans. The study aimed to gain detailed information on the efficacy of a competitive exclusion culture (CE culture) against EEC exposure in very young chicks. Administration of only 104 cfu/bird on day 2 of life with seven EEC strains (different ESBL and AmpC genes) induced a high and rapid intestinal colonisation in untreated chicks which lasted until an age of 5 weeks. Pretreatment of the birds with a CE culture resulted in a relevant reduction (about 4.0 log10 units) of the different EEC strains. A considerable protective effect (reduction of 2.0 log10 units) by the CE culture could be detected after exposure of the chicks with very high doses of 106 to 108 cfu/bird. Invasion of the liver by EEC organisms was completely prevented by the CE culture even in case of very high challenge doses. The CE culture of undefined composition used here resulted in a substantial decrease of caecal colonisation of EEC strains in young chickens over a lifetime of broilers. Because of the different protective effects against the single EEC strains, modifications in the composition of undefined CE cultures or the development of defined cultures effective against EEC might improve the efficacy of gut flora preparations.

Prototheca species and Pithomyces chartarum as Causative Agents of Rhinitis and/or Sinusitis in Horses.

Pyogranulomatous rhinitis associated with an algal infection was diagnosed in a 25-year-old gelding and a 23-year-old mare had necrotizing sinusitis with intralesional algae and pigmented fungi. Algae were identified immunohistochemically in both cases as Prototheca spp. In the gelding, further characterization by polymerase chain reaction and sequencing revealed that the organism was Prototheca zopfii genotype 2. Fungi from the mare were identified as Pithomyces chartarum by molecular analysis. Prototheca species are achlorophyllous algae and P. chartarum represents a dematiaceous fungus; they are saprophytes and facultative pathogens. Prototheca spp. and P. chartarum should be considered as rare respiratory pathogens of horses.

Within-herd prevalence thresholds for the detection of Mycobacterium avium subspecies paratuberculosis-positive dairy herds using boot swabs and liquid manure samples.

The control of Johne's disease requires the identification of Mycobacterium avium ssp. paratuberculosis (MAP)-positive herds. Boot swabs and liquid manure samples have been suggested as an easy-to-use alternative to sampling individual animals in order to diagnose subclinical Johne's disease at the herd level, but there is a need to evaluate performance of this approach in the field. Using a logistic regression model, this study aimed to calculate the threshold level of the apparent within-herd prevalence as determined by individual faecal culture, thus allowing the detection of whether a herd is MAP positive. A total of 77 boot swabs and 75 liquid manure samples were taken from 19 certified negative and 58 positive dairy herds. Faecal culture, three different polymerase chain reaction (PCR) methods and the combination of faecal culture with PCR were applied in order to detect MAP. For 50% probability of detection, a within-herd prevalence threshold of 1·5% was calculated for testing both matrices simultaneously by faecal culture and PCR, with the threshold increased to 4·0% for 90% probability of detection. The results encourage the use of boot swabs or liquid manure samples, or a combination both, for identifying MAP-positive herds and, to a certain extent, for monitoring certified Johne's disease-negative cattle herds.

Prototheca zopfii Genotype 2-induced Nasal Dermatitis in a Cat.

Few published cases of feline protothecosis exist; all of these were restricted to the skin and speciation of the causative organism revealed an infection with Prototheca wickerhamii in each case. This report describes Prototheca zopfii genotype 2-induced inflammation of the nasal skin and cutaneous mucosa of the right nostril in a 14-year-old neutered female domestic shorthair cat. Microscopical examination revealed marked pyogranulomatous inflammation with numerous intralesional algae. These had a round to ovoid shape, were 8-21 µm in diameter, formed endospores and displayed a positive immunoreaction for Prototheca zopfii antigen. By 18S rRNA gene amplification and sequencing the intralesional algae were confirmed as Prototheca zopfii and further characterized as Prototheca zopfii genotype 2. This case report reveals Prototheca zopfii as an additional Prototheca species associated with feline protothecosis.

Transmission of ESBL/AmpC-producing Escherichia coli from broiler chicken farms to surrounding areas.

Although previous studies have demonstrated high carriage of ESBL/AmpC-producing Escherichia coli in livestock, especially in broiler chickens, data on emission sources of these bacteria into the environment are still rare. Therefore, this study was designed to systematically investigate the occurrence of ESBL/AmpC-producing E. coli in slurry, air (inside animal houses), ambient air (outside animal houses) and on soil surfaces in the areas surrounding of seven ESBL/AmpC-positive broiler chicken fattening farms, including investigation of the possible spread of these bacteria via the faecal route and/or exhaust air into the environment. Seven German broiler fattening farms were each investigated at three points in time (3-36 h after restocking, 14-18 and 26-35 days after housing) during one fattening period. The occurrence of ESBL/AmpC genes in the investigated samples was confirmed by PCR, detecting blaCTX-M, blaSHV, blaTEM and blaCMY-genes, and, if necessary, by sequencing and/or the disc diffusion method. The results showed a wide spread of ESBL/AmpC-producing E. coli in broiler farms, as well as emissions into the surroundings. 12 out of 14 (86%) slurry samples were positive for ESBL/AmpC-producing E. coli. Additionally, 28.8% (n=23/80) of boot swabs taken from various surfaces in the areas surrounding of the farms as well as 7.5% (n=3/40) of the exhaust air samples turned out to be positive for these microorganisms. Moreover, a small proportion of air samples from inside the barns were ESBL/AmpC-positive. By comparing selected isolates using pulsed field gel electrophoresis, we proved that faecal and airborne transfer of ESBL/AmpC-producing microorganisms from broiler fattening farms to the surrounding areas is possible. Two isolates from farm G2 (slurry and boot swab 50 m downwind), two isolates from farm G3 (slurry and individual animal swab) as well as two isolates from farm G6 (air sample in the barn and air sample 50 m downwind) showed 100% similarity in PFGE analysis.

Occurrence of livestock-associated methicillin-resistant Staphylococcus aureus in Turkey and broiler barns and contamination of air and soil surfaces in their vicinity.

The emission of microorganisms, especially resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), from poultry farms is of public interest, and its occurrence and relevance are controversially discussed. So far, there are limited data on this issue. In this study, we investigated the occurrence of livestock-associated (LA)-MRSA inside and outside previously tested MRSA-positive poultry barns in Germany. In total, five turkey and two broiler fattening farms were investigated four and three times, respectively. In a longitudinal study during one fattening period, samples were collected from animals, the animals' environment inside the barn, including the air, and the barns' surroundings, such as ambient air and boot swabs of ground surfaces at different distances from the barn. Moreover, a cross-sectional study was carried out once inside the barns on five turkey and four broiler farms during the last third of the fatting period. In the cross-sectional study, LA-MRSA was detected in the air of most barns (7 of 9, 77.8%), as well as in many samples originating from animals, with detections levels of 50 to 54% in broiler and 62 to 77% in turkey farms. In the longitudinal study, LA-MRSA was found in the ambient air outside two turkey barns and on the ground surface on the downwind side of many (44.4%) turkey and broiler farms. The same spa types of isolates were observed inside and outside the barns. Transmission of MRSA within poultry farms, as well as emission via the airborne route, seems to be possible.

Prevalence of anti-Yersinia outer protein antibodies in goats in lower saxony.

Little is known about the prevalence of caprine yersiniosis in Germany. Only few cases are reported every year. The intention of the survey was to provide representative data on the seroprevalence of anti-Yersinia antibodies in goats in the German state of Lower Saxony. A commercially available Western blot kit was used to identify caprine and ovine anti-Yersinia antibodies against five proteins [YopM, H, D, E and V-antigen (V-Ag)]. Of the 681 investigated goat sera, 449 (66%) had anti-Yop/V-Ag antibodies. Only two of 28 animal holdings housed sero-negative goats. Boxplot analysis showed that the number of non-reactive animals is correlated to the size of a herd and the fact of milk production, respectively. A tendency was observed that various management factors may influence the anti-Yersinia antibody status. No statement was possible on the impact of keeping additional carrier animals such as pigs, cows or sheep on a farm or the type of husbandry on the seroprevalence of anti-Yersinia antibodies. This study provides trend-setting data for yersiniosis in goat-holdings. The impact on consumer health, i.e. especially for risk groups-like people allergic to cow milk and the impact on the profit of a farm will have to be elucidated in further studies.

Impact of invA-PCR and culture detection methods on occurrence and survival of salmonella in the flesh, internal organs and lymphoid tissues of experimentally infected pigs.

This study evaluated the suitability of invA gene amplification by PCR as an effective means of detecting Salmonella species in pigs experimentally infected with S. Typhimurium DT104. A controlled infection study using 24 pigs was performed in order to compare efficacy, precision and detection rates of the invA-based PCR method originally described by Rahn, K. De Grandis, S.A., Clarke, R.C., McEwan, S.A., Galan, J.E., Ginocchio, C., Curtiss, R. 3rd, C.L. Gyles, (Mol. Cell. Probes 1992; 6: 271-279) as a new in-house invA-based PCR method for the specific detection of Salmonella spp. in pork and different tissue samples of slaughter pigs. Finally, PCR results were compared with culture detection rates obtained by isolation procedures following the ISO 6579:2000, the 'gold standard'. After slaughtering, 14 different tissue samples of each pig were investigated to verify the usefulness of the two invA-based PCR methods in different matrices of slaughter pigs. The results demonstrate that the application of the widely used invA-based primer pair (139 + 141) may result in questionable products if samples gained from selective enrichment in the Rappaport-Vassiliadis medium were investigated. These questionable products can lead to false-positive results, if no additional hybridization procedure is attached or if unspecialized persons use this method in routine laboratory practice. The newly developed in-house PCR method used is based on the 3'-prime region of invA, especially designed and harmonized for the detection of Salmonella in different matrices of slaughtered pigs after bacterial enriched broth culture. In this study, this PCR revealed no questionable products and, furthermore, the specificity of the amplificate could be tested by means of the restriction enzyme NdeI. In comparison with the culture detection procedure, the new PCR method has a sensitivity of 100% and a specificity of 96%. Thus, this method might be used as a meaningful tool in eliminating Salmonella-positive carcasses at slaughterhouse level and thus, keeping them out of the food chain.

[Eradication of Prototheca zopfii infection in a dairy cattle herd]. / Sanierung eines Prototheca zopfii-infizierten Milchviehbestandes.

Protothecosis is a severe form of mastitis in dairy cows caused by colorless algae of the genus Prototheca. Since P. zopfii is highly resistant to all known chemotherapeutics, infected cows must be removed from the herd. Eradication measures are difficult since many chronically infected cows may become intermittent shedders. Therefore, cultural methods are insufficient for control measures. In order to eradicate Prototheca zopfii-mastitis in dairy cattle herds, two isotype specific indirect ELISA for detection of IgA and IgG1 in whey were used in a dairy herd highly affected with protothecal mastitis. All cows (n = 313) were tested four times in intervals of six months. Milk specimens were examined in parallel by cultivation and serologically using two indirect ELISA systems for specific IgA and IgG1 in whey. Cows tested Prototheca positive were consequently separated from the herd and slaughtered. At the first examination, 15.6% of the animals were found positive by culture, and 23.3% were positive in at least one of the ELISA systems. Within two years, protothecal prevalence and incidence decreased to zero indicating that the eradication strategy used was successful. In summary, serological identification of P. zopfii-infected lactating cows is an useful tool to eradicate protothecal bovine mastitis in infected herds.

A chi(2) test for model determination and sublevel detection in ion channel analysis.

A chi(2) test is proposed that provides a means of discriminating between different Markov models used for the description of a measured (patch clamp) time series. It is based on a test statistic constructed from the measured and the predicted number of transitions between the current levels. With a certain probability, this test statistic is below a threshold if the model with a reduced number of degrees of freedom is compatible with the data. A second criterion is provided by the dependence of the test statistic on the number of data points. For data generated by the alternative model it increases linearly. The applicability of this test for verifying and rejecting models is illustrated by means of time series generated by two distinct channels with different conductances and by time series generated by one channel with two conductance levels. For noisy data, a noise correction is proposed, which eliminates noise-induced false jumps that would interfere with the test. It is shown that the test can also be extended to aggregated Markov models.

Influence of long-time transportation stress on re-activation of Salmonella typhimurium DT104 in experimentally infected pigs.

In this study a Salmonella Typhimurium infection model in swine was used in order to investigate the influence of pre-mortal stress induced by long time period transportation on the re-activation of Salmonella in experimentally infected pigs. Salmonella free pigs were exposed to a highly virulent strain of Salmonella Typhimurium DT104 by direct intragastrical administration. Clinical parameters were monitored and the shedding rate in faeces was qualitatively and quantitatively determined by standard bacteriological procedures for 21 days. The distribution of the challenge organism in 14 different internal organs of transported and nontransported animals was determined. All infected animals developed clinical signs of salmonellosis 12 to 24 hours post infection. About 88 to 100% of the fecal samples were culture-positive up to post exposure day 6, and then varied from 71 to 92% until slaughter, respectively. At necropsy S. Typhimurium was recovered most frequently from caecum and ileocolic lymph nodes (83%), colon (79%), palatine tonsils (71%) and mandibular lymph nodes (62.5%). A negative impact of transportation stress on the shedding rate and the general condition of the animals was observed.

Improvement of an invA-based PCR for the specific detection of Salmonella typhimurium in organs of pigs.

The aim of this study was to investigate the suitability of the invA-based polymerase chain reaction (PCR) assay for the specific detection of Salmonella in organs of experimentally infected pigs and to compare these results to classical bacterial culture. While the PCR conditions specified in the "Deutsche Industrie Norm", DIN 10135 (section 35 LMBG, 1999), cutle based on the publication of Rahn et al. 1992, revealed various unspecific amplification products, modifications of the PCR conditions allowed the specific amplification of the invA fragment from inner organs. The modified PCR assay correlates exactly with cultivation results (as required by DIN Norm 6579) and enables the detection of Salmonella within 48 hours with equal sensitivity compared to routine cultivation.

Optimizing deconvolution techniques by the application of the Münchhausen meta algorithm.

A deconvolution applied to disturbed data often gives poor results, due to fundamental difficulties associated with ill-posed problems. Many numerical and theoretical methods have been invented to circumvent this phenomenon. Their performance varies, depending on the given problem and data. The main aim of this paper is to provide a decision rule for choosing a method for deconvolution and application of this method to the same data. We have called this meta-algorithm Münchhausen. In this paper we introduce and describe for the first time the basic principle of artificial disturbance of the data in the set-up of deconvolution. We demonstrate some interesting features of the random procedure Münchhausen, such as the non parametric set-up, robustness to disturbance of the data and last but not least good performance.

Errors of the backextrapolation method in determination of the blood volume.

Backextrapolation is an empirical method to calculate the central volume of distribution (for example the blood volume). It is based on the compartment model, which says that after an injection the substance is distributed instantaneously in the central volume with no time delay. The occurrence of recirculation is not taken into account. The change of concentration with time of indocyanine green (ICG) was observed in an in vitro model, in which the volume was recirculating in 60 s and the clearance of the ICG could be varied. It was found that the higher the elimination of ICG, the higher was the error of the backextrapolation method. The theoretical consideration of Schröder et al (Biomed. Tech. 42 (1997) 7-11) was proved. If the injected substance is eliminated somewhere in the body (i.e. not by radioactive decay), the backextrapolation method produces large errors.

Simulation of the initial concentration-time course after intravenous application of the drug.

In this paper we present a widely applicable computational method for the description of the initial concentration-time-course after intravenous injection of a substance. The intravascular concentration-time course, r, is described as r = c0 + g x r, where the asterisk denotes the convolution operation, c0 is the concentration-time course during the first passage of the substance and g is the transport function of the body. If the body transport function is known, then the concentration-time course of a substance can be predicted. The site of interest can be chosen arbitrarily, i.e. the concentration-time course in the arterial circulation supplying any organ can be described. This might be of special interest for the optimal design of intravenous injections of contrast media, where initial concentrations at the region of interest determine the success of the diagnostic procedure.

[Asymptotic behavior of calculated concentration time curves]. / Das asymptotische Verhalten von gemessenen Konzentrationszeitkurven.

The measured concentration time curve of an injected substance is often used as a basis for calculating the distribution volume. For the first time, the present paper describes a generally applicable formula for calculating the asymptote of a concentration time curve in medical applications. With a knowledge of this formula, previously unexplained phenomena (varying results obtained from two different methods of calculating the distribution volume) can now be understood. At the same time, errors of methodology (choice of injection and measuring sites) can be avoided.

[LOGNORMAL-NLSQ-technique. Evaluation of a new mathematical method for determining blood volume]. / LOGNORMAL-NLSQ-Technik. Uberprüfung einer neuen mathematischen Methode zur Bestimmung des Blutvolumens.

This paper describes the investigation of a new mathematical method of calculating blood volume. The new method determines the blood volume by calculating the product of the mean circulation transit time. The mean transit time is calculated from the body transport function. To examine the accuracy of the LOGNORMAL-NLSQ technique, 45 concentration time curves were measured in an in vitro recirculation model with variable clearance. The calculated volume was 4% smaller than the actual volume. This may be attributed to the functional dead space within the model, and is tolerable for clinical situations. The LOGNORMAL-NLSQ technique might acquire considerable importance in future, especially since it provides accurate results very quickly.

Computation of the initial distribution of a drug by repetitive convolution with a circulatory transport function.

Hereby we present a widely applicable computational method for the description of recirculation and distribution phenomena occurring immediately after intravenous injection of a substance. The intravascular concentration-time course, r, is described as r = c0 + g * r, where the asterisk denotes the convolution operation, c0 is the concentration-time course during the first passage of the substance at an arterial measuring site and g is the transport function of the body. If the body transport function is known, then the arterial concentration-time course of a substance can be predicted for different amounts, injection times and elimination rates. The site of interest can be chosen arbitrarily, i.e. the concentration-time course in the arterial circulation supplying any organ can be described. This might be of special interest for the optimal design of intravenous injections of contrast media, where initial concentrations at the region of interest determine the success of the diagnostic procedure.

[Dynamic blood volume determination using the body transport function]. / Dynamische Blutvolumenbestimmung mit Hilfe der Körpertransportfunktion.

This paper describes a dynamic blood volume determination which is faster and more accurate than the classic method. The new method determines blood volume by means of the product of the mean transit time of the circulation and the cardiac output. The mean transit time is calculated from the body transport function. To examine the precision of the dynamic method the blood volume of 24 patients was determined in both the dynamic and the classical way, using radioactively labelled erythrocytes. The comparison of the two methods resulted in a correlation coefficient of r = 0.77. The dynamic method of blood volume determination will be helpful especially in risk patients to accurately determine the quantities of fluids to be administered.

Simulation of arterial drug concentration after intravenous application.

The aim of this study was to develop a widely applicable model for circulatory indicator dispersion which could describe the pharmacokinetics of early drug distribution. The model assumes that the substance is injected into the right atrium and measured in the aorta. The dilution curve results from the dispersion and recirculation of the indicator in the body. The concentration time curve in the aorta, r, can be described as r = c0 + g* r, where g is the transport function of the body and c0 is the concentration time course, which is measured for the first time in the aorta. If the body transport function is known, then the aortic dilution curve of a drug can be predicted for different elimination rates and injection times. The site of interest can be chosen arbitrarily, i.e. the concentration of inflow into the kidney or any other organ can be described.