Abundance and seasonal activity of questing Ixodes ricinus ticks in their natural habitats in southern Germany in 2011.

Questing ticks were sampled monthly over a period of 11 months from February, 2011 to December, 2011 at 13 sites in southern Germany using the flagging method. The ticks were identified to species, gender, and stadium. Although both I. ricinus and D. reticulatus were sampled, this study concentrated on I. ricinus, since it was the most abundant tick to be found. Additional weather data (air and soil temperature, relative air humidity, precipitation, sunshine duration) were recorded on each sampling site and the local vegetation described. A total of 14, 394 ticks was collected (7,862 larvae, 5,568 nymphs, 964 adults) and their activity was recorded in order to determine the seasonal activity pattern over different periods of the year. In contrast to the widely accepted pattern of a bimodal seasonal activity in moderate areas with a dominant peak in spring and a minor peak in autumn, a unimodal activity pattern was found for all development stages on six of the 12 sampling sites. Tick abundance was compared to weather variables. Tick host-seeking activity was found to be significantly dependent on the temperature at ground level, precipitation, and sunshine duration as well as relative air humidity. Adult ticks showed a positive correlation with the duration of sunshine, whereas nymphs were mostly unaffected by this phenomenon.

Diversity of Babesia and Rickettsia species in questing Ixodes ricinus: a longitudinal study in urban, pasture, and natural habitats.

In a previous study, our group investigated the Babesia spp. prevalence in questing Ixodes ricinus ticks from nine city parks in South Germany in the years 2009 and 2010. We showed predominant prevalence of B. venatorum (in previous literature also known as Babesia sp. EU1), especially in those parks in a more natural condition and with occurrence of large wild animals, such as roe deer. To obtain longitudinal data and to broaden the knowledge about this pathogen, further investigations were carried out in 2011 and 2012 in four of those city parks. Two additional habitat types were chosen for comparison of prevalence data and species analysis focusing on occurrence of potential reservoir hosts. A total of 10,303 questing I. ricinus were collected in four city parks, a pasture, and a natural area in Bavaria, and a representative number of samples were investigated for prevalence of DNA of Babesia spp. (n=4381) and Rickettsia spp. (n=2186) by PCR. In the natural and pasture area, a significantly higher Babesia spp. prevalence compared to the urban area was detected. The natural area revealed sequences of B. microti, B. venatorum, and B. capreoli. In the pasture and urban habitat, predominantly B. venatorum was found, whereas B. capreoli was less frequent and only one B. microti-infected tick was found. All B. microti sequences were 100% identical to the zoonotic Jena/Germany strain. For Rickettsia spp., the significantly highest prevalence was also detected in the natural and pasture areas, whereas lower prevalence was found in the urban area. Sequence analysis revealed R. helvetica (98%) and R. monacensis (2%). Prevalence rates and occurrence of Babesia spp. and Rickettsia spp. differed in urban, pasture and natural sites, most likely depending on the habitat structure (natural or cultivated) and therefore on the appearance and availability of reservoir hosts like roe deer or small mammals.

Detection of tick-borne pathogens in roe deer (Capreolus capreolus), in questing ticks (Ixodes ricinus), and in ticks infesting roe deer in southern Germany.

The hard tick Ixodes ricinus is the most common tick in Central Europe and plays an important role as a vector of several pathogens. In the complex life cycles of these pathogens, the role of wild animals as natural reservoirs has been discussed. The aims of this study were to investigate the role of roe deer (Capreolus capreolus) as a potential reservoir host for Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp. Therefore, we explored the differences in the infection rates of roe deer and engorged and questing ticks with these pathogens from a single forest site with special attention to coinfection. Blood, spleen, and skin samples of a total of 95 roe deer individuals were screened by molecular methods for these pathogens from September 2010 to January 2012 in the 'Angelberger Forst' (Bavaria, Germany). Moreover, 331 engorged ticks from 44 roe deer individuals and 199 host-seeking ticks from the same area were screened. Altogether, the following prevalence rates and a high diversity of species were detected for the respective pathogens in individual animals and ticks: (i) Babesia spp.: roe deer, 89.5%; engorged ticks, 7.3%; questing ticks: adults, 2.5%, nymphs, 3.3%. Sequencing revealed B. venatorum, B. capreoli, and B. microti. (ii) A. phagocytophilum: roe deer 98.9%; engorged ticks, 86.1%; questing ticks: adults, 8.9%, nymphs, 0.8%. (iii) Rickettsia spp.: roe deer, 0%; engorged ticks, 16.6%; questing ticks: adults, 13.9%, nymphs, 17.5%. Sequencing revealed R. helvetica. Furthermore, several coinfections were detected in both roe deer and ticks. The high prevalence rates of B. capreoli and A. phagocytophilum in roe deer support their role as reservoir hosts for these pathogens, but no evidence for a role of roe deer in the life cycle of R. helvetica could be provided.

Anaplasma phagocytophilum in questing Ixodes ricinus ticks: comparison of prevalences and partial 16S rRNA gene variants in urban, pasture, and natural habitats.

Urban, natural, and pasture areas were investigated for prevalences and 16S rRNA gene variants of Anaplasma phagocytophilum in questing Ixodes ricinus ticks. The prevalences differed significantly between habitat types, and year-to-year variations in prevalence and habitat-dependent occurrence of 16S rRNA gene variants were detected.

Candidatus Neoehrlichia mikurensis in rodents in an area with sympatric existence of the hard ticks Ixodes ricinus and Dermacentor reticulatus, Germany.

BACKGROUND: Candidatus Neoehrlichia mikurensis (CNM) has been described in the hard tick Ixodes ricinus and rodents as well as in some severe cases of human disease. The aims of this study were to identify DNA of CNM in small mammals, the ticks parasitizing them and questing ticks in areas with sympatric existence of Ixodes ricinus and Dermacentor reticulatus in Germany. METHODS: Blood, transudate and organ samples (spleen, kidney, liver, skin) of 91 small mammals and host-attached ticks from altogether 50 small mammals as well as questing I. ricinus ticks (n=782) were screened with a real-time PCR for DNA of CNM. RESULTS: 52.7% of the small mammals were positive for CNM-DNA. The majority of the infected animals were yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus). Small mammals with tick infestation were more often infected with CNM than small mammals without ticks. Compared with the prevalence of ~25% in the questing I. ricinus ticks, twice the prevalence in the rodents provides evidence for their role as reservoir hosts for CNM. CONCLUSION: The high prevalence of this pathogen in the investigated areas in both rodents and ticks points towards the need for more specific investigation on its role as a human pathogen.

Babesia spp. and Anaplasma phagocytophilum in questing ticks, ticks parasitizing rodents and the parasitized rodents--analyzing the host-pathogen-vector interface in a metropolitan area.

BACKGROUND: The aims of this study were to evaluate the host-tick-pathogen interface of Babesia spp. and Anaplasma phagocytophilum in restored areas in both questing and host-attached Ixodes ricinus and Dermacentor reticulatus and their small mammalian hosts. METHODS: Questing ticks were collected from 5 sites within the city of Leipzig, Germany, in 2009. Small mammals were trapped at 3 of the 5 sites during 2010 and 2011. DNA extracts of questing and host-attached I. ricinus and D. reticulatus and of several tissue types of small mammals (the majority bank voles and yellow-necked mice), were investigated by PCR followed by sequencing for the occurrence of DNA of Babesia spp. and by real-time PCR for A. phagocytophilum. A selected number of samples positive for A. phagocytophilum were further investigated for variants of the partial 16S rRNA gene. Co-infection with Rickettsia spp. in the questing ticks was additionally investigated. RESULTS: 4.1% of questing I. ricinus ticks, but no D. reticulatus, were positive for Babesia sp. and 8.7% of I. ricinus for A. phagocytophilum. Sequencing revealed B. microti, B. capreoli and Babesia spp. EU1 in Leipzig and sequence analysis of the partial 16S RNA gene of A. phagocytophilum revealed variants either rarely reported in human cases or associated with cervid hosts. The statistical analysis revealed significantly less ticks infected with A. phagocytophilum in a city park in Leipzig as compared to the other sampling sites. A. phagocytophilum-DNA was detected in 2 bank voles, DNA of B. microti in 1 striped field-mouse and of Babesia sp. EU1 in the skin tissue of a mole. Co-infections were detected. CONCLUSION: Our results show the involvement of small mammals in the natural endemic cycles of tick-borne pathogens. A more thorough understanding of the interactions of ticks, pathogens and hosts is the essential basis for effective preventive control measures.

Dynamics of tick infestations in foxes in Thuringia, Germany.

This study aimed to provide up-to-date information on the dynamics of tick infestations on foxes in Thuringia, as the most recent information available was published in 1997. Fox carcasses that had been sent to the Thuringian State Authority for Food Safety and Consumer Protection (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz - TLLV), between January 1st and December 31st, 2009, were examined for the presence of ticks. All ticks collected were stored at -20 °C before being identified and classified according to their developmental stage and sex. Out of a total of 1286 foxes examined, 989 (76.9%) were infested with ticks. A total of 13,227 ticks were collected from the foxes. The stage most frequently found was the larva (48.1%), followed by the adult (34.1%), and the nymphal stage (17.8%). Regarding the adult stage, Ixodes ricinus was the most frequent tick species detected (82.2%), followed by I. canisuga (10.8%) and I. hexagonus (6.7%). Dermacentor reticulatus ticks were very rare (0.3%). With regard to nymphs, I. canisuga and I. hexagonus were the most frequent tick species found, and this was also assumed for the larval stage. The results indicate the occurrence of tick infestations in foxes throughout the year, mainly by I. ricinus, I. canisuga, and I. hexagonus, with seasonal variations. Foxes were infested by I. ricinus ticks significantly more frequently from April to September. This applied to all tick developmental stages, but especially to adults. In contrast to I. ricinus, the infestation of foxes with I. canisuga and I. hexagonus was significantly higher from January to March and from October to December, especially with the immature developmental stages.

Prevalence of Anaplasma phagocytophilum in Ixodes ricinus in Bavarian public parks, Germany.

Previous studies have shown that public parks provide extraordinary habitats for ticks and tick-borne pathogens. Therefore, the aim of this study was to investigate the tick abundance (ticks/100 m(2)) in urban areas and the prevalence of Anaplasma phagocytophilum. Collection of ticks was performed by the flagging method in selected Bavarian public parks in a 2-year survey. DNA from 30 ticks of each developmental stage, sampling site and month was extracted and screened by specific real-time PCR for A. phagocytophilum. Selected positive samples were further genetically differentiated by nested PCR, targeting the 16S rRNA gene. A total of 13,403 Ixodes ricinus were collected with an average tick abundance of 15-53 ticks/100 m(2) in 2009 and 15-35 ticks/100 m(2) in 2010, depending on the sampling site. DNA of A. phagocytophilum was detected in 11.6% (n=146) and 8.5% (n=50) of adult females, 13.3% (n=164) and 9.2% (n=51) of adult males as well as 5% (n=60) and 3.9% (n=29) of nymphs for 2009 (n=3685) and 2010 (n=1884), respectively. 16S rRNA gene analysis revealed 6 different genetic variants, 2 of which were 100% identical to known sequences in humans. The results give strong evidence that the occurrence of A. phagocytophilum in I. ricinus is highly variable in different habitat types due to geographical, climatic, and biological factors as well as different genetic variants of A. phagocytophilum circulated in ticks with a noticeable habitat and host tropism.

Occurrence of Babesia spp., Rickettsia spp. and Bartonella spp. in Ixodes ricinus in Bavarian public parks, Germany.

BACKGROUND: Only limited information is available about the occurrence of ticks and tick-borne pathogens in public parks, which are areas strongly influenced by human beings. For this reason, Ixodes ricinus were collected in public parks of different Bavarian cities in a 2-year survey (2009 and 2010) and screened for DNA of Babesia spp., Rickettsia spp. and Bartonella spp. by PCR. Species identification was performed by sequence analysis and alignment with existing sequences in GenBank. Additionally, coinfections with Anaplasma phagocytophilum were investigated. RESULTS: The following prevalences were detected: Babesia spp.: 0.4% (n = 17, including one pool of two larvae) in 2009 and 0.5 to 0.7% (n = 11, including one pool of five larvae) in 2010; Rickettsia spp.: 6.4 to 7.7% (n = 285, including 16 pools of 76 larvae) in 2009. DNA of Bartonella spp. in I. ricinus in Bavarian public parks could not be identified. Sequence analysis revealed the following species: Babesia sp. EU1 (n = 25), B. divergens (n = 1), B. divergens/capreoli (n = 1), B. gibsoni-like (n = 1), R. helvetica (n = 272), R. monacensis IrR/Munich (n = 12) and unspecified R. monacensis (n = 1). The majority of coinfections were R. helvetica with A. phagocytophilum (n = 27), but coinfections between Babesia spp. and A. phagocytophilum, or Babesia spp. and R. helvetica were also detected. CONCLUSIONS: I. ricinus ticks in urban areas of Germany harbor several tick-borne pathogens and coinfections were also observed. Public parks are of particularly great interest regarding the epidemiology of tick-borne pathogens, because of differences in both the prevalence of pathogens in ticks as well as a varying species arrangement when compared to woodland areas. The record of DNA of a Babesia gibsoni-like pathogen detected in I. ricinus suggests that I. ricinus may harbor and transmit more Babesia spp. than previously known. Because of their high recreational value for human beings, urban green areas are likely to remain in the research focus on public health issues.

Evaluation of N-terminal pro-B-type natriuretic peptide as a diagnostic marker of various stages of cardiomyopathy in Doberman Pinschers.

OBJECTIVE: To evaluate the diagnostic value of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations in Doberman Pinschers in various stages of dilated cardiomyopathy (DCM). ANIMALS: 328 Doberman Pinschers. PROCEDURES: Staging of DCM was determined via analysis of results of physical examinations, 24-hour ambulatory ECG (Holter) recordings, and echocardiographic evaluations. Plasma samples for NT-proBNP assays were obtained at each examination. Concentrations of NT-proBNP were measured in 337 samples obtained from 196 healthy Doberman Pinschers (control dogs) and in 195 samples obtained from 132 Doberman Pinschers in various stages of DCM. These included dogs that had ventricular premature contractions (VPCs; 79 samples), echocardiographic changes (23 samples), or both (51 samples); 16 samples were from dogs with overt DCM, and 26 were from dogs that were considered normal during initial examination but developed DCM within 1.5 years after this assessment. Receiver operating characteristic curves were analyzed to determine sensitivity and specificity of NT-proBNP concentrations for detection of DCM. RESULTS: NT-proBNP concentrations in dogs that had or developed DCM were significantly higher than those of control dogs. Sensitivity and specificity of NT-proBNP concentrations (cutoff value, > 400 pmol/L) to detect all stages of DCM were 81.1 % and 75.0%, respectively; sensitivity was 90.0% and specificity was 75.0% to predict echocardiographic changes. Specificity to detect echocardiographic changes was 90.4% at a cutoff value of 550 pmol/L. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma concentrations of NT-proBNP were increased in dogs with DCM and in apparently healthy dogs that developed DCM within 1.5 years after samples were obtained, compared with concentrations in control dogs.

Utility of measuring plasma N-terminal pro-brain natriuretic peptide in detecting hypertrophic cardiomyopathy and differentiating grades of severity in cats.

BACKGROUND: Cats with hypertrophic cardiomyopathy (HCM) often have no clinical signs or subtle signs. Measurement of N-terminal pro-brain natriuretic peptide (NT-proBNP) has been demonstrated in people to be highly specific for heart disease and also correlates with severity of HCM. NT-proBNP may also be valuable in detecting and grading HCM in cats, but results to date have been equivocal. OBJECTIVES: The aims of this study were to evaluate NT-proBNP as a screening test for diagnosis of HCM in cats and determine an appropriate cut-off value and to determine if NT-proBNP concentrations correlated with severity of HCM in cats. METHODS: Plasma NT-proBNP concentrations were measured in 201 cats using an ELISA designed for use in cats. Cats were classified using echocardiography as clinically healthy controls (n=99) or cats with equivocal (n=9), mild (n=15), moderate (n=17), or severe (n=61) HCM. RESULTS: NT-proBNP concentrations (median; 25th-75th interquartile percentiles) in mildly (216.1; 87.6-392.5 pmol/L), moderately (282.7; 131.9-466.6 pmol/L), and severely (839.5; 655.3-1046.4 pmol/L) affected cats were significantly higher than those in healthy controls (18.9; 3.4-62.4 pmol/L). Concentrations in severely affected cats were significantly higher than in cats from other HCM groups. There was no significant difference between mild and moderate HCM. Cut-off values >49 pmol/L had a sensitivity of 97.8% and specificity of 66.7%; >100 pmol/L had a sensitivity of 92.4% and specificity of 93.9%; and >150 pmol/L had a sensitivity of 88% and a specificity of 100%. CONCLUSIONS: NT-proBNP with a cut-off value of >100 pmol/L was useful in detecting even mild HCM. Cats with increased NT-proBNP concentrations should be examined by echocardiography.

The accuracy of the FAMACHA-method in detecting anaemia and haemonchosis in goat flocks in Switzerland under field conditions.

In this study, goats from six farms in Central Switzerland were examined for the evaluation of the FAMACHA-method under middle European conditions. Individual faecal egg counts were determined at a 4-week interval for a period of 6 months and the gastrointestinal nematode (GIN) genera were differentiated using larval culture. Simultaneously, the goats were bled for packed cell volume (PCV) determination and scored for anaemia of the conjunctiva according to the FAMACHA-method. The three methods used for evaluating haemonchosis, namely FEC, PCV and FAMACHA-score, were compared to test the FAMACHA-method for its accuracy and efficacy in detecting haemonchosis in Switzerland. PCV and FAMACHA-score correlated significantly during the entire period of 6 months, whereas PCV and FEC correlated significantly in four study months. The FAMACHA-score and FEC correlated significantly in June only. PCV served as the gold standard for evaluating the accuracy of FAMACHA-method in detecting anaemic goats. The sensitivity of FAMACHA in detecting anaemic goats was 93%, using the anaemia criteria cut-offs FAMACHA-categories >or=3 and PCV <22%. The applicability of the method for detecting goats which needed treatment was tested with FEC >300 epg and >600 epg as cut-off values for treatment. The sensitivity of the method for detecting goats which needed a treatment was 76%, with regard to FEC of Haemonchus contortus (treatment cut-offs: FAMACHA >or=3 and FEC >300 epg). The percentage of false negatives (FEC-Hc-portion) was less than 11%. In addition, the use of FAMACHA categories >or=3, as a treatment indicator, revealed that 64% of the animals were recommended for treatment. These results indicate the suitability of FAMACHA as an additional part of an integrated anthelmintic control of goat flocks in Switzerland.

Anthelminthic resistance of Haemonchus contortus in small ruminants in Switzerland and Southern Germany.

Two goat and two sheep flocks have been found to be suspicious of a clinically evident reduced anthelmintic efficacy, i.e. lacking improvement of gastrointestinal disorders, insufficient weight gain and continuing inappetence after anthelmintic treatments. In order to conduct an appropriate evaluation of the efficacy the following trials were performed: the faecal egg count reduction test on the studied goats of the two herds revealed a reduction of the egg-excretion after the eprinomectin-treatment (1 mg/kg BW, pour-on) of 17.4% and 27.5%, respectively, which clearly confirms the occurrence of anthelmintic resistance against eprinomectin in these two herds. The alternatively administered moxidectin-treatment (1 mg/kg BW, pour-on) of one flock resulted in a 99.1% faecal egg count reduction. In both sheep flocks, 30 randomly selected sheep were divided in three groups and each group was treated with a different anthelmintic, according to the instructions for use. The faecal egg count reductions for the various groups treated orally with benzimidazoles were 70.8% and 55.3% (albendazole), 52.4% (fenbendazole) and 47.3% (oxfendazole). The two moxidectin-treated groups (0.2 mg/kg BW, oral) showed an EpG-reduction of 100% and 44.3%, respectively, thus also demonstrating resistance against macrocyclic lactones. Pre- and post-treatment faecal larval cultures revealed Haemonchus contortus as the predominant resistant species.