Evaluation of a commercial coproantigen immunoassay for the detection of Toxocara cati and Ancylostoma tubaeforme in cats and Uncinaria stenocephala in dogs.

Coproantigen immunoassays (IDEXX Fecal Dx® antigen tests) were evaluated for their ability to identify Toxocara cati and Ancylostoma tubaeforme infections in cats and Uncinaria stenocephala infection in dogs. Five cats were experimentally infected with 500 embryonated eggs of T. cati, eight cats with 500 third-stage larvae (L3) of A. tubaeforme and seven dogs with 500 L3 of U. stenocephala. In addition to the three coproantigen tests, the course of infection was monitored by a combined sedimentation-flotation method with ZnSO4 as flotation medium (specific gravity: 1.28-1.30) and a modified McMaster method in case of copromicroscopically positive samples. Eggs of T. cati were first observed between 28 and 54 days post infection (dpi) in four of the five infected cats. In these four cats, positive roundworm coproantigen signals were obtained between 16 and 44 dpi. Positive coproantigen signal always preceded egg observations, but the interval varied between 6 and 30 days. Hookworm-specific positive coproantigen signals were detected in seven of the eight A. tubaeforme infected cats between 10 and 52 dpi, while consecutive egg excretion was observed in three cats between day 26 and 54 pi. Of these three, coproantigen signal preceded egg observation by 12 to 24 days. Four cats had positive coproantigen results in the absence of egg excretion, and one cat never achieved a positive result for egg or coproantigen. In six of seven U. stenocephala infected dogs, infection was confirmed by copromicroscopy between 16 and 24 dpi as well as for hookworm coproantigen between 10 and 14 dpi. Coproantigen signal was detected prior to egg observation by 2 to 14 days. No cross-reactions between the roundworm, hookworm und whipworm tests occurred in study animals. The results of this study demonstrate the ability of the coproantigen tests to detect the common roundworm and hookworm infections in cats and U. stenocephala infections in dogs as well as the ability to detect the prepatent stage of infection.

Do All Roads Lead to Rome? The Potential of Different Approaches to Diagnose Aelurostrongylus abstrusus Infection in Cats.

An infection with the cat lungworm, Aelurostrongylus abstrusus, can be subclinical, but it can also cause severe respiratory clinical signs. Larvae excretion, antibody levels, clinical assessment findings of the respiratory system and diagnostic imaging findings were recorded and compared for six cats with experimental aelurostrongylosis. In five cats, patency started 33-47 days post infection (pi), but two cats excreted larvae only in long intervals and low numbers. Positive ELISA results were observed in four cats with patent aelurostrongylosis, starting between five days before and 85 days after onset of patency. One seropositive cat remained copromicroscopically negative. Mild respiratory signs were observed in all cats examined. A computed tomographic (CT) examination of the lungs displayed distinct alterations, even in absence of evident clinical signs or when larvae excretion was low or negative. The thoracic radiograph evaluation correlated with the CT results, but CT was more distinctive. After anthelmintic treatment in the 25th week post infection, pulmonary imaging findings improved back to normal within 6-24 weeks. This study shows that a multifaceted approach, including diagnostic imaging, can provide a clearer diagnosis and monitoring of disease progression. Furthermore, a CT examination provides an alternative to post mortem examination and worm counts in anthelmintic efficacy studies.

Regional, seasonal, biennial and landscape-associated distribution of Anaplasma phagocytophilum and Rickettsia spp. infections in Ixodes ticks in northern Germany and implications for risk assessment at larger spatial scales.

Tick-associated Rickettsiales are important pathogens with relevance for public and animal health; therefore, knowledge regarding their distribution is essential for risk assessment and disease prevention. To investigate the prevalence of Anaplasma phagocytophilum and Rickettsia spp. in northern Germany, Ixodes ticks were flagged monthly from April to October in 2018 and 2019 at three collection sites each in the regions of Bremen, Emsland, Hanover, Kassel and Uelzen. A total of 3150 ticks (1052 females, 1048 males and 1050 nymphs) were individually examined for rickettsial infections using probe-based quantitative real-time PCR. Overall prevalence of A. phagocytophilum was 6.4 % (202/3150; 6.7 % [71/1052] in females, 7.5 % [79/1048] in males and 5.0 % [52/1050] in nymphs). For Rickettsia spp., the overall prevalence was 29.6 % (931/3150; 33.4 % [351/1052] in females, 28.3 % [297/1048] in males and 27.0 % [283/1050] in nymphs). Rickettsia species identification by real-time pyrosequencing on a subset of 409 positive samples was successful in 407 cases (99.5 %). Rickettsia helvetica was the predominant species with a detection rate of 99.8 % (406/407). Additionally, Rickettsia monacensis was detected in one tick (0.2 %). Generalized linear mixed models showed significant regional as well as monthly differences regarding the prevalence of both pathogens. In addition, the prevalence of both pathogens was significantly higher in 2018 (A. phagocytophilum: 8.0 % [126/1575], Rickettsia spp.: 35.4 % [558/1575]) than in 2019 (A. phagocytophilum: 4.8 % [76/1575], Rickettsia spp.: 23.9 % [373/1575]). In contrast, no effect of landscape type on pathogen prevalence was found. As Rickettsia spp.-detection was based on the single-copy gene gltA, it was possible to calculate the individual pathogen load per tick, which was significantly higher in female ticks than in nymphs (mean values: 8.19 × 104 vs. 9.58 × 103). Regional, seasonal and biennial prevalence differences of tick-transmitted Rickettsiales show the necessity to investigate ticks from multiple locations, over several months and in more than one year to reliably assess the infection risk on a larger geographical scale.

Efficacy of Bravecto® Plus spot-on solution for cats (280 mg/ml fluralaner and 14 mg/ml moxidectin) for the prevention of aelurostrongylosis in experimentally infected cats.

BACKGROUND: The feline lungworm Aelurostrongylus abstrusus affects the lower respiratory tract in cats worldwide. As infections may lead to chronic respiratory changes or even death, preventive treatment in cats with outdoor access is warranted. METHODS: The preventive efficacy of a spot-on solution (Bravecto® Plus spot-on solution for cats, MSD) against cat aelurostrongylosis was evaluated using three different preventive treatment regimes in a negative controlled, randomized and partially blinded laboratory efficacy study with 31 purposed-bred cats. The minimum recommended dose of 2.0 mg moxidectin + 40 mg fluralaner/kg bodyweight was applied once 12 (Group [G]1), 8 (G2) or 4 (G3) weeks before experimental infection with 300 third-stage larvae (L3) of A. abstrusus. Another group served as untreated control (G4). Individual faecal samples were analysed as of day 30 post infection (pi) to monitor larvae excretion. Necropsy was performed at days 47-50 pi. The lungs were examined macroscopically for pathological findings and (pre-)adult worms were counted to assess preventive efficacy. RESULTS: Beginning at day 32-40 pi, all cats of the control group were constantly shedding larvae of A. abstrusus, whereas only one animal of G1 excreted larvae at several consecutive days. In addition, two cats of G1 and G3 and three of G2 were positive on a single occasion. The geometric mean (GM) of the maximum number of excreted larvae was 7574.29 in the control group compared to 1.10 (G1), 1.19 (G2) and 0.53 (G3), resulting in a GM reduction of > 99.9% in all treatment groups. All lungs of the control animals showed severe or very severe alterations at necropsy, while in 94.44% of the treated cats lung pathology was rated as absent or mild. The GM number of (pre-)adult A. abstrusus retrieved from the lungs was 26.57 in the control group, 0.09 in G1 and 0.00 in G2 and G3. Thus, GM worm count reduction was 99.66% in G1 and 100% in G2 and G3. CONCLUSIONS: A single application of Bravecto® Plus spot-on solution at a dose of 2.0 mg moxidectin + 40 mg fluralaner/kg bodyweight reliably prevents cat aelurostrongylosis for at least 12 weeks.

Two-year monitoring of tick abundance and influencing factors in an urban area (city of Hanover, Germany).

Ticks may transmit a variety of human and animal pathogens. Prevalence of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum in ticks has been monitored in the city of Hanover, Germany, since 2005. However, to determine the infection risk for humans and animals, not only pathogen prevalence, but also tick abundance and seasonality need to be taken into account. Therefore, the aim of this study was to investigate tick abundance at ten different collection sites in the city of Hanover, Germany. Collection of questing ticks was performed by the flagging method in the first and second half of each month during the tick season (April-October) in 2017 and 2018. At each 200 m² collection site, one of four 50 m² fields was sampled per visit on a rotational basis, resulting in 100 m² sampled per month. In addition, data on weather conditions, near-ground temperature, relative humidity and vegetation composition were noted at each collection event. In 2017, a total of 1770 ticks were collected, while 1866 ticks were collected in 2018. Ixodes ricinus was the most prevalent species (97.0 % of all ticks, 98.0 % of nymphs, 91.6 % of adults) followed by I. inopinatus (2.3 % of all ticks, 1.1 % of nymphs, 8.0 % of adults), I. frontalis (0.6 % of all ticks, 0.6 % of nymphs, 0.3 % of adults) and I. hexagonus (0.03 % of all ticks, 0.03 % of nymphs, 0.0 % of adults). Using generalized linear mixed modeling, density of I. ricinus and I. inopinatus in 2017 was significantly higher than in 2018. Regarding different landscape types, ticks were significantly more abundant in mixed forests than in parks, with more than 50 ticks/100 m² on average in both years. In urban parks, average tick density amounted to 15 ticks/100 m² in 2017 and 11 ticks/100 m² in 2018 and in broad-leaved forests average tick density was 13 and 18 ticks/100 m² in 2017 and 2018, respectively. Tick density showed a marked peak in June 2017 and in May 2018 at most sites, whereas a less pronounced peak was recognizable in September. Tick density varied considerably between collection sites. However, no statistically significant effect of (micro-)climatic variables, including near-ground temperature, relative humidity and saturation deficit, was found. Thus, further factors, such as the abundance of wildlife hosts, need to be considered in future studies to explain the differences between collection sites.

Transovarial transmission of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus under field conditions extrapolated from DNA detection in questing larvae.

BACKGROUND: Ixodes ricinus constitutes the main European vector tick for the Lyme borreliosis pathogen Borrelia burgdorferi (sensu lato), the relapsing fever borrelia Borrelia miyamotoi, as well as Anaplasma phagocytophilum and several Rickettsia species. Under laboratory conditions, a transovarial transmission to the next tick generation is described for Rickettsia spp. and Borrelia spp., especially regarding B. miyamotoi, whereas the efficiency of transovarial transfer under field conditions is largely unstudied. METHODS: In order to better estimate the potential infection risk by tick larvae for humans and animals, 1500 I. ricinus larvae from 50 collected "nests" (larvae adhering to the flag in a clumped manner) were individually examined for Borrelia, Rickettsia and A. phagocytophilum DNA using quantitative real-time PCR (qPCR). RESULTS: Thirty-nine of 50 nests each (78.0%, 95% CI: 64.0-88.5%) were positive for Borrelia spp. and Rickettsia spp. DNA, and in three nests (6.0%, 95% CI: 1.3-16.5%) A. phagocytophilum DNA was detected. Overall, DNA from at least one pathogen could be detected in 90.0% (45/50, 95% CI: 78.2-96.7%) of the nests. Of the 1500 larvae, 137 were positive for Borrelia spp. DNA (9.1%, 95% CI: 7.7-10.7%), 341 for Rickettsia spp. DNA (22.7%, 95% CI: 20.6-24.9%) and three for A. phagocytophilum DNA (0.2%, 95% CI: 0-0.6%). Quantity of Borrelia spp. and Anaplasma spp. DNA in positive larvae was low, with 2.7 × 100Borrelia 5S-23S gene copies and 2.4 × 101A. phagocytophilum msp2/p44 gene copies detected on average, while Rickettsia-positive samples contained on average 5.4 × 102gltA gene copies. Coinfections were found in 66.0% (33/50, 95% CI: 51.2-78.8%) of the nests and 8.6% (38/443, 95% CI: 6.1-11.6%) of positive larvae. In fact, larvae had a significantly higher probability of being infected with Borrelia spp. or Rickettsia spp. when both pathogens were present in the nest. CONCLUSIONS: This study provides evidence for transovarial transmission of Rickettsia spp. and Borrelia spp. in I. ricinus under field conditions, possibly facilitating pathogen persistence in the ecosystem and reducing the dependence on the presence of suitable reservoir hosts. Further studies are needed to prove transovarial transmission and to explain the surprisingly high proportion of nests containing Rickettsia and/or Borrelia DNA-positive larvae compared to infection rates in adult ticks commonly reported in other studies.

Ixodes inopinatus in northern Germany: occurrence and potential vector role for Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum in comparison with Ixodes ricinus.

In 2014, a new tick species, Ixodes inopinatus, was described, which is closely related to Ixodes ricinus. So far, I. inopinatus has been found in Tunisia, Morocco, Spain, Portugal, Romania, Austria, and southern Germany. No data is yet available regarding occurrence of I. inopinatus in northern Germany and the potential role of I. inopinatus as a vector for tick-borne pathogens. Therefore, 3845 DNA samples from Ixodes ticks collected for prevalence studies on Borrelia spp., Rickettsia spp., and Anaplasma phagocytophilum during the years 2010-2015 in the northern German cities of Hamburg and Hanover were differentiated into I. ricinus or I. inopinatus by sequencing a part of the 16S rRNA gene. In total, 4% (137/3845) of the sequenced ticks were assigned to the species I. inopinatus and 96% (3708/3845) to I. ricinus. The prevalence of Borrelia spp., Rickettsia spp., and A. phagocytophilum DNA in I. inopinatus was 34% (46/137), 46% (63/137), and 3% (4/137), respectively, whereas the prevalence of these bacteria in I. ricinus was 25% (919/3708), 47% (1729/3708), and 4% (135/3708), respectively. Compared with I. ricinus, significantly more I. inopinatus ticks tested positive for Borrelia. To the best of our knowledge, this is the first report of I. inopinatus in northern Germany. Detection of the DNA of Borrelia spp., Rickettsia spp., and A. phagocytophilum in questing I. inopinatus indicates a potential role of this tick species as a vector of these pathogens, which needs to be confirmed by transmission experiments.

Ixodes frontalis: a neglected but ubiquitous tick species in Germany.

The ornithophilic tick species Ixodes frontalis (Panzer) is spatially distributed in Europe, Asia and northern Africa. It can serve as a carrier of different bacteria and viruses, but little is known of its actual vector competence. In addition, adult females of this species are associated with the avian "tick-related syndrome" (TRS). Like most ornithophilic tick species, I. frontalis is usually collected from bird nests or directly from their hosts. Reports of I. frontalis being collected with the flagging method are scarce. In Germany, the species is considered as very rare. In the few reports that are available, the majority of ticks has been discovered on migrating birds. Therefore, knowledge of the actual distribution of this species in Germany was strictly limited. In this study, we report the finding of 1084 individuals of I. frontalis collected with the flagging method in urban areas and gardens in several regions of Germany. Furthermore, the species was discovered on 35 dead Eurasian blackbirds (Turdus merula) from all over Germany. Finally, we report of five new possible cases of TRS, three in free-ranging birds and two in captive birds, including a Harris's hawk (Parabuteo unicinctus) kept for falconry.