[How widespread is resistance to invermectin among gastrointestinal nematodes in sheep in The Netherlands?]. / Hoe wijd verspreid is resistentie tegen ivermectine van maagdarmwormen bij het schaap in Nederland?

In Autumn 2009, a faecal egg count reduction test (FERCT) was carried out on three sheep farms. Groups of 8-11 lambs were treated with ivermectin or moxidectin, with a 14-day interval between treatment and sampling. Ivermectin resistance was present on all three farms. Treatment with ivermectin resulted in a reduction in faecal egg numbers of 94.6%, 63%, and 59%. On two farms, 14 days after treatment pooled faecal samples yielded predominantly larvae of Hamonchus contortus (100% and 98%, respectively). On the third farm, H. contortus and (probably) Teladorsagia circumcincta were resistant to ivermectin (64% and 36% of the larvae, respectively). Treatment with moxidectin resulted in a 100% reduction in egg output in sheep on all three farms. More sensitive culture techniques failed to detect any larvae in samples taken from two farms, but a few Ostertagia-type larvae, probably of T. circumcincta, were detected in samples from the third farm. It can be concluded that gastrointestinal nematodes in sheep from these three farms were resistant to ivermectin, whereas resistance to moxidectin was not detected.

Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species.

BACKGROUND: Hard ticks have been identified as important vectors of rickettsiae causing the spotted fever syndrome. Tick-borne rickettsiae are considered to be emerging, but only limited data are available about their presence in Western Europe, their natural life cycle and their reservoir hosts. Ixodes ricinus, the most prevalent tick species, were collected and tested from different vegetation types and from potential reservoir hosts. In one biotope area, the annual and seasonal variability of rickettsiae infections of the different tick stages were determined for 9 years. RESULTS: The DNA of the human pathogen R. conorii as well as R. helvetica, R. sp. IRS and R. bellii-like were found. Unexpectedly, the DNA of the highly pathogenic R. typhi and R. prowazekii and 4 other uncharacterized Rickettsia spp. related to the typhus group were also detected in I. ricinus. The presence of R. helvetica in fleas isolated from small rodents supported our hypothesis that cross-infection can occur under natural conditions, since R. typhi/prowazekii and R. helvetica as well as their vectors share rodents as reservoir hosts. In one biotope, the infection rate with R. helvetica was ~66% for 9 years, and was comparable between larvae, nymphs, and adults. Larvae caught by flagging generally have not yet taken a blood meal from a vertebrate host. The simplest explanation for the comparable prevalence of R. helvetica between the defined tick stages is, that R. helvetica is vertically transmitted through the next generation with high efficiency. The DNA of R. helvetica was also present in whole blood from mice, deer and wild boar. CONCLUSION: Besides R. helvetica, unexpected rickettsiae are found in I. ricinus ticks. We propose that I. ricinus is a major reservoir host for R. helvetica, and that vertebrate hosts play important roles in the further geographical dispersion of rickettsiae.

Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes.

The ability to reliably detect anthelmintic resistance is a crucial part of resistance management. If data between countries are to be compared, the same test should give the same results in each laboratory. As the egg hatch test for benzimidazole resistance is used for both research and surveys, the ability of different laboratories to obtain similar results was studied through testing of known isolates of cyathostomins, Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora in programs supported by the EU (Cost B16 and FP6-PARASOL). Initial results showed difficulties in obtaining reproducible and similar data within and between laboratories. A series of ring tests, i.e., simultaneous and coordinated rounds of testing of nematode isolates in different laboratories was subsequently performed. By adopting identical protocols, especially the use of deionized water and making dilutions of thiabendazole in dimethyl sulfoxide in the final ring test, laboratories correctly identified both susceptible and resistant isolates. The protocols for the test and preparation of solutions of thiabendazole are described.

Import of Parafilaria bovicola in the Netherlands.

In March 2007 cutaneous nodules were observed in the neck-, shoulder- and back-region of a breeding bull, imported from France in November 2006. The nodules opened spontaneously and produced a haemorrhagic exudate. After suspicion of parafilariosis, the bull was treated with moxidectin, but without effect. Treatment with ivermectin stopped the bleedings. To prevent spread of this imported infection, the bull was euthanised and necropsied. Worms were found in the subcutis which could be identified as Parafilaria bovicola. This is the first diagnosed case of parafilariosis in the Netherlands. In the following period, all cattle on the farm were carefully examined for signs of parafilariosis and provided with permethrin containing ear tags. Till July 2008, no new cases on the farm were observed.

Persistent detection of Babesia EU1 and Babesia microti in Ixodes ricinus in the Netherlands during a 5-year surveillance: 2003-2007.

We report the finding of Babesia EU1 and Babesia microti in Ixodes ricinus ticks in the Netherlands. During 5 years of surveillance between 2003 and 2007, 1488 ticks were collected in a dune forest area near the North Sea and were screened for Babesia infections. In 17 ticks, DNA of the protozoan parasite genus Babesia was detected using a Babesia-specific 18S rRNA polymerase chain reaction. Further, reverse line blot analysis and DNA sequence analysis showed that 13 of these ticks carried Babesia EU1, two ticks carried B. microti, and one tick carried B. divergens. This study shows that the human pathogenic species Babesia EU1 and B. microti can complete their life cycle in the Netherlands.

[Reintroduction of E. granulosus by import of cows in the Netherlands]. / Herintroductie van echinococcose via runderimport in Nederland.

Since East European countries joined the EU, the import of both dairy and beef cows from these countries increased considerably. Based on the identification and registration system it turned out that in the period from May until December 2007 about 200 cows per month were imported from Romania. These animals were either slaughtered immediately or in autumn. In autumn, cysts were noticed both in slaughtered cows during meat inspection and in deceased animals (originated from Romania) during postmortem investigation performed by the Animal Health Service. Because cysts were strongly reminiscent of Echinococcus granulosus hydatid cysts, samples were sent to the authorized laboratory (National Reference Laboratory of Parasitology), where the reintroduction of this potentially zoonotic parasitic infection has been confirmed. The risks of reintroduction of E. granulosus in the Netherlands are described.

The efficacy of an ivermectin/closantel injection against experimentally induced infections and field infections with gastrointestinal nematodes and liver fluke in cattle.

Three studies were performed to test the efficacy of an ivermectin/closantel injection (200 microg/kg(-1) ivermectin and 5 mg/kg(-1) closantel) in cattle. Two were experimentally induced infections of Ostertagia ostertagi, Cooperia oncophora and Fasciola hepatica in calves, and the third had natural field infections in cattle with several species of gastrointestinal nematodes and F. hepatica. In the two studies with artificial infections, four groups of 8 calves were used. All calves were infected with metacercariae on Day 0. Infection with the nematodes took place on Day 33 in groups 1 and 2 and on Day 54 in groups 3 and 4. Treatment was given to calves of group 1 on Day 63 and to calves of group 3 on Day 84. Calves of groups 2 and 4 served as untreated control groups. Calves of groups 1 and 2 were sacrificed on Day 84, calves of groups 3 and 4 on Day 105. The field study was carried out on a commercial farm in the Netherlands. Six groups of cattle were used. Groups A and B consisted of 10 parasite free calves, introduced to the farm and grazed for four weeks on pastures naturally infected with gastrointestinal nematode larvae and liver fluke metacercariae. Group C were the farmers own calves (15), group D heifers (10), group E dry cows (6) and group F milking cows (20). Treatment was given to animals of group A, C, D and E 10 weeks after housing of group A and B. Animals of groups B and F served as untreated controls. Calves of groups A and B were sacrificed 14 days after treatment. The efficacy of the treatment was calculated on basis of the post-mortem fluke and nematode worm counts in the first two studies and on a combination of post-mortem fluke and nematode worm counts and faecal egg output in the field study. In the two experimental studies, the efficacy of the treatment against F. hepatica was 99.2% and 94.5% for 9-week-old flukes and 98.4% and 99.5% for 12-week-old flukes. For O. ostertagi in both studies efficacy was 100% and against C. oncophora in both Groups 1 efficacy was 84.9% and 99.0% and in Groups 3 85.0% and 99.4%. In the field study, based on the post mortem fluke and nematode worm counts in groups A and B, efficacy against F. hepatica was 98.4%, O. ostertagi 100%, C. oncophora 99.4%, C. punctata 100%, Nematodirus helvetianus 60.8%, Trichuris spp. 100% and against larval intestinal nematodes 100%. The results of the faecal examinations 14 days after treatment confirmed the post-mortem results with 100% reduction of egg output for O. ostertagi, C. punctata, Trichostrongylus spp. and Trichuris spp. and low egg output of C. oncophora and N. helvetianus.

Doramectin and albendazole resistance in sheep in The Netherlands.

A faecal egg count reduction test was conducted on a sheep farm with suspected avermectin resistance. Five groups of 10 sheep were formed. Group 1 was the untreated control group. Groups 2-5 were treated according to weight with the recommended dose of, respectively, levamisole, doramectin, moxidectin or albendazole. Resistance was found in the sheep treated with doramectin (15% efficacy) and albendazole (87% efficacy). Levamisole and moxidectin were 100 and 99% effective, respectively. Larval identification of the faecal cultures of the doramectin-treated sheep revealed 100% Haemonchus contortus larvae. After albendazole treatment, 77% of the cultured larvae were H. contortus and 23% Teladorsagia/Trichostrongylus. Because there is a lot of trade in sheep on the farm, it is probable that the resistant worms were introduced with livestock from other farms than being selected on the farm.

Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in The Netherlands.

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.

The musk rat (Ondatra zibethicus) as intermediate host of cestodes in the Netherlands.

An investigation on the presence of larval cestodes in musk rats (Ondatra zibethicus) was carried out in two regions of the Netherlands (east Groningen and south Limburg) where in a earlier study foxes with Echinococcus multilocularis were found. A total of 1726 musk rats were dissected (1200 in Groningen, 526 in Limburg). Larval stages of Taenia taeniaeformis were most frequently found (total 44.8%: Groningen 42%, Limburg 51.3%), followed by T. martis (total 6.1%: Groningen 0.7%, Limburg 18.6%). Infections with T. crassiceps (total 0.3%: Groningen 0%, Limburg 1.0%), T. polyacantha (total 0.2%; Groningen 0.3%, Limburg 0%) and E. multilocularis (0.1%: Groningen 0.1%, Limburg 0%) were rare. Infections with T. taeniaeformis were more frequent in adults (71.8%) than in juveniles (34.2%). The same was found for T. martis: adults 15.3%, juveniles 2.5%. This difference was also reflected in the relation between weight of the animals and presence of infection. Heavier animals (>1000 g) were more often infected with T. taeniaeformis (74.1%) than animals less than 1000 g (34.8%). In musk rats weighing less than 500 g (n=155) only 5.2% were infected, but above 1200 g, 82.6%. The highest number of T. taeniaeformis was 28, of T. martis 13, of T. crassiceps >1000 and of T. polyacantha 24. The E. multilocularis was in a very young stage, a few white spots in the liver. Although E. multilocularis infections were exceptional, it is expected that with a rise in the number of infected foxes in the Netherlands the number of infected musk rats will increase.