Performance of seven serological assays for diagnosing tularemia.

BACKGROUND: Tularemia is a rare zoonotic disease caused by the Gram-negative bacterium Francisella tularensis. Serology is frequently the preferred diagnostic approach, because the pathogen is highly infectious and difficult to cultivate. The aim of this retrospective study was to determine the diagnostic accuracy of tularemia specific tests. METHODS: The Serazym®Anti-Francisella tularensis ELISA, Serion ELISA classic Francisella tularensis IgG/IgM, an in-house ELISA, the VIRapid® Tularemia immunochromatographic test, an in-house antigen microarray, and a Western Blot (WB) assay were evaluated. The diagnosis tularemia was established using a standard micro-agglutination assay. In total, 135 sera from a series of 110 consecutive tularemia patients were tested. RESULTS: The diagnostic sensitivity and diagnostic specificity of the tests were VIRapid (97.0% and 84.0%), Serion IgG (96.3% and 96.8%), Serion IgM (94.8% and 96.8%), Serazym (97.0% and 91.5%), in-house ELISA (95.6% and 76.6%), WB (93.3% and 83.0%), microarray (91.1% and 97.9%). CONCLUSIONS: The diagnostic value of the commercial assays was proven, because the diagnostic accuracy was >90%. The diagnostic sensitivity of the in-house ELISA and the WB were acceptable, but the diagnostic accuracy was <90%. Interestingly, the antigen microarray test was very specific and had a very good positive predictive value.

High frequency of chlamydial co-infections in clinically healthy sheep flocks.

BACKGROUND: The epidemiological situation of ovine chlamydial infections in continental Europe, especially Germany is poorly characterised. Using the German state of Thuringia as a model example, the chlamydial sero- and antigen prevalence was estimated in thirty-two randomly selected sheep flocks with an average abortion rate lower than 1%. Seven vaccinated flocks were reviewed separately. RESULTS: A wide range of samples from 32 flocks were examined. Assumption of a seroprevalence of 10% (CI 95%) at flock level, revealed that 94% of the tested flocks were serologically positive with ongoing infection (i.e. animals with seroconversion) in nearly half (47%) of the flocks. On the basis of an estimated 25% antigen prevalence (CI 95%), PCR and DNA microarray testing, together with sequencing revealed the presence of chlamydiae in 78% of the flocks. The species most frequently found was Chlamydophila (C.) abortus (50%) followed by C. pecorum (47%) and C. psittaci genotype A (25%). Mixed infections occurred in 25% of the tested flocks. Samples obtained from the vaccinated flocks revealed the presence of C. abortus field samples in 4/7 flocks. C. pecorum was isolated from 2/7 flocks and the presence of seroconversion was determined in 3/7 flocks. CONCLUSIONS: The results imply that chlamydial infections occur frequently in German sheep flocks, even in the absence of elevated abortion rates. The fact that C. pecorum and the potentially zoonotic C. psittaci were found alongside the classical abortifacient agent C. abortus, raise questions about the significance of this reservoir for animal and human health and underline the necessity for regular monitoring. Further studies are needed to identify the possible role of C. psittaci infections in sheep.

Detection of rotaviruses and intestinal lesions in broiler chicks from flocks with runting and stunting syndrome (RSS).

The intestinal tract and intestinal contents were collected from 34 stunted, 5-to-14-day-old broiler chicks from eight flocks with runting and stunting syndrome (RSS) in Northern Germany to investigate intestinal lesions and the presence of enteric pathogens with a special focus on rotaviruses (RVs). Seven chicks from a healthy flock were used as controls. Severe villous atrophy was seen in chicks from six flocks with RSS but not in the control flock. Lesions were often "regionally" distributed in the middle-to-distal small intestine. Transmission electron microscopy (TEM), polyacrylamide-gel electrophoresis (PAGE), reverse-transcriptase polymerase chain reaction (RT-PCR), and seminested RT-PCR were used for detection and characterization of RVs. The PAGE allows discrimination of different RV groups, and the RT-PCR was used to verify the presence of group (gp) A RVs. RVs were detected (by all methods) in 32 of 34 chicks from the flocks with RSS. By TEM (negative staining), RV particles were observed in intestinal contents of 28 chicks from the flocks with RSS. PAGE analysis showed four RV groups: gpA, gpD, gpF, and gpG. Group A RVs were detected in four chicks from two flocks with RSS, without intestinal lesions. GpD RVs were detected in 12 chicks of five flocks with RSS, 10 of them with severe villous atrophy. GpF RVs were confirmed in four chicks from three flocks with RSS and in two birds in the control flock. GpG RVs were verified in two chicks from two flocks with RSS, one with, and one without, intestinal lesions. At present, PCR methods are only available for detection of gpA RVs. Using RT-PCR, gpA RVs were identified in samples from 22 chicks including samples of two chicks from the control flock. Statistical analysis revealed a positive correlation between presence of gpD RV and severe villous atrophy in flocks with RSS. The results suggest that gpD RV plays a major role in the pathogenesis of RSS.

Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial.

Diagnostic differentiation between the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis is known to be problematic when only conventional serological and biochemical tests are used. The main reason for this is that both agents share a considerable number of related proteins and common epitopes. DNA-based detection methods offer advantages in terms of specificity and sensitivity. However, there is an urgent need to compare currently used PCR assays because they target different genomic regions and, therefore, may perform differently. In the present work, five laboratories, which use PCR routinely, evaluated the specificity of four different PCR systems for M. agalactiae and three systems for M. bovis on a total of 41 strains of the two Mycoplasma species including six previously unidentified strains. As the vast majority of PCR examinations (97.1% of all tests) correctly identified the strains the specificity of all seven detection systems appears to be high. In four cases, incorrect identification by conventional diagnostic methods was rectified by PCR. Isolates from non-typical hosts, i.e. three M. bovis strains from small ruminants and two M. agalactiae strains from cattle, were characterised by sequencing the 16S and part of the 23S ribosomal RNA genes.

Detection of Chlamydia suis from clinical specimens: comparison of PCR, antigen ELISA, and culture.

Cell culture is still widely regarded as the gold standard in chlamydial diagnosis despite its well-known limitations in terms of sensitivity. On the other hand, the polymerase chain reaction (PCR) has emerged as a promising alternative because of rapidity and high sensitivity. However, validation of methodologies is required before the issue of standardization can be addressed. In the present study, 109 clinical samples (organ tissue, nasal, and faecal swabs) from pigs experimentally infected with Chlamydia suis were examined by cell culture, nested PCR in the ompA gene region, and two different antigen enzyme-linked immunosorbent assays (ELISAs) in order to compare the diagnostic performance of these methods. Culture and PCR produced the highest proportion of concordant results (kappa coefficient 0.712). Among 99 samples, 34 were positive in both assays, 51 were negative in both assays, 12 culture-negatives were positive in PCR, and only 2 culture-positives were negative in PCR. Thus, the sensitivity and specificity of PCR vs. culture as standard were 94.4% and 81.0%, respectively, whereas the corresponding values for culture vs. PCR as standard were 73.9% and 96.2%, respectively. Both ELISA tests performed considerably weaker. The data underline the potential of PCR as a powerful detection method for chlamydiae.

Ochratoxin A and some of its derivatives modulate radical formation of porcine blood monocytes and granulocytes.

Modulating effects of ochratoxin A (OTA) and some of its derivatives on viability and oxidative burst activity of porcine monocytes and granulocytes have been studied. The formation of free oxygen radicals by monocytes was suppressed by OTA and ochratoxin C (OTC) at concentrations between 10 and 1000 ng/ml. Intracellular radical formation of granulocytes was in part already significantly reduced at 1 ng/ml of these mycotoxins. Conversely, the intracellular formation of radicals in monocytes of individual pigs was stimulated by the toxins at 1-100 ng/ml. A biologically active fraction of the crude toxin (RE2) which had been identified as OTC had a stronger effect than all other derivatives of ochratoxin A. Whether these modulating effects of OTA and OTC on phagocyte functions are of significance in the pathogenesis of infectious diseases, needs to be studied in more detail. In this context, the occurrence of OTC in food and feeds should be examined more closely.

Effects of the mycotoxin ochratoxin A and some of its metabolites on the human cell line THP-1.

The immunomodulatory effects of ochratoxin A (OTA) and some of its metabolites on the human monocyte/macrophage line THP-1 are described. Metabolic activity, cell proliferation, cell membrane integrity, cell differentiation, phagocytic behaviour, nitrogen oxide synthesis and cell surface markers were largely suppressed by these mycotoxins at concentrations between 10 and 1000 ng/ml, in individual cases already at 1 ng/ml. After analysis of a crude toxin, a substance designated RE2 could be isolated besides OTA, which was identified as ochratoxin C (OTC). The latter showed a stronger suppressive effect on most functions studied than all other metabolites of OTA. Because of the immunomodulatory effects of OTA and OTC, more attention should be paid to their immunopathogenic importance in addition to their known cytotoxic and genotoxic effects. The occurrence and importance of the mycotoxin OTC should be more closely examined in this context.

Serological investigation of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicator animals for circulation of Francisella tularensis in Germany.

Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment.

Efficacy testing of several Ixodes ricinus tick repellents: different results with different assays.

In the European Union (EU), tick repellents for humans need to be registered and approved by the authorities in order to be marketed. As there are currently no specific technical guidelines for product evaluation, we compared 3 different test methods: the mechanical moving object bioassay (MOB), and 2 assays involving human volunteers. For the latter, procedures according to the U.S. Environmental Protection Agency (EPA) and the Stiftung Warentest (StiWa), a German consumer care organization, were used. Two repellents, Autan(®) (AU), based on 20% Picaridin [2-(2-hydroxyethyl)-1-piperidinecarboxylic acid-1-methylpropyl ester], and ZeckWeck (ZW), based on 12.5g/100g Citriodiol™ (main compound: p-menthane-3,8-diol) were tested with all 3 assays. Three repellents, Anti Brumm(®) naturelle, based on 20% Citriodiol™ (main compound: p-menthane-3,8-diol), G090141, based on 20% EBAAP (ethyl buthyl acetyl aminopropionate), and G090152, based on 10% decanoic acid (capric acid), which is contained in Zanzarin(®), were tested according to the EPA and the StiWa procedures. The EPA assay indicated a significantly higher repellency of the products AU and G090141 than the StiWa test, but no difference between assays could be detected for the remaining 3 products. Also the corresponding protection times were significantly longer (approximately 4h) when determined according to EPA versus to StiWa for 3 of the products, whilst the difference was insignificant for ZW and G090152. Additionally, significantly lower numbers of ticks initially walked onto the repellent-treated skin when tested according to EPA versus to StiWa in all products except ZW and G090152. Thus, the StiWa protocol appears to pose higher demands on a repellent than the EPA method. Contrary to expectation, the MOB showed the same or even lower product efficacy when compared to the EPA and StiWa tests. Particularly, the percentage of ticks clinging to repellent-treated filter paper was significantly higher than the proportion of ticks walking onto treated skin in the other assays. This could mean that in nature more ticks may probably cling to a human protected by a given repellent than the EPA or the StiWa assay might suggest. Nevertheless, the MOB produced results that are quite similar to the tests involving human volunteers.

Goats and sheep as sentinels for tick-borne encephalitis (TBE) virus--epidemiological studies in areas endemic and non-endemic for TBE virus in Germany.

The aim of the study was to examine grazing goats and sheep as specific sentinels for characterization of the tick-borne encephalitis virus (TBEV)-related risk in an area by means of serosurveillance tests in the German federal states Baden-Wuerttemberg, Bavaria, Thuringia, North Rhine-Westphalia, Lower Saxony, Schleswig-Holstein, and Mecklenburg-West Pomerania. A total of 3590 sheep sera and 3793 goat sera was collected in 2003 and 2006-2009 and were examined by ELISA screening and confirmed by serum neutralization test. Considerable differences in seroprevalence were seen between single flocks in districts in Baden-Wuerttemberg, Bavaria, and Thuringia with values between 0 and 43% which confirmed the patchy pattern of TBEV foci that can range in size from very small to large. The here described serological screening may be a helpful tool for an early warning system of a potential TBEV risk. Testing of 1700 ticks by real-time RT-PCR in two districts in Baden-Wuerttemberg revealed only one positive tick, thus illustrating the problems of expensive and time-consuming tick collection.

Prevalence of Coxiella burnetii in clinically healthy German sheep flocks.

BACKGROUND: Current epidemiological data on the situation of Coxiella (C.) burnetii infections in sheep are missing, making risk assessment and the implementation of counteractive measures difficult. Using the German state of Thuringia as a model example, the estimated sero-, and antigen prevalence of C. burnetii (10% and 25%, respectively) was assessed at flock level in 39/252 randomly selected clinically healthy sheep flocks with more than 100 ewes and unknown abortion rate. RESULTS: The CHECKIT™ Q-fever Test Kit identified 11 (28%) antibody positive herds, whereas real-time PCR revealed the presence of C. burnetii DNA in 2 (5%) of the flocks. Multiple-locus variable number of tandem repeats analysis of 9 isolates obtained from one flock revealed identical profiles. All isolates contained the plasmid QpH1. CONCLUSIONS: The results demonstrate that C. burnetii is present in clinically inconspicuous sheep flocks and sporadic flare-ups do occur as the notifications to the German animal disease reporting system show. Although C. burnetii infections are not a primary veterinary concern due to the lack of significant clinical impact on animal health (with the exception of goats), the eminent zoonotic risk for humans should not be underestimated. Therefore, strategies combining the interests of public and veterinary public health should include monitoring of flocks, the identification and culling of shedders as well as the administration of protective vaccines.

Divergent cytokine responses of macrophages to Mycobacterium avium subsp. paratuberculosis strains of Types II and III in a standardized in vitro model.

Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1ß, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1ß and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1ß and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1ß mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis.

Use of a novel real-time PCR technique to monitor and quantitate Mycoplasma bovis infection in cattle herds with mastitis and respiratory disease.

Mycoplasma bovis infection of cattle tends to persist in affected herds and can be resistant to treatment. Given that the level of shedding of the organism can reflect the state of ongoing infection, a study was established to quantify the degree of such shedding in milk samples from infected herds using a novel real-time PCR technique. While the M. bovis load in herds with clinical disease was significantly higher than in disease-free herds (P<0.001), infection persisted in the latter. Similarly, M. bovis was detected more frequently in conjunctival swabs from calves with respiratory disease when compared with samples from animals that did not exhibit such clinical signs. This novel real-time PCR assay is specific for M. bovis and can detect bacterial loads as low as 100 colony forming units/mL of milk.

Evaluation of five ELISA test kits for the measurement of antibodies against Mycobacterium avium subspecies paratuberculosis in bovine serum.

Five commercially available ELISA tests for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in bovine serum were evaluated at the individual animal level using sera from 286 paratuberculosis-free and 110 paratuberculosis-infected dairy cattle. Sensitivity (Se) and specificity (Sp) of the tests were estimated after determination of the cut-off dtheta by TG-ROC analysis or using the cut-off values recommended by the manufacturers, respectively. When the dtheta cut-off values were applied, the five ELISA tests showed sub-optimal Se and Sp. Adopting the cut-offs recommended by the manufacturers, the Sp of four of the five ELISA increased, two tests reaching Sp > or = 99.0%. Test sensitivity clearly depended on the disease state of the animals examined. Se was significantly higher in clinically diseased than in latently infected dairy cattle. Calculation of the positive and negative predictive values indicated that, depending on the test, a considerable proportion of false positive and false negative results have to be expected. Therefore, the suitability of antibody detection for the diagnosis of paratuberculosis in individual animals is questioned.

[Occurence of salmonellae in laying hens in different housing systems and inferences for control]. / Zum Vorkommen von Salmonellen bei Legehennen in unterschiedlichen Haltungsformen und Schlussfolgerungen für die Bekämpfung.

As eggs represent now as ever the most important source for Salmonella infection in human beings and because of the currently occurring shift in housing conditions for laying hens from conventional cages to alternative systems it was studied whether the Salmonella prevalence in layers is influenced by the housing system. Following systems were considered: organic farming with free range management systems, floor management systems with free range, floor management systems without free range, conventional cages. 453 pooled faecal samples as single or double examination per herd from 329 flocks in different housing systems for table egg production from three Federal Lander were examined bacteriologically. The share of layer flocks which were Salmonella positive at least once independently of the housing system amounted to 32.2%. Analysis of the Salmonella findings in the single housing systems revealed that the share of Salmonella positive flocks was higher in conventional cage systems (46.3%) than in alternative housing systems (32.996% in organic farming with free range management systems, 21.9% in floor management systems with free range, 23.4% in floor management systems without free range). The results of the study clearly show that Salmonella Enteritidis (mostly phage type 4, other phage types rarely) presents with a share of 78% the dominant serovar in laying hens. The total number of all other serovars (apart from Salmonella Enteritidis and subspecies I rough) reached a share of ca. 14%, however, no other single serovar was dominant. As Salmonella Enteritidis is the predominant serovar in laying hens it is strongly recommended to use Salmonella Enteritidis vaccines for immunisation programmes of chickens during the rearing period. Because of the high prevalence of Salmonella organisms in the different housing systems, detailed information on the epidemiology of Salmonella in laying hens are needed to introduce effective control measures. Of particular interest is the question whether the Salmonella findings in laying flocks are the result of multiplication of already existing Salmonella organisms in the animals or whether the bacteria are introduced only during the laying period.

TBE incidence versus virus prevalence and increased prevalence of the TBE virus in Ixodes ricinus removed from humans.

Traditionally, the classification of risk areas of tick-borne encephalitis (TBE) is based on the recording of autochthonous cases of the disease. In Germany, an extension of these areas over the years and an increasing virus prevalence in ticks have been observed in recent years. Registration of foci with autochthonous TBE cases, recording of disease incidence and virus prevalence in ticks are all proven epidemiological methods to characterize TBE risk areas. These data are necessary for a scientifically proven recommendation of TBE vaccines, and they need to be updated regularly. These epidemiological methods have advantages and disadvantages with respect to the risk assessment of TBE areas. Despite the fact that these methods are suitable for risk assessment in practice, disease incidence (new cases per year/100,000 inhabitants) and virus prevalence in questing ticks did not correlate. Using nested RT-PCR we were able to demonstrate that the prevalence of TBE virus (TBEV) in ticks removed from humans was significantly higher than in unfed, free-living Ixodes ricinus of the same area. The 561 ticks collected from humans in doctors' surgeries in Bavaria in 2002 were examined by nRT-PCR. The estimated overall virus prevalence in tested ticks was 8.8% (95% CI: 6.45-11.57%). The removed ticks examined were classified according to the sites of exposure of the patients in the individual districts. Peak values were measured in the district of Regen with 20.6% and in the district of Freyung-Grafenau with 18.3%. In recent studies on unfed I. ricinus (nymphs, adults), the average TBEV prevalence in ticks in Bavarian risk areas was between 0.5% and 2%.