Vaccination with recombinant paramyosin against the bovine lungworm Dictyocaulus viviparus considerably reduces worm burden and larvae shedding.

BACKGROUND: The lungworm Dictyocaulus viviparus, causing parasitic bronchitis in cattle, induces a temporary protective immunity that prevents clinical disease. A radiation-attenuated larvae based vaccine is commercially available in a few European countries, but has the disadvantages of a live vaccine. As a recombinant subunit vaccine would overcome these disadvantages, the parasite's muscle protein paramyosin (PMY) was tested as a recombinant vaccine antigen. METHODS: D. viviparus-PMY was recombinantly expressed in Escherichia coli as a glutathione-S-transferase (GST)-fused protein. Emulsified in adjuvant Saponin Quil A, the protein was given intramuscularly into calves. Two independent recombinant PMY (rPMY) vaccination trials with negative control groups (first trial: adjuvant only; second trial: non-fused GST) as well as an additional positive control group in the second trial, using the Bovilis Dictol live vaccine to verify vaccination results, were performed. To determine the vaccination success, shedding of larvae as well as worm burden and worm sizes were analyzed. Additionally, ELISA-based determination of development of immunglobulins IgM, IgA, IgE, IgG as well as the subclasses IgG1 and IgG2 was performed. To analyze PMY localization in the bovine lungworm, immunohistochemical staining of adult worms was carried out. RESULTS: Immunohistochemical staining revealed that PMY is part of the bovine lungworm's pharyngeal and body wall muscles. Vaccination with rPMY resulted in 47% [geometric mean: 67%] and 57% (geometric mean: 71%) reduction of larvae shedding in the first and second vaccination trial, respectively. Worm burden was reduced by 54% (geometric mean: 86%) and 31% (geometric mean: 68%), respectively, and worms of rPMY-vaccinated cattle were significantly shorter in both trials. Furthermore, ELISAs showed a clear antibody response towards rPMY with exception of IgE for which titers could not be detected. After challenge infection, rPMY antibodies were only exceptionally elevated among study animals indicating PMY to be a hidden antigen. CONCLUSIONS: Even though vaccination with the attenuated live vaccine was with 94% (geometric mean: 95%) reduction in larvae shedding and 93% (geometric mean: 94%) reduction in worm burden superior to rPMY vaccination, results using the latter are promising and show the potential for further development of a recombinant PMY-based vaccine against the bovine lungworm.

Neurotoxocarosis: marked preference of Toxocara canis for the cerebrum and T. cati for the cerebellum in the paratenic model host mouse.

BACKGROUND: Infective larvae of the worldwide occurring zoonotic roundworm T. canis exhibit a marked affinity to the nervous tissues of paratenic hosts. In humans, most cases of neurotoxocarosis are considered to be caused by larvae of T. canis as T. cati larvae have rarely been found in the CNS in previous studies. However, direct comparison of studies is difficult as larval migration depends on a variety of factors including mouse strains and inoculation doses. Therefore, the present study aims to provide a direct comparison of both roundworm species in mice as a model for paratenic hosts with specific focus on the CNS during the acute and chronic phase of disease to provide a basis for further studies dealing with neurotoxocarosis. METHODS: C57Bl/6J mice were infected with 2000 embryonated T. canis and T. cati eggs, respectively as well as Balb/c mice infected with T. cati eggs only. On 8 time points post infection, organs were removed and microscopically examined for respective larvae. Special focus was put on the CNS, including analysis of larval distribution in the cerebrum and cerebellum, right and left hemisphere as well as eyes and spinal cord. Additionally, brains of all infection groups as well as uninfected controls were examined histopathologically to characterize neurostructural damage. RESULTS: Significant differences in larval distribution were observed between and within the infection groups during the course of infection. As expected, significantly higher recovery rates of T. canis than T. cati larvae were determined in the brain. Surprisingly, significantly more T. canis larvae could be found in cerebra of infected mice whereas T. cati larvae were mainly located in the cerebellum. Structural damage in brain tissue could be observed in all infection groups, being more severe in brains of T. canis infected mice. CONCLUSIONS: The data obtained provides an extensive characterization of migrational routes of T. canis and T. cati in the paratenic host mouse in direct comparison. Even though to a lesser extent, structural damage in the brain was also caused by T. cati larvae and therefore, the potential as pathogenic agents should not be underestimated.

Lungworm Infections in German dairy cattle herds--seroprevalence and GIS-supported risk factor analysis.

In November 2008, a total of 19,910 bulk tank milk (BTM) samples were obtained from dairy farms from all over Germany, corresponding to about 20% of all German dairy herds, and analysed for antibodies against the bovine lungworm Dictyocaulus viviparus by use of the recombinant MSP-ELISA. A total number of 3,397 (17.1%; n = 19,910) BTM samples tested seropositive. The prevalences in individual German federal states varied between 0.0% and 31.2% positive herds. A geospatial map was drawn to show the distribution of seropositive and seronegative herds per postal code area. ELISA results were further analysed for associations with land-use and climate data. Bivariate statistical analysis was used to identify potential spatial risk factors for dictyocaulosis. Statistically significant positive associations were found between lungworm seropositive herds and the proportion of water bodies and grassed area per postal code area. Variables that showed a statistically significant association with a positive BTM test were included in a logistic regression model, which was further refined by controlled stepwise selection of variables. The low Pseudo R(2) values (0.08 for the full model and 0.06 for the final model) and further evaluation of the model by ROC analysis indicate that additional, unrecorded factors (e.g. management factors) or random effects may substantially contribute to lungworm infections in dairy cows. Veterinarians should include lungworms in the differential diagnosis of respiratory disease in dairy cattle, particularly those at pasture. Monitoring of herds through BTM screening for antibodies can help farmers and veterinarians plan and implement appropriate control measures.

Seroprevalence and GIS-supported risk factor analysis of Fasciola hepatica infections in dairy herds in Germany.

A total of 20 749 bulk tank milk (BTM) samples was collected in November 2008 from all over Germany, corresponding to 20.9% of all German dairy herds. The BTM samples were analysed for antibodies against Fasciola hepatica using the excretory-secretory (ES) ELISA. A geospatial map was drawn to show herd prevalences per postal code area. Various spatial risk factors were tested for potential statistical associations with the ELISA results in logistic regression supported by a geographical information system (GIS). The mean seroprevalence was 23.6% and prevalences in different German federal states varied between 2.6% and 38.4%. GIS analysis revealed statistically significant positive associations between the proportion of grassed area and water bodies per postal code area and positive BTM ELISA results. This can be explained by the biology of the intermediate host, the amphibious snail Galba (Lymnea) truncatula and the pasture-borne nature of fasciolosis. The full logistic regression model had a Pseudo-R 2 of 22%, while the final model obtained by controlled stepwise model building revealed a Pseudo-R 2 of 14%, indicating that additional, unrecorded factors and random effects contributed substantially to the occurrence of positive ELISA results. Considering the high seroprevalences in some areas and the economic impact of fasciolosis, farmers and veterinarians should be strongly advised to implement effective liver fluke control programmes.

Evaluation of a recombinant cathepsin L1 ELISA and comparison with the Pourquier and ES ELISA for the detection of antibodies against Fasciola hepatica.

The liver fluke Fasciola hepatica is one of the most important parasites in cattle farming worldwide and responsible for serious economic losses. In this study, three ELISAs for the detection of antibodies against F. hepatica were compared by analysis of serum antibody levels of 20 calves infected with different doses (50-1000 metacercariae) of F. hepatica metacercariae. The commercially available Pourquier ELISA was tested against an in-house ELISA based on excretory-secretory (ES) products as well as an ELISA based on recombinant cathepsin L1 (rCL1). During the period 4-21 weeks post infection (pi) the Pourquier ELISA showed 100% sensitivity (Se) and 100% specificity (Sp). ROC-analyses revealed a cut-off value of 0.090 ODR for the ES and 0.096 ODR for the rCL1 ELISA. Se of the ES ELISA was 90% and 95% in weeks 4 and 5 pi, respectively, and reached 100% during the period 6-12 weeks pi. Between weeks 13-21 pi Se varied from 90 to 100%. Sp of the ES ELISA was 99.3%. By comparison, calculated rCL1 ELISA Se values were rather low during prepatency (20-85% during weeks 4-7 pi), but increased with early beginning of patency and reached 100% during weeks 10-15 pi. Afterwards, Se ranged between 90% and 100%. With regards to different doses of F. hepatica metacercariae, Spearman rank correlation showed a significant relationship between the infection dose and measured antibody levels for the Pourquier ELISA but not the ES and rCL1 ELISA. Overall, the present study revealed the Pourquier ELISA as the superior test followed by the ES ELISA. Nonetheless, even if rCL1 ELISA was obviously inferior during prepatency, its test parameters associated with patent infections make it a convincing ELISA to diagnose patent fluke infections in cattle.

Bulk milk-estimated seroprevalence of Fasciola hepatica in dairy herds and collecting of risk factor data in East Frisia, northern Germany.

The liver fluke Fasciola (F) hepatica is one of the most important trematodes in cattle farming worldwide. Fasciolosis in dairy cows is leading to production losses due to decreased milk yield, liver condemnation and impaired reproduction. The treatment of dairy cows is unsatisfactory, because available drugs are either effective against adult flukes only or have long withdrawal times or in some countries may not be used at all. In the present study the prevalence of F. hepatica in dairy farms located in East Frisia, which is part of the federal state Lower Saxony, was investigated. East Frisia is considered a high risk area for Fasciola hepatica infections, because of its coastal location, high precipitation and moist pastures. About 750 bulk milk samples were collected in January and November 2006 and analysed for F. hepatica antibodies using the Pourquier ELISA. In addition, questionnaires, which were answered by 260 of the participating farmers, were evaluated to analyse management-related factors associated with fasciolosis. In January and November, 52.1% and 53.6% of the bulk milk samples, respectively, showed positive results. Thereby, 88.1% of the examined farms showed an unchanged infection status, whereas 6.2% of the farms became seropositive during the grazing season and 5.8% of the dairy herds turned seronegative. Statistical analysis revealed a significant negative association between average annual milk production and the frequency of infections with F. hepatica.

Treatment of third-stage larvae of Toxocara cati with milbemycin oxime plus praziquantel tablets and emodepside plus praziquantel spot-on formulation in experimentally infected cats.

Toxocara cati is the most prevalent gastrointestinal helminth in cats worldwide, with cats of all ages at risk of infection. An anthelminthic treatment that not only affects the gut-dwelling stages of this parasite but is also effective against developmental stages in the tissue has the advantage that the pathology caused by migrating larvae is minimized and the need for repeated treatments is reduced. This study was conducted to evaluate the efficacy of milbemycin oxime/praziquantel tablets (Milbemax®, Novartis) against third-stage larvae of T. cati in comparison to a spot-on formulation of emodepside and praziquantel (Profender®, Bayer). Twenty-four kittens were experimentally infected with T. cati and randomly allocated to three study groups. Treatments were performed at the minimum therapeutic dosage 5 days after the experimental infection. The development of patent infections was monitored and all cats were dewormed 50 days post-infection. Efficacies were calculated based on counts of excreted worms in the treated groups compared to a negative control group. Seven of the eight cats in the negative control group developed a patent T. cati infection and all cats were excreting worms at the end of the study (geometric mean worm count 18.1). No efficacy could be observed for the milbemycin oxime-treated animals. All cats developed a patent infection and excreted worms (geometric mean worm count 27.7). The treatment with Profender® was 98.5 % effective against L3 of T. cati. One cat developed a patent infection and was excreting worms at the end of the study (geometric mean worm count 0.3). No adverse reactions were noted in either treatment group.

Rickettsia spp. and coinfections with other pathogenic microorganisms in hard ticks from northern Germany.

Rickettsia species are the causative agent of different forms of spotted fever and thus, monitored in a number of prevalence studies. The current study examined the status of ticks from the city of Hanover, Northern Germany, regarding the presence of Rickettsia spp. and coinfections with Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum. In total, 1,089 questing Ixodes ricinus L. ticks were analyzed using quantitative real time polymerase chain reaction. A duplex quantitative real time polymerase chain reaction for simultaneous detection of Rickettsia spp. and Ixodes spp.-DNA as positive control for successful DNA-isolation was established. Rickettsia spp. were detected in 363 (33.3%) of the 1,089 investigated ticks. Quantification of Rickettsia showed that larvae contained up to 50,000 bacteria, nymphs up to 85 million and adults up to 200 million per tick. Species differentiation was possible in 178 out of 363 Rickettsia positive samples and resulted in a predominant occurrence of R. helvetica (98.9%, 176/178), whereas R. monacensis was rarely found (1.1%, 2/178). Besides detection of Rickettsia, positive ticks were compared with results from previous studies to examine coinfections with B. burgdorferi sl and A. phagocytophilum. The resulting coinfection rates were 9.1% (99/1,089) for B. burgdorferi sl and 2.8% (11/391) for A. phagocytophilum. Triple-infection with Rickettsia spp., B. burgdorferi sl, and A. phagocytophilum occurred in 5 (1.3%) out of 391 ticks. The current study is the first presenting quantitative data concerning the load of Ixodes ticks with Rickettsia individuals.

Validation of a Dictyocaulus viviparus MSP-ELISA and cut-off adjustment in a one-year longitudinal field study in dairy cattle herds.

A one-year field study analysing lungworm seropositivity by use of the MSP-ELISA was performed (1) to investigate the antibody dynamics in individual milk samples following field (re-)infections of dairy cows with the bovine lungworm Dictyocaulus viviparus, (2) to investigate the correlation between individual and bulk tank milk (BTM) antibody titres and (3) to review the current individual as well as BTM cut-off value, which was extrapolated from dilution experiments (Fiedor et al., 2009). Over a one-year period individual and BTM samples were collected monthly on 15 dairy farms. Following a critical review of previous cut-off values, individual and BTM samples were subjected to different cut-off thresholds. Following Receiver-Operating-Characteristics (ROC) analysis, individual milk samples were assessed with the cut-off value 0.573, previously shown to be associated with each 100% sensitivity and specificity. In addition, the present study enabled BTM cut-off adjustment based on field data. To ensure reliable detection of herds with an in-herd prevalence of ≥20% the BTM cut-off was lowered from 0.493 to 0.410, corresponding to 100% sensitivity and 97.32% specificity. Regression analysis showed that the percentage of seropositive animals related to the corresponding BTM ODR correlated moderately (r=0.581, P<0.001), whereas a strong correlation (r=0.764, P<0.001) was found between mean individual and BTM ODR per herd and sampling month. Seasonal antibody pattern became obvious in a single-peaked antibody curve in late summer/early autumn for individual milk whilst BTM showed a two-peaked distribution with an additional spring peak besides the late summer/early autumn peak. This leads to the conclusion that the BTM-ELISA could be a useful tool to detect and control pasture contamination in the spring, following sexual maturation of hypobiotic lungworm larvae harboured by clinically asymptomatic carrier animals. In addition to the knowledge gained on antibody patterns in dairy herds and the relationship of individual and BTM, the present study enabled sensitivity and specificity calculations for the obsolete BTM cut-off value 0.493 to be performed.

Seasonal pattern of Fasciola hepatica antibodies in dairy herds in Northern Germany.

Fasciolosis, caused by the liver fluke Fasciola hepatica, is one of the most important parasitoses in cattle farming worldwide. In dairy cows, the trematode leads to economic losses due to decreased milk yield, a negative impact on reproduction parameters, and liver condemnations. In the present study, the seasonal patterns of F. hepatica antibodies in bulk-tank milk from dairy herds located in East Frisia, a region of the federal state Lower Saxony in the north of Germany, were investigated. This region was chosen since it is known as a high risk area for fluke infections due to its coastal location at the North Sea with the consequence of rather moist pastures. Between 669 and 868 bulk-tank milk samples were collected in January, September and November 2008 and 2010, respectively, and analysed for antibodies against F. hepatica with an in-house ELISA based on excretory-secretory antigens of the liver fluke. The overall East Frisian prevalence was 49.1%, 57.1% and 53.9% in January, September and November 2008 and 45.1%, 49.5% and 48.4% in 2010. From a number of 606 farms, which were sampled in all six investigated months, 34.5% of the farms continued to remain positive, whereas 30.9% continued to remain negative. A percentage of 69.1% (419 farms) were positive on at least one sampling occasion during the study period. The distributions of optical density ratio (ODR) values were skewed to the left but showed a second, lower peak in a high ODR range. Statistical analysis revealed a significant difference concerning the prevalence increase from January to September 2008. Furthermore, the prevalence decrease from September as well as November 2008 to these months in 2010 was significantly different, what might result from a more frequent use of anthelminthics or different climatic conditions.

Strongyloides papillosus: changes in transcript levels of lysozyme and aspartic protease 2 in percutaneously migrated larvae.

The infection of the host is the crucial event in the life-cycle of parasites. To understand the molecular mechanisms of this important step, different methods are used in present studies. For analysis of changes in transcript levels the most sensitive method is the quantitative real-time PCR (qPCR). For an accurate analysis the evaluation of a set of adequate reference genes is necessary. The present study aimed to analyse the transcriptional levels of two genes of interest, the putative aspartic protease Spa-asp-2 and the putative lysozyme Spa-lys, in infective, free-living larvae of Strongyloides papillosus at different ages and from long-term and short-term infections and percutaneously migrated ("parasitic") larvae. Percutaneously migrated larvae were collected using the PERL chamber system and ovine skin in vitro. Reference genes identified as most suitable for transcriptional analysis according to geNorm analysis were genes for the eukaryotic translation elongation factor 1 alpha (Spa-eft-2), actin variation 2 (Spa-act-v2) and beta tubulin (Spa-tbb-1). Transcriptional analysis of the genes in percutaneously migrated larvae showed an upregulation of Spa-asp-2, while Spa-lys was downregulated. Data from the presented study provide a first glance into the changes of transcript levels of S. papillosus induced by percutaneous migration.

In vitro studies on the sexual maturation of the bovine lungworm Dictyocaulus viviparus during the development of preadult larvae to adult worms.

The bovine lungworm Dictyocaulus viviparus is one of the most important parasites in grazing cattle. However, not much is known about morphology and molecular aspects of sexual maturation occurring during development of preadult larvae (L5) to adults. Since studies in the pulmonary compartments are infeasible, an in vitro cultivation method was established. The study was conducted with L5 during in vitro cultivation, assessing longitudinal growth and sexual maturation. Best results were achieved with RPMI-1640 medium with L-glutamine, 50% fetal bovine serum, amphotericin B (0.25 mg/ml), penicillin (10,000 U/ml), and streptomycin (10 mg/ml) at 39°C and 5% atmospheric CO2. During cultivation, individuals grew from an average length of 4.64 to 9.88 mm independent of their density per setup. Regarding sexual maturation, female individuals started to lay eggs, whereas the testes of male individuals were filled with spermatozoa. Consequently, adult female and adult male worms developed. However, no copulation was observable and eggs did not embryonate. Development was further investigated by quantitative real-time PCR transcriptional analysis of major sperm protein (msp) and vitellogenin (vit) representing male and female sexual development, respectively. Male msp transcription peaked after 5 days of cultivation [corresponding to 20 days post infection (dpi)] and decreased gradually afterwards. Female vit transcription showed the highest rate after 15 days of cultivation (30 dpi), however it never reached the transcription rate in female adults isolated from the host. All in all, the present study gives not only insights into morphological differentiation but provides data lightening molecular aspects of sexual maturation in D. viviparus.

A survey on Dictyocaulus viviparus antibodies in bulk milk of dairy herds in Northern Germany.

Parasitic bronchitis caused by the bovine lungworm, Dictyocaulus viviparus, occurs worldwide in temperate areas. The parasite is found predominantly in calves and heifers, but dairy cattle can suffer from lungworms when they become infected for the first time or if they have lost immunity due to lack of exposure to lungworm larvae during the grazing season. The present study was performed to determine the D. viviparus bulk milk antibody prevalence in dairy herds in the East Frisian region of northwestern Germany, Lower Saxony, by analysing bulk milk samples collected in January (860 samples), September (866 samples) and November (860 samples) 2008, thereby representing 906 dairy farms. These samples were tested for antibodies against D. viviparus by a milk ELISA. This test detects patent infections only since it is based on recombinant major sperm protein as antigen. While in January 12.8% of dairy farms were positive for D. viviparus antibodies, the bulk milk samples collected in September and November revealed 6.9% and 6.6% positive dairy herds. From the 906 dairy farms included in the study, 191 (21.1%) tested positive at least once for antibodies against lungworm. From 810 dairy farms from which bulk milk samples were obtained during all three samplings, 146 (18.0%) farms were positive at one sampling date, 27 (3.3%) at two, and 4 (0.5%) on all three sampling dates. The majority of the farms represented in the study belonged to four districts of East Frisia, which showed no significant difference in the proportion of positive dairy farms.

Transcriptional differences between hypobiotic and non-hypobiotic preadult larvae of the bovine lungworm Dictyocaulus viviparus.

The survival of the bovine lungworm Dictyocaulus viviparus, one of the most important parasites in cattle, inside the host is ensured by arrested development during adverse environmental conditions, commonly referred to as hypobiosis. In the present study, a subtractive hybridization approach was used to compare the transcription profiles of hypobiotic and non-hypobiotic larvae (L5hyp and L5, respectively). Thereby, 75 L5hyp-enriched and 58 L5-enriched representative ESTs (rESTs) were identified. Subsequent sequence similarity search revealed that 28 L5hyp-rESTs and 11 L5-rESTs were homologous to known transcripts, whereas 47 L5hyp-rESTs and 47 L5-rESTs showed no homologies with published sequences, thus possibly representing parasitic or even Dictyocaulus-specific genes. The differential transcripts were predicted to be involved in nucleic acid synthesis, DNA binding, metabolic pathways and signal transduction. Overall, data presented in this paper provide a first basis for further characterization and analysis of genes driving normal as well as arrested (hypobiotic) parasite development.

Use of fluorescent lectin binding to distinguish Teladorsagia circumcincta and Haemonchus contortus eggs, third-stage larvae and adult worms.

Lectin binding to carbohydrates on parasite surfaces has been investigated as a method of distinguishing adult worms, eggs and sheathed and exsheathed L3 of Teladorsagia circumcincta and Haemonchus contortus, economically important abomasal parasites in temperate climates. Both species were maintained as pure laboratory cultures of field isolates from New Zealand. Each of the four life cycle stages could be distinguished by the binding of at least one lectin: adult worms by Sambucus nigra agglutinin (SNA); eggs by peanut agglutinin (PNA), ConcavalinA and Lens culinaris agglutinin (LCA); exsheathed L3 by Griffonia simplicifolia-I lectin (GSL-I) and Lotus tetragonolobus lectin (LTL) and sheathed L3 by Aleuria aurantia lectin (AAL). The whole surface of both adult T. circumcincta and H. contortus strongly bound lectins specific for N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), mannose and fucose, but the two species could be distinguished by SNA binding only to T. circumcincta. Eggs could be distinguished by the binding of mannose-specific PNA to H. contortus and GalNAc-specific LCA and PSA to T. circumcincta eggs. GalNAc, GlcNAc and mannose lectins bound to the cuticle and over the excretory pores of a large proportion of sheathed L3 of both species, but only the H. contortus surface had exposed fucose or sialic acid complexes. The distinguishing lectin for sheathed L3 was AAL, which did not bind to T. circumcincta, but bound weakly to the head region of all fresh H. contortus and to 50-90% after 3 months storage. The cuticle of exsheathed L3 was unresponsive to all 19 lectins, and any binding was restricted to the head and tail regions. L3 exsheathed after 2-4 months storage could be distinguished by the binding of GSL-I and LTL to H. contortus but not to T. circumcincta. Lectin binding could be a useful adjunct in identifying L3, but lacked the consistency to be definitive, whereas it could be further developed as a practical method of distinguishing parasitic nematodes at other stages in the life cycle, particularly the eggs.

Borrelia burgdorferi sensu lato and Rickettsia spp. infections in hard ticks (Ixodes ricinus) in the region of Hanover (Germany).

In a total of 605 Ixodes (I.) ricinus ticks collected in the spring-months March, April and May 2005, quantitative real time PCR (qPCR) revealed 26.6% Borrelia (B.) burgdorferi sensu lato (sl)-positive ticks, i. e. divided by sex and stage into 31.9% positive adults (34.8% females and 29.0% males) and 18.5% positive nymphs. Mono-infections with genospecies from the B. burgdorferi sl-complex were found in over two thirds of the positive individuals, whereas almost one third showed double- or even triple-infections. Genospecies-specific conventional PCR determined B. afzelii as the most frequent genospecies followed by B. garinii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). Rickettsia spp. were found in 34.2% of the collected ticks, divided into 37.6% adults (42.5% females and 32.8% males) and 29.0% nymphs. Co-infections of Rickettsia-positive ticks with B. burgdorferi sl spirochaetes were present in 10.1% of the ticks. Thereby, adult ticks exhibited a co-infection rate of 13.4% (15.5% females and 11.3% males) and nymphs of 5.0%. Independently of the above mentioned study, 3939 Ixodes ticks, sent in between 2006 and 2010 for B. burgdorferi sl-diagnostic, were examined by qPCR exclusively for B. burgdorferi sl. The resulting B. burgdorferi sl prevalence was 23.1% and 24.4% in 2006 and 2007, respectively, followed by a continuous decrease to 12.8% in 2010. To analyse whether this observed decrease in infection frequency is due to sampling bias, in a current study randomly sampled ticks collected from defined sites equally distributed over the city of Hanover are investigated in a statistically relevant sample size.

Prevalence of Anaplasma phagocytophilum and coinfection with Borrelia burgdorferi sensu lato in the hard tick Ixodes ricinus in the city of Hanover (Germany).

The castor bean tick Ixodes ricinus has been found to be the main vector for Lyme borreliosis spirochetes and Anaplasma phagocytophilum in Central Europe. 1646 I. ricinus ticks from Hanover, a city located in Northern Germany, were examined for infection with A. phagocytophilum and coinfection with Borrelia burgdorferi sensu lato (sl) to obtain so far missing prevalence data for this region. The total A. phagocytophilum infection rate was 3.2% (52/1646 ticks), divided into 4.1% (32/777) adults and 2.3% (20/869) nymphs. Coinfections with B. burgdorferi sl were found in 0.9% of all tick stages. The detected genospecies were B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), and B. garinii, which was the most frequent species in coinfected ticks.

GTP-cyclohydrolase and development in Teladorsagia circumcincta and Dictyocaulus viviparus (Nematoda: Strongylida).

GTP-Cyclohydrolase (GTP-CH) is necessary for the production of tetrahydrobiopterin, a required cofactor for the three aromatic amino acid hydroxylases and nitric oxide synthases. The gene encoding GTP-CH is transcribed at high levels in infective third larval stages of a number of parasitic trichostrongylid nematodes. We explore the potential role of GTP-CH within the processes of nematode development and environmentally-induced hypobiosis. For two species of parasitic nematode that are of major economic and welfare importance to livestock in temperate regions, Teladorsagia circumcincta and Dictyocaulus viviparus, we have demonstrated that each of the pre-parasitic larval stages transcribe high mean levels of cat-4 (the gene encoding GTP-CH). Using quantitative real-time polymerase chain reaction analysis and two different isolates of D. viviparus, only one of which is capable of entering hypobiosis, we have shown that there were only minor differences between these isolates in mean cat-4 transcript levels, both during the parasitic stages and during the earlier environmental life cycle stages (L(1)-L(3)). Taken together, these data indicate that, although both species of nematode produce high levels of cat-4 transcript in pre-parasitic larval stages, GTP-CH levels are unlikely to be involved in the induction of parasite hypobiosis. Alternative roles for GTP-CH in larval development are discussed.

Dynamics of distribution and efficacy of different spot-on permethrin formulations in dogs artificially infested with Dermacentor reticulatus.

BACKGROUND: Varying reports concerning the duration and reliability of different permethrin preparations' efficacy can be found in the literature. The aim of this study was to investigate the dynamics of the distribution and efficacy of four different spot-on formulations with permethrin as the active ingredient formulated with different solvents. To examine the influence of these solvents on the speed of distribution and the acaricidal activity of permethrin in the coat, an in vivo study under laboratory conditions was performed. Six dogs per test period were treated with the recommended dose and 1, 14 and 28 days after treatment dogs were infested with Dermacentor reticulatus ticks: a) on the back, near the application site, and b) on the hind leg, the greatest possible distance from the application site. Efficacies were determined 6 hours after tick infestation to examine the repellent effect and the speed of kill of the products which plays an important role in the context of tick transmitted diseases. RESULTS: After six hours of exposure, a significant acaricidal efficacy (p < 0.001) could be observed in all treated groups over the whole duration of the study, regardless of which product was used. While the arithmetic mean of attached ticks was < 3 on Day 1, numbers increased over the course of the study to a mean of > 9 on Day 28. However, most of these ticks were dead even 28 days after treatment, as the mean of live attached ticks was still < 2. Significant differences could neither be found between the different permethrin spot-on formulations, nor between the two parts of the body (p > 0.05). CONCLUSIONS: All products were able to kill ticks within six hours following infestation from Day 1 to Day 28 after treatment. Additionally, no significant difference between the tick numbers on the back and the hind leg could be found at any time, which implies a homogenous distribution of permethrin over the body. The efficacy of all four products was comparable during the whole study period, showing that the different solvents do not significantly affect the dynamics of distribution.