Plasma pharmacokinetics of clorsulon following administration of a single subcutaneous or intravenous injection to cattle.

The benzenedisulfonamide derivative clorsulon is a potent fasciolicide which is marketed in fixed combination injectables, typically combined with the macrocyclic lactone ivermectin. In the presented pharmacokinetic study, the plasma profile of clorsulon in 32 healthy, young Brown Swiss cattle was administered a single intravenous dose at 3 mg/kg body weight or subcutaneously at 3, 6 or 12 mg/kg body weight (4 intact male and 4 female animals per treatment) as a 30% w/v clorsulon injection formulation. Serial blood samples were collected up to 24 days after administration to establish the pharmacokinetics, bioavailability and dose proportionality of clorsulon. Following a single intravenous injection of clorsulon at 3 mg/kg body weight, the area under the concentration versus time curve from the start of dose administration to the time of the last quantifiable concentration (AUClast ) was 4830 ± 941 day*ng/mL, and half-live was 2.37 ± 0.98 days. The back extrapolated concentration at time 0 was 38,500 ± 6070 ng/mL. The volume of distribution at steady state and clearance were 685 ± 107 mL/kg and 664 ± 127 mL/day/kg, respectively. In the groups dosed at 3, 6 or 12 mg/kg body weight by subcutaneous injection, clorsulon plasma concentrations rose to maximum within 0.5 day and decreased to the last sample point. For these groups, the maximum plasma clorsulon concentrations were 3100 ± 838, 5250 ± 1220 and 10,800 ± 1730 ng/mL, respectively, and the AUClast was 5330 ± 925, 9630 ± 1300 and 21,500 ± 3320 day*ng/mL, respectively. Half-lives, 2.01 ± 0.62, 3.84 ± 1.42 and 5.36 ± 0.60 days, respectively, increased significantly with dose, likely related to increasing dose volume. Clorsulon was well absorbed and fully bioavailable (103%-114%) after subcutaneous injection. No gender-related difference in systemic exposure was observed. Assessment of Cmax and AUClast demonstrated a proportional increase in systemic exposure to the clorsulon subcutaneous doses over the range of 3-12 mg/kg body weight.

Host-pathogen associations revealed by genotyping of European strains of Anaplasma phagocytophilum to describe natural endemic cycles.

BACKGROUND: The zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied. METHODS: Sequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets. RESULTS: The highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals. CONCLUSIONS: In this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host-pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.

Multispecies helminth parasitism of grazing dairy cows in Germany and Austria, examined in the housing period.

Helminth composition and burden data for dairy cows have not been reported for >40 years for Germany and even less information is available for Austria. In the context of two recent studies, helminth parasitism was studied in 32 cows (23 from six farms in Bavaria and Tyrol; 9 from one farm in Saxony) from pasture-based dairy farms necropsied during the housing period. Helminths were enumerated and identified based on morphological characters (all helminths but rumen flukes) or molecular techniques (rumen flukes). Thirteen species of gastrointestinal nematodes and two species each of liver flukes (Fasciola hepatica, Dicrocoelium dendriticum) and rumen flukes (Calicophoron daubneyi, Paramphistomum leydeni) were recorded; no lungworms were recovered from any cow. Early fourth-stage (inhibited) larval Ostertagia species nematodes (210 to 140,600) were recovered from all cows, 31 each had adult Ostertagia ostertagi/Ostertagia lyrata (40 to 2020) and Trichostrongylus axei (10 to 53,400), 23 Oesophagostomum radiatum (1 to 242) and 20 Cooperia punctata (10 to 3330). Other nematodes present in descending order of prevalence were: Cooperia oncophora/Cooperia surnabada, Ostertagia leptospicularis/Ostertagia kolchida, Oesophagostomum venulosum, Bunostomum phlebotomum, Chabertia ovina, Nematodirus helvetianus, Trichostrongylus longispicularis, Haemonchus contortus and Aonchotheca bilobata. The cows from Bavaria and Tyrol harbored more total gastrointestinal nematodes than that from Saxony (geometric mean adult plus inhibited larval nematodes, 6510 vs. 2051, respectively). However, in both cohorts of cows abomasal nematodes accounted for ∼97% of the total nematode burden with inhibited larval Ostertagia species nematodes contributing over 70% of the total gastrointestinal nematode burden and âˆ¼ 96% of the Ostertagia species burden. Approximately 44%, 37% and 19% of the cows harbored <5000, 5000 to 10,000 or > 10,000 total gastrointestinal nematodes, respectively. Fecal nematode egg and coproculture nematode larval counts significantly correlated with the cows' total adult nematode burden (rs = 0.354, p < 0.05, and rs = 0.608, p < 0.001, respectively). Although the magnitude of nematode burden to exert production effects on dairy cows is not well defined and may vary relative to several factors including nutritional supplementation, the level of mixed parasitism found in this investigation supports consideration of grazing dairy cows in helminth control measures, especially at the time of housing in autumn.

Efficacy of eprinomectin topical solution and eprinomectin extended-release injection treatments against developing larval and adult Chabertia ovina and Oesophagostomum venulosum - two less common cattle nematode parasites.

The efficacy of the topical and injection formulations of eprinomectin (0.5 % w/v topical solution, EPRINEX®; 5 % w/v extended-release injection, LONGRANGE®; Boehringer Ingelheim) against larval and adult stages of the rare bovine large intestine nematodes Chabertia ovina and Oesophagostomum venulosum was evaluated in one Good Clinical Practice study compliant to World Association for the Advancement of Veterinary Parasitology and International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products anthelmintic guidelines. Forty nematode naïve induced infected Brown Swiss calves were allocated into five groups of eight completely at random. Inoculation with isolates obtained in Germany was staggered so that the parasites of the two species were, when the treatments were administered, concurrently fourth-stage larval or adult nematodes, respectively. One group served as untreated control, two groups each received eprinomectin 0.5 % w/v topical solution or eprinomectin 5 % w/v extended-release injection per product label. Animals were necropsied at one and the same day (corresponding to six weeks or two weeks after administration of treatments against fourth-stage larval or adult nematodes) for parasite recovery and count. Both C. ovina (range, 39-604) and O. venulosum (range, 3-23) were recovered from all eight controls while among the 32 calves which were administered topical solution or extended-release injection eprinomectin, only a single C. ovina specimen was isolated from one animal which received the injection product when the nematodes were adult. Eprinomectin treatments were > 99.9 % effective against fourth-stage larval or adult C. ovina and O. venulosum nematodes (p 0.001 at α = 0.05). All animals accepted the treatments well. No adverse events or other health problems were observed in the study animals.

Feasibility of establishing a veterinary marker to total residue in edible tissues with non-radiolabeled study using high-resolution mass spectrometry.

Traditionally, in vivo metabolism and total residue studies in veterinary drug research were conducted using radiolabeled drug where information on metabolite profiles and marker residue to total residue ratio is obtained. The Veterinary International Conference on Harmonisation (VICH) guideline GL46 indicates that the metabolism and residue kinetics in food-producing animals may be documented by an alternative approach, one other than the traditional radiolabeled study. High-resolution mass spectrometry (HRMS) has been widely used in human pharmaceutical R&D from metabolite profiling and identification in early drug discovery to first-in-human (FIH) studies in development. Recent advances in data mining tools have greatly improved the metabolite profiling capability with HRMS. It is now routine to study metabolism using non-radiolabeled samples without missing any major metabolites. In the current paper, we explored the feasibility of conducting non-radiolabeled marker residue studies to obtain metabolism information using HRMS. Metabolite profiles of gamithromycin in edible tissues of sheep treated with 6 mg/kg body weight subcutaneous injections were obtained with HRMS. The semi-quantitative relationship between the level of gamithromycin and the total treatment-related residues was established by determining the percentages of extracted ion chromatograms for metabolites and parent compound residues in each tissue. Major components (gamithromycin and its metabolite, declad) were measured quantitatively using a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. Metabolite profiles in excreta were also obtained and the major components measured quantitatively with a LC-MS/MS method to ensure no major metabolite was missing. Combining previous knowledge of marker residue studies in cattle and swine, as well as an in vitro comparative metabolism study with metabolite data across various species, gamithromycin was designated as the marker residue in sheep edible tissues. The marker to total residue ratios were established using a combination of the semi-quantitative HRMS results and quantitative results with the major components: the marker residue and declad. The pros and cons of the HRMS method as well as the appropriate use of the method for marker residue studies are discussed.

Eprinomectin 5% w/v extended-release injection for the treatment of larval and/or adult Bunostomum phlebotomum, Haemonchus contortus, H. placei, Nematodirus helvetianus, Oesophagostomum radiatum, and Trichostrongylus colubriformis infections in cattle.

In an effort to generate data for regulatory purposes on the therapeutic efficacy of eprinomectin 5% w/v extended-release injection (Eprinomectin ERI) (LongRange®, Boehringer Ingelheim) against infections of immature and adult stages of some nematode species (or stages) which are generally less common or predominantly seen in younger cattle, nine studies (minimum two per parasite and stage) were conducted in the USA and Germany. A total of 218 young cattle were included in seven experimentally induced infection studies (180 animals) and two studies with naturally acquired nematode infections (38 animals), which were compliant with WAAVP and VICH guidelines. In each study, cattle were randomly assigned into groups which received 1 mL per 50 kg body weight of either saline (controls) or Eprinomectin ERI (equivalent to 1 mg eprinomectin per kg body weight) via subcutaneous injection when the parasites were developing fourth-stage larval (L4) or adult nematodes. Following necropsy and parasite recovery, percentage efficacy was calculated based on the comparison of geometric mean nematode counts of the Eprinomectin ERI- vs. the saline-treated animals. Eprinomectin ERI-treated cattle had significantly (p < 0.05) lower counts of each species and stage of nematodes than the controls. Eprinomectin ERI treatment was demonstrated to be efficacious (> 90%) against L4 and adult Bunostomum phlebotomum and Nematodirus helvetianus; against L4 Haemonchus placei, Oesophagostomum radiatum, and Trichostrongylus colubriformis; and against adult H. contortus. Percentage efficacy against L4 H. contortus was variable (69.6 to 100%). All treatments were well accepted, and no treatment-related health problems were observed in any study.

Naturally acquired ovine-adapted nematode infections in young cattle and their treatment with eprinomectin 5% w/v extended-release injection.

The objective of this controlled study was to determine the anthelmintic efficacy of eprinomectin 5% w/v extended-release injection (Eprinomectin ERI; LONGRANGE®, Boehringer Ingelheim Animal Health) against primarily ovine-adapted gastrointestinal strongylid nematode (GIN) parasites in naturally infected young cattle. Eighteen calves which grazed on a sheep-dominated mixed sheep-cattle farm were randomly allocated into two equal groups (saline [control] or Eprinomectin ERI, administered each at 1 mL/50 kg body weight once subcutaneously), treated and euthanized 14 days thereafter for a differential GIN count. Ten species of strongylid nematodes were recovered from the control calves (ovine-adapted Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Cooperia curticei, Nematodirus battus, Chabertia ovina; bovine-adapted Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus; 'generalist' Trichostrongylus axei). Adult GIN counts ranged from 1,540 to 5,244 for the control calves and from zero to 110 for the Eprinomectin ERI-treated calves. Accepting the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products anthelmintic guidelines criteria on adequacy of infections to be demonstrated in the control animals, this study showed that Ch. ovina, C. curticei, H. contortus, N. battus, T. axei, and T. colubriformis were reduced significantly (p < 0.0001) by ≥ 98.7% in the animals treated with Eprinomectin ERI. In conclusion, Eprinomectin ERI treatment was efficacious against a range of ovine-adapted nematode parasites in naturally infected young cattle.

Pharmacokinetic-pharmacodynamic relationships of pour-on administered eprinomectin in nematode-infected lactating female and male castrated dairy breed goats.

Eprinomectin (EPM), a macrocyclic lactone with low excretion in milk and high efficacy against endoparasites and ectoparasites, is widely used in veterinary medicine. In this paper, EPM pharmacokinetics and anthelmintic efficacy previously established in one study with lactating female goats and three studies with male castrated growing dairy breed goats (all with induced mixed adult gastrointestinal nematode parasitism and treated with a single 1-mg/kg pour-on administration of EPM) were retrospectively evaluated using pharmacokinetic-pharmacodynamic (PK-PD) modeling. The PK-PD analyses between EPM exposure (Cmax and AUClast) and anthelmintic response (percent efficacy) were performed for lactating female goats only and pooled lactating female and male castrated goats. The Cmax and AUClast showed no significant difference between lactating female goats and combined male castrated goats. PK-PD modeling demonstrated Trichostrongylus colubriformis, a small-intestine nematode, as a suitable indicator of the EPM nematocidal efficacy. The EC90 values obtained by modeling Cmax vs T. colubriformis were 3.50 and 2.43 ng/mL for lactating female goats only and pooled lactating female and male castrated goats, respectively. The values of AUClast needed for 90% efficacy of T. colubriformis were 25.4 and 21.1 day*ng/mL for lactating female goats only and pooled lactating female and male castrated goats, respectively. Overall, the predicted pharmacological response against T. colubriformis is similar for lactating goats only and pooled lactating female and male castrated goats and correlates with observed efficacy. In conclusion, a dosage of 1-mg/kg EPM as a pour-on is sufficient to ensure efficacy against common nematodes in both lactating female and male castrated goats.

Treatment of natural Protostrongylus rufescens lungworm infection in sheep with eprinomectin 5 mg/mL topical solution.

Parasitic respiratory infections in domestic sheep and goats are caused by Dictyocaulus filaria and various species belonging to the Protostrongylidae family of nematodes which frequently occur in mixed infections. Although the parasitism with protostrongylid lungworms is generally considered to be of low pathogenicity, there are reports of clinical disease including cases associated with Protostrongylus rufescens infection. The efficacy against P. rufescens of eprinomectin 5 mg/mL topical solution (EPRINEX® Multi, Boehringer Ingelheim) was thus evaluated in a clinical study compliant with GCP and VICH anthelmintic efficacy testing guidelines in adult sheep with naturally acquired infection. Following ranking on pre-treatment Protostrongylus fecal larval counts and forming into blocks of two animals, the sheep were randomly allocated to either remain untreated (control) or to be administered eprinomectin 5 mg/mL topical solution at 1 mL/5 kg body weight (equivalent to 1 mg eprinomectin per kg body weight) once as a pour-on. Fecal samples of the sheep were examined to monitor the larval excretion weekly for five weeks after treatment; then the animals were necropsied for lungworm recovery and count to determine the efficacy of the treatment. After treatment, Protostrongylus larval excretion decreased to zero within three weeks. Nematode counts demonstrated that the efficacy of the treatment with eprinomectin 5 mg/mL topical solution was 100 % against P. rufescens: no lungworms were recovered from any treated sheep while all controls harbored P. rufescens (range, 17-406) (p < 0.001). The treatment was well accepted; no treatment-related health problems were observed.

Gamithromycin in sheep: Pharmacokinetics and clinical evaluation against ovine footrot.

In the context of a development program to obtain the market authorization of injectable gamithromycin 15% w/v solution (Zactran®, Boehringer Ingelheim) for use in sheep against footrot, the pharmacokinetic profile of gamithromycin was established and the safety and efficacy of the treatment were confirmed in a multicenter field study in Europe. The basic pharmacokinetic parameters established in healthy young Merino sheep administered gamithromycin at 6 mg/kg body weight based on the analysis of plasma samples which were collected in intervals up to 12 days after subcutaneous injection were: area under the curve until last quantifiable concentration, 8.88 ± 2.33 µg*h/mL; maximum plasma concentration, 448 ± 180 ng/mL; terminal half-life, 42.5 ± 5.25 h. The safety and clinical efficacy against footrot of gamithromycin 15% w/v solution were evaluated in comparison to tilmicosin 30% w/v solution (Micotil®, Elanco) treatment in 364 sheep of various breeds, sex and age from commercial farms in the United Kingdom (2 sites), Germany (3 sites) and France (1 site). Animals were enrolled based on lesions characteristic of footrot and lameness associated with the presence of footrot-related bacterial pathogens and were randomly allocated and treated in a 1:1 ratio with a single subcutaneous dose of gamithromycin or tilmicosin at label dosage (6 or 10 mg/kg body weight, respectively). Lameness and footrot lesions were evaluated at five and 21 days after treatment; the injection site in all animals was examined the day after treatment and followed up daily in the animals with injection site reaction until complete injection site reaction resolution. Samples of 310 and 120 animals tested positive for Dichelobacter nodosus and Fusobacterium necrophorum, respectively, at inclusion, and data of 359 animals were included into the combined analyses (5 animals excluded for unintentional overdosing [1], lack of follow-up [1], concurrent antibiotic medication for non-footrot conditions [3]). Lameness scores at 21 days after treatment demonstrated a significantly (p = 0.0396) better success for the gamithromycin treatment compared to the tilmicosin treatment (97.8% vs. 93.3%). Post-dosing footrot lesion scores followed similar trends of rapid and marked decrease (improvement) for both treatments with similar (p = 0.127) treatment success for the gamithromycin and tilmicosin treatments (97.8% and 96.0%, respectively). Both treatments were safe; injection site reactions noted in 19 gamithromycin- and 25 tilmicosin-treated animals resolved within five days or six days of treatment, respectively. Gamithromycin 15% w/v solution administered once to sheep by subcutaneous injection at 6 mg/kg body weight demonstrated a pharmacokinetic profile similar to that reported previously in sheep and cattle and was confirmed to be a safe and efficacious treatment for naturally occurring ovine footrot in a multicenter clinical field study conducted in Europe.

Pour-on administration of eprinomectin to lactating dairy goats: Pharmacokinetics and anthelmintic efficacy.

Lactation is discussed as a physiological covariate which may influence the exposure characteristics of systemically acting drugs including macrocyclic lactones and potentially alter their pharmacological response. This study characterizes for the first time in the same study, the plasma profile and therapeutic anthelmintic efficacy of eprinomectin 5 mg/ml solution (EPRINEX® Multi, Boehringer Ingelheim) administered as a pour-on at 1 mg per kg body weight to lactating dairy goats. The study was conducted in compliance with VICH GCP and anthelmintic efficacy evaluation guidelines and included 20 goats harboring induced adult gastrointestinal and pulmonary nematode infections. The goats were blocked on pre-treatment body weight and randomly allocated either to remain untreated (control) or to be eprinomectin-treated. Plasma samples to determine the plasma disposition kinetics of eprinomectin (B1a component) were obtained at intervals up to 14 days following treatment when the animals were necropsied for parasite enumeration and identification. Basic pharmacokinetic parameters of eprinomectin determined in the ten eprinomectin-treated goats were as follows: AUClast , 23.8 ± 9.7 day*ng/ml and Cmax , 5.35 ± 2.27 ng/ml; individual maximum plasma concentrations were observed from 8 to 48 h after treatment (median Tmax , 0.5 days). Topical eprinomectin treatment efficacy, based on significant (p < .01) worm burden reductions in eprinomectin-treated animals relative to untreated controls, was ≥97% to 100% against adult Dictyocaulus filaria, Haemonchus contortus, Teladorsagia circumcincta(pinnata/trifurcata), Trichostrongylus axei, T. colubriformis, Cooperia curticei, Nematodirus battus, and Oesophagostomum venulosum. Both pharmacokinetic parameters and anthelmintic activity in lactating dairy goats were similar to those observed in parasitized young growing and adult female non-lactating dairy goats treated with eprinomectin administered as a pour-on.

Gamithromycin in swine: Pharmacokinetics and clinical evaluation against swine respiratory disease.

The pharmacokinetics of gamithromycin were evaluated in 26 male castrated and female crossbred swine administered gamithromycin 15% w/v (Zactran®, Boehringer Ingelheim) intravenously at 6 mg/kg bodyweight or intramuscularly at 3, 6 or 12 mg/kg bodyweight. Blood samples were collected up to Day 10 to establish the plasma profile of gamithromycin, bioavailability and dose proportionality. When administered by intramuscular injection at 6 mg/kg BWT, pharmacokinetic parameters were as follows: area under the curve until last quantifiable plasma concentration, 5.13 ± 0.957 µg*hours/ml; maximum plasma concentration, 960 ± 153 ng/ml at 5 to 15 min; terminal half-life of 94.1 ± 20.4 hr. Absolute bioavailability was 92.2%. Increase in systemic exposure was proportional to the gamithromycin dose level over the range 3-12 mg/kg BWT. No gender-related statistically significant difference in exposure was observed. For clinical evaluation of Zactran® against swine respiratory disease, 305 pigs from six commercial farms in three countries in Europe with signs associated with Actinobacillus pleuropneumoniae and/or Haemophilus parasuis and/or Pasteurella multocida and/or Bordetella bronchiseptica were used. At each site, animals were treated once in a 1:1 ratio with a single intramuscular dose of Zactran® (6 mg gamithromycin/kg bodyweight) or Zuprevo® (4% w/v tildipirosin at 4 mg/kg bodyweight; MSD Animal Health) at the recommended dose respectively. Animals were observed and scored daily for 10 consecutive days for signs of swine respiratory disease (depression, respiration and rectal temperature), and animals presenting signs of clinical swine respiratory disease (Depression Score 3 and/or Respiratory Score 3 associated with Rectal Temperature > 40.0°C) were removed from the study and considered as treatment failure. Animals which remained in the study were individually assessed for 'treatment success' or 'treatment failure' (Depression Score ≥ 1 and Rectal Temperature > 40.0°C or Respiratory Score ≥ 1 and Rectal Temperature > 40.0°C). Using a non-inferiority hypothesis test (non-inferiority margin = 0.10), the proportion of treatment successes in the Zactran® group (97%) was equivalent to or better than that in the Zuprevo® group (93%).

Anaplasma phagocytophilum and Babesia Species of Sympatric Roe Deer (Capreolus capreolus), Fallow Deer (Dama dama), Sika Deer (Cervus nippon) and Red Deer (Cervus elaphus) in Germany.

(1) Background: Wild cervids play an important role in transmission cycles of tick-borne pathogens; however, investigations of tick-borne pathogens in sika deer in Germany are lacking. (2) Methods: Spleen tissue of 74 sympatric wild cervids (30 roe deer, 7 fallow deer, 22 sika deer, 15 red deer) and of 27 red deer from a farm from southeastern Germany were analyzed by molecular methods for the presence of Anaplasma phagocytophilum and Babesia species. (3) Results: Anaplasma phagocytophilum and Babesia DNA was demonstrated in 90.5% and 47.3% of the 74 combined wild cervids and 14.8% and 18.5% of the farmed deer, respectively. Twelve 16S rRNA variants of A. phagocytophilum were delineated. While the infection rate for A. phagocytophilum among the four cervid species was similar (71.4% to 100%), it varied significantly for Babesia between roe deer (73.3%), fallow deer (14.3%), sika deer (27.3%) and red deer (40.0%). Deer ≤2 years of age tested significantly more often positive than the older deer for both A. phagocytophilum and Babesia species. (4) Conclusions: This study confirms the widespread occurrence of A. phagocytophilum and Babesia species in wild cervids and farmed red deer in Germany and documents the co-occurrence of the two tick-borne pathogens in free-ranging sika deer.

Tick-borne pathogens in the European polecat, Mustela putorius and in attached Ixodes hexagonus ticks from Germany.

The European polecat, Mustela putorius, occurs almost throughout Europe. However, there is a lack of data on the ectoparasite fauna and the potential role in the circulation of tick-borne pathogens (TBP) of this mustelid species. The aim of this study was to investigate whether M. putorius contributes to the maintenance of TBP in Germany. DNA samples extracted from spleen tissue of 117 M. putorius, which had been collected mainly in North-western Germany from 2012 to 2015, were tested by real-time and conventional PCRs for Anaplasma phagocytophilum, Babesia spp., Bartonella spp., Candidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. In addition, 200 randomly selected engorged Ixodes hexagonus ticks (100 females and 100 nymphs) collected from 39 of the 88 M. putorius were tested for these TBPs, except for Hepatozoon spp., and additionally for Borrelia spp. and Rickettsia spp. Three of six pathogens were detected in the spleen tissue of the 117 M. putorius: A. phagocytophilum (n = 5; 4.3%), Babesia cf. odocoilei (n = 1; 0.9%) and CNM (n = 1; 0.9%), including one case of co-infection (A. phagocytophilum and CNM). Ixodes hexagonus ticks tested positive only for Bartonella spp. (26/200 ticks; 13.0%) which were detected exclusively in adult female ticks. Sequencing revealed the presence of Bartonella taylorii and uncultured Bartonella spp. The results suggest that M. putorius neither seems to serve as the main reservoir nor plays an important role in maintaining TBPs in Germany but may rather contribute to the dilution of these pathogens. However, M. putorius contributes to the maintenance of tick populations, especially of I. hexagonus. The high prevalence of Bartonella spp. in I. hexagonus ticks may suggest a certain importance of this tick species in the maintenance of these bacteria in nature.

Eprinomectin pour-on: Prevention of gastrointestinal and pulmonary nematode infections in sheep.

The present study was conducted to further characterize the anthelmintic activity of the 0.5% w/v topical formulation of eprinomectin (EPRINEX® Pour-on, Merial) when administered at 1 mg/kg body weight to sheep in preventing the establishment of induced gastrointestinal and pulmonary nematode infections. Thirty-six female Merino sheep (∼4 months of age, weighing 27.0-36.0 kg) were blocked by pre-treatment body weight to form blocks of four animals. Within blocks, the animals were randomly allocated to either remain untreated (control) or be treated once with EPRINEX® either on Day 0, Day 7 or Day 14. Starting on Day 15, the sheep were given trickle infections with infective larvae of seven species of gastrointestinal nematodes and Dictyocaulus filaria lungworms daily for seven consecutive days. Five weeks after completion of the daily challenge (Day 56), the animals were necropsied for parasite recovery and count. Treatment with EPRINEX® prevented the establishment (>90%, p ≤ 0.027) of D. filaria, Teladorsagia circumcincta (pinnata/trifurcata), Cooperia curticei, Nematodirus battus, Trichostrongylus colubriformis and Oesophagostomum venulosum for at least 21 days, and of Haemonchus contortus and Trichostrongylus axei for at least 14 days. Sheep in the groups treated with EPRINEX® at Days 7 and 14 had significantly (p ≤ 0.018) higher Day -1 to Day 56 wt gains than the untreated controls. No treatment-related health problems or any other health problems were observed throughout the study.

Eprinomectin pour-on (EPRINEX® Pour-on, Merial): efficacy against gastrointestinal and pulmonary nematodes and pharmacokinetics in sheep.

BACKGROUND: The anthelmintic efficacy of the 0.5% w/v topical formulation of eprinomectin (EPN), EPRINEX® Pour-on (Merial) when administered at 1 mg/kg body weight was evaluated in sheep in two dose confirmation laboratory studies and one multicenter field study. In addition, the pharmacokinetics of EPN when administered at that dosage to adult sheep was determined. RESULTS: In the two dose confirmation studies, which included 10 sheep each, sheep treated with topical EPN had significantly (p < 0.05) fewer of the following nematodes than the untreated sheep with overall reduction of nematode counts by >99%: adult Dictyocaulus filaria, Haemonchus contortus, Teladorsagia circumcincta(pinnata/trifurcata), Trichostrongylus axei, T. colubriformis, T. vitrinus, Cooperia curticei, Nematodirus battus, Strongyloides papillosus, Chabertia ovina and Oesophagostomum venulosum, and inhibited fourth-stage Teladorsagia larvae. A total of 196 sheep harboring naturally acquired gastrointestinal nematode infections were included in the field efficacy study at two sites each in Germany (48 Merino x Ile de France lambs, 52 adult Merino females) and in Italy (adult male and female Bagnolese, Lacaune, Lacaune x Bagnolese, Bagnolese x Sarda sheep; 48 animals per site). Animals were blocked on pre-treatment body weight and within each block, one animal was randomly assigned to the control (untreated) group and three animals were randomly assigned to be treated with topical EPN. Examination of feces 14 days after treatment demonstrated that, relative to the controls, topical EPN-treated sheep had significantly (p < 0.0001) lower strongylid egg counts. Reduction was ≥97% at each site and 98.6% across all sites. Pharmacokinetics of EPN following single treatment with topical EPN were determined in eight ~4.5 year old female Merino cross sheep based on the analysis of plasma samples which were collected from two hours to 21 days following treatment. The main pharmacokinetic parameters were: Cmax 6.20 ± 1.71 ng/mL, AUClast 48.8 ± 19.2 day*ng/mL, Tmax 3.13 ± 2.99 days and T1/2 6.40 ± 2.95 days. No treatment-related health problems or adverse drug events were observed in any study. CONCLUSION: These studies demonstrated 0.5% w/v EPN administered topically at 1 mg/kg body weight to be highly efficacious against a broad range of ovine gastrointestinal nematodes and D. filaria lungworms and well tolerated by sheep of different ages, breeds, gender and physiological status.

Anaplasma phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon) in Germany.

Infections with the tick-borne pathogens Anaplasma phagocytophilum and Babesia spp. can cause febrile disease in several mammalian species, including humans. Wild ruminants in Europe are suggested to serve as reservoir hosts for particular strains or species of these pathogens. The aims of this study were to investigate the occurrence of A. phagocytophilum and Babesia spp. in roe deer (Capreolus capreolus), fallow deer (Dama dama) and mouflon (Ovis musimon orientalis) in Germany, and the diversity and host association of genetic variants of A. phagocytophilum and Babesia species. From 2009 to 2010, 364 spleen samples from 153 roe deer, 43 fallow deer and 168 mouflon from 13 locations in Germany were tested for DNA of A. phagocytophilum and Babesia spp. by real-time PCR or conventional PCR, respectively. Variants of A. phagocytophilum were investigated with a nested PCR targeting the partial 16S rRNA gene, and species of piroplasms were identified by sequencing. DNA of A. phagocytophilum was detected in 303 (83.2%) samples: roe deer, 96.1% (147/153); fallow deer, 72.1% (31/43); and mouflon, 74.4% (125/168). Sequence analysis of 16S rRNA-PCR products revealed the presence of nine different genetic variants. DNA of Babesia spp. was found in 113 (31.0%) samples: roe deer, 62.8% (96/153); fallow deer, 16.3% (6/43); and mouflon, 6.5% (11/168). Babesia capreoli, Babesia sp. EU1 (referred to also as B. venatorum), B. odocoilei-like and a Theileria species were identified. Co-infections with A. phagocytophilum and Babesia spp. were detected in 30.0% of the animals which were tested positive for A. phagocytophilum and/or Babesia spp. Roe deer had a significantly higher percentage of co-infections (60.8%), followed by fallow deer (14.0%) and mouflon (6.5%). Thus, the results suggest that roe deer plays a key role in the endemic cycles of the pathogens investigated.

Efficacy of oral afoxolaner plus milbemycin oxime chewables against induced gastrointestinal nematode infections in dogs.

The efficacy of oral afoxolaner plus milbemycin oxime combination chewables against induced gastrointestinal nematode infections in dogs was evaluated in six separate studies. Two studies were performed to evaluate the efficacy of the product against Toxocara canis, two studies evaluated the efficacy against Toxascaris leonina, one study evaluated the efficacy against Ancylostoma braziliense, and one study evaluated the efficacy against Ancylostoma caninum. In the A. caninum study, the efficacy of milbemycin oxime alone and afoxolaner alone was also evaluated. Dogs in all studies were inoculated with infective eggs or larvae and confirmed to have patent infections based on a fecal examination prior to allocation to study group and treatment. Each study utilized a randomized block design with blocks based on pre-treatment body weight. All dogs were assigned to blocks based on body weight, and then each dog within a block was randomly assigned to treatment group. There were two groups of 10 dogs each in the T. canis, T. leonina, and A. braziliense studies: 1) an untreated (control) group and 2) a group treated with afoxolaner plus milbemycin oxime chewables (NexGard Spectra(®), Merial). This group was treated at a dose as close as possible to the minimum effective dose of afoxolaner and milbemycin oxime (2.5mg+0.5mg per kg body weight, respectively) once on Day 0 using whole chews. There were four groups of 10 dogs each in the A. caninum study: 1) untreated (control), 2) NexGard Spectra(®) as described above, 3) milbemycin oxime alone (dose of at least 0.5mg per kg of body weight) and 4) afoxalaner alone (dose of at least 2.5mg per kg body weight). For parasite recovery and counts, dogs were euthanized humanely and necropsied seven days after treatment. The efficacy of the afoxolaner plus milbemycin oxime combination was ≥98% against T. canis, ≥95.8% against T. leonina, and 90.2% against A. braziliense. Efficacy of the combination against A. caninum was 99.7%, while the efficacy of milbemycin oxime alone was 99.6% and the efficacy of afoxolaner alone was 2.1%. Dogs treated with afoxolaner plus milbemycin oxime chewables had significantly (p≤0.0002) fewer nematodes than the untreated controls in all studies. There were no adverse events or other health problems that were related to treatment with Nexgard Spectra(®) in these studies. The results of these controlled studies demonstrate the high efficacy of the afoxolaner plus milbemycin oxime chewables against a broad range of canine intestinal nematode infections.

Parasites and vector-borne pathogens in client-owned dogs in Albania. Blood pathogens and seroprevalences of parasitic and other infectious agents.

Knowledge on the epidemiology of parasitic and vector-borne infections is still very limited for Albania, a country located in the Balkan Peninsula in southeast Europe. Recent publications indicated prevalence rates of up to 52% for vector-borne infections in less-cared dogs in Albania. To provide data on the epidemiological situation in dogs under veterinary care, a total of 602 client-owned dogs presented to four small animal clinics between March 2010 and April 2011 in Tirana, Albania, were screened by examination of Giemsa-stained blood smears, PCR, and serological methods for the presence of arthropod-borne infections, as well as Neospora caninum and Toxoplasma gondii. Eight different pathogens, namely Babesia vogeli, Hepatozoon canis, Leishmania infantum, Dirofilaria immitis, Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, and Mycoplasma haemocanis, were detected by direct methods with prevalence rates ranging from 1 to 9%. Seroprevalence for Babesia spp., L. infantum, Anaplasma spp., and E. canis were 6.6, 5.1, 24.1, and 20.8%, respectively. Dogs >1 year of age were positive for vector-borne infections significantly more often than younger dogs (p = 0.003). More than half (51.7%) of the dogs were seroreactive to T. gondii and 18.3% to N. caninum. This is the first report on the detection of A. phagocytophilum, A. platys, E. canis, and M. haemocanis by PCR as well as the serological confirmation of exposure of dogs to N. caninum and T. gondii in Albania. The spectrum of pathogens and the seroprevalences for N. caninum and T. gondii in client-owned dogs from Tirana, Albania, are comparable to that reported in other countries in the Mediterranean Basin. The prevalence rates of vector-borne pathogens are at the lower range of that reported in studies from this geographical region. This is probably due to increased awareness of the owners of pet dogs, including better husbandry conditions and ectoparasiticidal treatment, thus limiting exposure of dogs to vectors.

Parasites and vector-borne diseases in client-owned dogs in Albania. Intestinal and pulmonary endoparasite infections.

From March 2010 to April 2011 inclusive, feces from 602 client-owned dogs visiting four small animal clinics in Tirana, Albania, were examined using standard coproscopical techniques including Giardia coproantigen ELISA and immunofluorescent staining of Giardia cysts. Overall, samples of 245 dogs (40.7 %, 95 % CI 36.6-45.6) tested positive for at least one type of fecal endoparasite (protozoan and/or helminth and/or pentastomid) stage, of which 180 (29.9 %, 95 % CI 26.3-33.7) and 129 (21.9 %, 95 % CI 18.2-24.9) tested positive for protozoan or nematode endoparasites, respectively. Fecal forms of at least 14 endoparasites were identified. The most frequently identified stages were those of Giardia (26.4 %), Trichuris (9.5 %), Toxocara (8.0 %), hookworms (7.1 %), Cystoisospora ohioensis (4.3 %), and Cystoisospora canis (3 %). For the first time for dogs in Albania, fecal examination indicated the occurrence of Hammondia/Neospora-like (0.2 %), Angiostrongylus lungworm (0.3 %), capillariid (2.8 %), and Linguatula (0.2 %) infections. Single and multiple infections with up to seven parasites concurrently were found in 152 (25.2 %, 95 % CI 21.8-28.9) and 93 dogs (15.4 %, 95 % CI 12.7-18.6), respectively. On univariate analysis, the dog's age, the dog's purpose (pet, hunting dog, working dog), the dog's habitat (city, suburban, rural), and environment (mainly indoors, indoors with regular outside walking, yard, kennel/run), presence/absence of other dogs and/or cats, history of anthelmintic use, and season of examination were identified as significant (p < 0.05) factors predisposing dogs to various types of endoparasitism while the variables breed (pure breed dogs vs. mixed-breed dogs), gender, and type of food were not significant predictors. Multivariate logistic regression analysis for factors associated with overall endoparasitism revealed that dogs >1 year of age (odds ratio [OR] = 0.64), dogs dewormed at least once per year (OR = 0.35), and dogs tested during spring, summer, and autumn (OR = 0.51, 0.15, and 0.20, respectively) had a significantly lower risk compared with ≤1 year old dogs, dogs not dewormed, or dogs tested during winter. The odds of a dog to be diagnosed positive for endoparasites was 1.56 times higher for dogs living together with other pets than that for a dog without other dogs or cats.