Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Prevalence and molecular detection of Eimeria species in different types of poultry in Greece and associated risk factors.

Coccidiosis is one of the most notable diseases in chickens having a high economic impact on the poultry industry worldwide. The present study is the first attempt to epidemiologically investigate Eimeria spp. distribution and associated risk factors under different housing and production systems in three major regions in Greece. Faecal samples were obtained from 42 operations (broilers, floor housed, free range and organic layers, backyard farms). A questionnaire was obtained from included operations to acquire additional information regarding farm management, location, production rate and diseases history. Positivity level was 85.7%. All seven Eimeria species were identified, and the most prevalent ones were E. acervulina (79.3%) and E. tenella (65.5%). Single-species and mixed infections were detected in 20.7% and 79.3% of the flocks, respectively. Flock size, type of outdoor area, production system and presence of respiratory disease proved significant risk factors. Flock size up to 10,000 birds correlated strongly (p = 0.02) with higher E. tenella quantities. A very strong correlation (p < 0.001) was found between the presence of respiratory disease and the average OPG level in broiler farms. Organic flocks showed higher prevalence of E. tenella (p = 0.023), while presence of vegetation at the outdoor area correlated strongly with E. brunetti (p < 0.001). Molecular analysis and correlation results in this survey give strong indications although more studies are needed to further understand the involvement of different Eimeria species in various husbandry, production and management systems, to gain more knowledge about the sustainable control of coccidia in poultry.

Anthelmintic resistance in ruminants: challenges and solutions.

Anthelmintic resistance (AR) is a growing concern for effective parasite control in farmed ruminants globally. Combatting AR will require intensified and integrated research efforts in the development of innovative diagnostic tests to detect helminth infections and AR, sustainable anthelmintic treatment strategies and the development of complementary control approaches such as vaccination and plant-based control. It will also require a better understanding of socio-economic drivers of anthelmintic treatment decisions, in order to support a behavioural shift and develop targeted communication strategies that promote the uptake of evidence-based sustainable solutions. Here, we review the state-of-the-art in these different fields of research activity related to AR in helminths of livestock ruminants in Europe and beyond. We conclude that in the advent of new challenges and solutions emerging from continuing spread of AR and intensified research efforts, respectively, there is a strong need for transnational multi-actor initiatives. These should involve all key stakeholders to develop indicators of infection and sustainable control, set targets and promote good practices to achieve them.

Following the route of veterinary antibiotics tiamulin and tilmicosin from livestock farms to agricultural soils.

Veterinary antibiotics (VAs) are not completely metabolized in the animal body. Hence, when animal excretes are used as soil manures, VA residues are dispersed with potential implications for environmental quality and human health. We studied the persistence of tiamulin (TIA) and tilmicosin (TLM) along their route from pig administration to fecal excretion and to agricultural soils. TLM was detected in feces at levels folds higher (4.27-749.6 µg g-1) than TIA (0.55-5.99 µg g-1). Different administration regimes (feed or water) showed different excretion patterns and residual levels for TIA and TLM, respectively. TIA and TLM (0.5, 5 and 50 µg g-1) dissipated gradually from feces when stored at ambient conditions (DT50 5.85-35.9 and 23.5-49.8 days respectively), while they persisted longer during anaerobic digestion (DT90 >365 days) with biomethanation being adversely affected at VA levels > 5 µg g-1. When applied directly in soils, TLM was more persistent than TIA with soil fumigation extending their persistence suggesting microbial degradation, while soil application through feces increased their persistence, probably due to increased sorption to the fecal organic matter. The use of TIA- and TLM-contaminated feces as manures is expected to lead to VAs dispersal with unexplored consequences for the environment and human health.

Bioaugmentation of animal feces as a mean to mitigate environmental contamination with anthelmintic benzimidazoles.

Anthelmintics are used to control infestations of ruminants by gastrointestinal nematodes. The limited metabolism of anthelmintics in animals result in their excretion in feces. These could be piled up in the floor of livestock farms, constituting a point source of environmental contamination, or used as manures in agricultural soils where they persist or move to water bodies. Hence the removal of anthelmintics from feces could mitigate environmental contamination. We hypothesized that a thiabendazole-degrading bacterial consortium would also degrade other benzimidazole anthelmintics like albendazole, fenbendazole, ricobendazole, mebendazole and flubendazole. In liquid culture tests the consortium was more effective in degrading compounds with smaller benzimidazole substituents (thiabendazole, albendazole, ricobendazole), rather than benzimidazoles with bulky substituents (fenbendazole, flubendazole, mebendazole). We then explored the bioaugmentation capacity of the consortium in sheep feces fortified with 5 and 50 mg kg-1 of thiabendazole, albendazole and fenbendazole. Bioaugmentation enhanced the degradation of all compounds and its efficiency was accelerated upon fumigation of feces, in the absence of the indigenous fecal microbial community. The latter contributes to anthelmintics degradation as suggested by the significantly lower DT50 values in fumigated vs non-fumigated, non-bioaugmented feces. Overall, bioaugmentation could be an efficient means to reduce environmental exposure to recalcitrant anthelmintic benzimidazoles.

Identifying human enteric parasitic infections in Greece, with focus on Giardia and Cryptosporidium.

A study was conducted in two different areas in Greece to investigate the presence of intestinal human parasitic infections (targeting healthy and individuals with diarrhoea). In total, 876 stool samples were collected from 822 adults and 54 children. Both sedimentation (acid/ether) and concentration/flotation techniques were performed in all samples to detect intestinal parasites. Additionally, a quantitative direct immunofluorescence assay was used specifically for the detection of Giardia and Cryptosporidium. PCR followed by sequencing was applied to genotype Giardia and Cryptosporidium positive samples. Thirty-five (4%) of the individuals examined harboured at least one species of intestinal parasite, the majority of which were protozoa (3.8%). The species found were Blastocystis hominis (1.8%), Giardia duodenalis (1.3%), Cryptosporidium spp. (0.6%), Entamoeba coli (0.2%) and E. histolytica/E. dispar (0.1%). Two persons were positive for Enterobius vermicularis. Genotyping results revealed the presence of G. duodenalis sub-assemblage AII, whereas sequencing was not successful for Cryptosporidium positive samples. A novel multi-locus genotype of G. duodenalis was identified, which has not been described in humans or animals previously. Overall, in the studied population, infection rates with intestinal parasites were low and similar to previous published data. As infection levels were low, no associations could be made between infection status and clinical relevance, risk factors or indication of potential sources of infection, apart from the fact that infections with Giardia were positively correlated to diarrhoea. Based on the parasite species and genotypes detected, there was no indication that animals were an important source of infection. Thus, it is suggested that Giardia infections were more likely to be acquired via human-to-human transmission, either involving indirect pathways such as contaminated food or water, or via direct contact.

Coccidiostatic effects of tannin-rich diets in rabbit production.

The potential anti-eimerial effect of tannin containing resources such as sainfoin and carob in rabbits was tested on does at pre-weaning and to growing rabbits in their feed. The trial began at parturition (D0), when 24 does and their litters were assigned into three groups. They were fed either with a control (Group CO), a carob (containing 10% carob pods meal) (Group CP) or a sainfoin diet (containing 34% dehydrated sainfoin pellets) (Group SA). All diets were made isoproteic and isoenergetic and also balanced for crude fibre but differed by their tannin content. Weaning occurred at D37, and growing rabbits remained in the same cage until D51. Then, they were transferred to fattening cages until the end of the trial (D104) and slaughtering. Weight gain of young rabbits among the three groups (mean = 31.2 g/day) did not differ statistically. The mortality rates were 10% (SA), 15% (CP) and 20% (CO), respectively, but the differences were not statistically significant. Post-weaning economical feed conversion ratio (FCR) was reduced between rabbits of group SA compared to CO and CP groups. Faecal oocyst count (FOC) in group SA was 60% lower than in CO and CP groups. Areas under the curve (AUCs) calculated between sampling days and FOC, after transfer to fattening cages, was 62% lower in group SA than in CO and CP groups. The main Eimeria species identified (from D59 to D83) was Eimeria magna (53% of oocysts). AUCs for E. magna did not differ according to diet. In conclusion, the diet containing sainfoin reduced oocyst excretion of Eimeria spp. by 60%, and improved the economical FCR.

Mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs.

Gastrointestinal (GI) nematode control has an important role to play in increasing livestock production from a limited natural resource base and to improve animal health and welfare. In this synthetic review, we identify key research priorities for GI nematode control in farmed ruminants and pigs, to support the development of roadmaps and strategic research agendas by governments, industry and policymakers. These priorities were derived from the DISCONTOOLS gap analysis for nematodes and follow-up discussions within the recently formed Livestock Helminth Research Alliance (LiHRA). In the face of ongoing spread of anthelmintic resistance (AR), we are increasingly faced with a failure of existing control methods against GI nematodes. Effective vaccines against GI nematodes are generally not available, and anthelmintic treatment will therefore remain a cornerstone for their effective control. At the same time, consumers and producers are increasingly concerned with environmental issues associated with chemical parasite control. To address current challenges in GI nematode control, it is crucial to deepen our insights into diverse aspects of epidemiology, AR, host immune mechanisms and the socio-psychological aspects of nematode control. This will enhance the development, and subsequent uptake, of the new diagnostics, vaccines, pharma-/nutraceuticals, control methods and decision support tools required to respond to the spread of AR and the shifting epidemiology of GI nematodes in response to climatic, land-use and farm husbandry changes. More emphasis needs to be placed on the upfront evaluation of the economic value of these innovations as well as the socio-psychological aspects to prioritize research and facilitate uptake of innovations in practice. Finally, targeted regulatory guidance is needed to create an innovation-supportive environment for industries and to accelerate the access to market of new control tools.

Species diversity and spatial distribution of ixodid ticks on small ruminants in Greece.

A cross sectional field study on the distribution of Ixodidae ticks was carried out over two consecutive tick seasons (2012 and 2013) in small ruminants (309 farms) located in mainland and five islands of Greece. A total of 2108 ticks (1199 females, 908 males, and 1 nymph) were collected from sheep and goats. Two species of Rhipicephalus, two of Ixodes, five of Hyalomma, three of Haemaphysalis, and one of Dermacentor were detected. The species with the widest distribution were Rhipicephalus sanguineus s.l. (64.8 %), R. bursa (25.9 %), and Dermacentor marginatus (4.1 %), whereas the least frequently collected species were Ixodes ricinus, I. gibbosus, Haemaphysalis parva, H. sulcata, H. punctata, Hyalomma marginatum, H. excavatum, H. dromedarii, H. rufipes, and H. impeltatum representing together less than 5.3 % of the collections. R. sanguineus s.l. was the predominant species, while D. marginatus and I. gibbosus were restricted to the northeastern Aegean Sea islands. This is the first survey on tick fauna from mainland and islands in Greece.

The effect of sainfoin (Onobrychis viciifolia) and carob pods (Ceratonia siliqua) feeding regimes on the control of lamb coccidiosis.

Recent research has suggested that plants containing condensed tannins may offer a promising alternative approach for the control of coccidiosis in lambs and goat kids. The present study aimed to examine the potential effect of condensed tannins in sainfoin (Onobrychis viciifolia) and carob pods (Ceratonia siliqua) incorporated in sheep rations against lamb coccidiosis. The above tannin-rich sources were studied in three independent feeding trials in which the animals (naturally infected by Eimeria spp. ewes and their lambs) were allocated (i) in the control group and received a tannin-free diet (lucerne hay), or (ii) in the treatment groups and received a tannin-rich diet based on sainfoin hay (in trials 1 and 2), or in carob pod meal and a combination of carob pod meal and sainfoin hay (in trial 3). In total, 95 newborn lambs (and their 73 ewes) were enrolled in all trials which started a month before lambing and ended 8-10 weeks after lambs were born (at weaning). The course of coccidial infection was monitored in lambs by faecal oocyst counts and consistencies which were recorded at weekly intervals. Moreover, lambs total weight gain was evaluated at the end of each trial. During all trials, 100 % of the animals got naturally infected by Eimeria species and the infection burden was higher in trials 2 and 3 compared to trial 1 but in all cases, severe signs of diarrhoea were not observed. Tannin-rich diets were well accepted by the animals not affecting their feed intake and body weight gain when compared to the controls. The results suggest that incorporation of both tannin-rich resources (especially sainfoin) in sheep rations can reduce Eimeria oocyst excretion rates by the lambs, which can decrease subsequently the contamination of the farm environment with the parasite. However, the high variability noted on the results is not allowing us to draw any definite conclusions at least until the potential of those plants is further investigated.

Echinococcus granulosus infection dynamics in livestock of Greece.

An epidemiological and molecular survey on the occurrence of Echinococcus hydatid cysts in livestock was conducted in Greece. In total 898 sheep, 483 goats, 38 buffaloes, 273 wild boars and 15 deer were examined and 30.2% (6.45% cyst fertility), 7.86% (3.2% cyst fertility), 42% (7.9% cyst fertility), 1.1% (0% cyst fertility), 0% of them were found infected, respectively. Infection rate in different geographical regions varied between 26.1 and 53.8% (cyst fertility 2.04 and 34.6%) in sheep, 7.33 and 13.3% (cyst fertility 0 and 3.2%) in goats. Genotyping, based on cox1 and nad1 analyses, demonstrated the predominance of E. granulosus s.s. (G1 genotype). The presence of one single genotype-complex within a relatively large spectrum of intermediate host species in Greece indicates the presence of a dominant transmission dog-sheep cycle involving additional host species which may act as disease reservoir for human infections.

Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock.

Parasitic infections with gastrointestinal nematodes (GINs) still represent a worldwide major pathological threat associated with the outdoor production of various livestock species. Because of the widespread resistance to synthetic chemical anthelmintics, there is a strong impetus to explore novel approaches for a more integrated management of these infections. The use of nutraceuticals in the control of GINs is one of the alternatives which has been widely studied for 20 years. The objectives of this review are: (i) to define and illustrate the concept of 'nutraceutical' in the context of veterinary parasitology based on data obtained on the most studied models to control GINs in small ruminants, the tannin-containing legumes (Fabaceae); (ii) to illustrate how the 'nutraceutical concept' could be expanded to other plants, other livestock production systems and other GI parasitic diseases, and (iii) to explain how this concept is opening up new research fields for better understanding the interactions between the host, the digestive parasites and the environment.

The occurrence and genetic characterization of Cryptosporidium and Giardia species in foals in Belgium, The Netherlands, Germany and Greece.

Faecal samples were collected from foals between the age of 1 week and 6 months in Belgium, The Netherlands, Germany and Greece. A quantitative direct immunofluorescence assay based on the commercial MERIFLUOR Cryptosporidium/Giardia kit was performed to evaluate the presence of (oo) cysts. Parasite positive samples were genotyped, based on the 18S ribosomal DNA gene and the heat shock protein (HSP70) gene for Cryptosporidium and on the ß-giardin gene and the triose phosphate isomerase (TPI) gene for Giardia. In total, 134 foals from Belgium, 44 foals from The Netherlands, 30 foals from Germany and 190 foals from Greece were examined. No Cryptosporidium oocysts were identified in faecal samples from foals in Germany and The Netherlands. In Belgium and Greece, 4.5% and 1.1% of the foals examined were Cryptosporidium positive, respectively, all with a low oocyst excretion ranging from 100 to 2450 oocysts per gram of faeces. For Giardia, 14.2%, 11.4%, 10.0% and 11.6% of the foals in Belgium, The Netherlands, Germany and Greece, respectively, were found to excrete cysts, with a range of 50 up to 4,000,000 cysts per gram of faeces. Younger animals secreted significantly more Giardia cysts than older horses (p<0.05), but no significant correlation between Giardia infection and diarrhoea was observed. Most Giardia positive samples belonged to assemblage AI and/or BIV, but also assemblage E was detected in two samples. Together with the identification of Cryptosporidium horse genotype, this suggests only a low risk for zoonotic transmission.

Improved method for screening mitochondrial cytochrome b markers to identify regional populations of the Old World screwworm fly and other myiasis agents.

A new protocol was developed to overcome obstacles to the high-throughput sequence analysis of the 716-717 nucleotides at the carboxyl terminal of the mitochondrial gene cytochrome b (cyt b) of the myiasis flies Chrysomya bezziana and Wohlfahrtia magnifica. For both of these obligate parasites, cyt b haplotypes provide diagnostic markers for phylogeographic populations, markers that identify the origins of emerging populations causing economically important myiasis in livestock and, in the case of C. bezziana (Old World screwworm fly), could help select reproductively-compatible populations for use in the Sterile insect technique as part of area wide integrated pest management. High sequence quality is important for unambiguously detecting the few mutations that are diagnostic for regional cyt b haplotypes and their lineages. A key innovation is the design of a new forward primer for the specific PCR amplification and high-quality sequencing of cyt b. The improved protocol will facilitate the use of this established comparative cyt b sequence analysis, not only by teams lacking the resources for whole genome sequencing (WGS) but also by those requiring reference sequences for developing comparative mitogenomics based on WGS.

Hepatogenous photosensitisation in Scottish sheep casued by Dicrocoelium dendriticum.

Dicrocoeliosis was identified as the probable predisposing cause of weight loss and hepatogenous photosensitisation affecting half of a group of 14 month-old ewe lambs on a farm on the Inner Hebridean island of Coll. Oral dosing of the ewe lambs with 15 mg/kg albendazole only achieved a 79.2% reduction in Dicrocoelium dendriticum egg count, 21 day post treatment. Thus, this report of dicrocoeliosis differs from the disease that occurs elsewhere, both in the severity and nature of its clinical signs, and in the response of the parasite to anthelmintic drug treatment. These differences could indicate the existence of a genetically divergent D. dendriticum population, which may have evolved within the unique and isolated biotope afforded by the machair on the island of Coll. Better understanding of these factors is a prerequisite for effective and sustainable disease control.

Cystic echinococcosis in Greece. Past and present.

Cystic echinococcosis is a zoonotic disease with a wide geographical distribution, Greece included, and is considered to be a serious problem for the public health and the livestock economy. Although the disease was widespread in Greece since ancient times, cystic echinococcosis was identified as a serious problem around 1970, and since then national surveillance programmes are running, based on meat inspection and stray dogs management. Ever since, there are official records of the parasite's prevalence in humans and livestock which show a continuous decline. More precisely, human hydatidosis, according to the official records, declines from an annual incidence of 14.8 per 100,000 inhabitants during 1967-1971 to 0.3 in 2008. Late surveys reveal that in Greece the prevalence of echinococcosis was 23-39.2% for sheep, 7.6-14.7% for goats, 0% in cattle and 0.6% in pigs, while further molecular analyses in Southern Greece showed the existence of the genotypes G1 and G3 in sheep and G7 in goats in that area. All data presented demonstrate that the parasite is still present in Greece. Surveillance is nowadays being performed under EU regulations but it is highly important to improve and adopt corrective and preventive measures to avoid animal and human infection.

Fleshflies in the flesh: epidemiology, population genetics and control of outbreaks of traumatic myiasis in the Mediterranean Basin.

Although the damaging effects on healthy tissues of its developing larvae were first described in 1770, the fleshfly Wohlfahrtia magnifica remains a serious pest for the livestock industry. Wohlfahrtiosis, the severe myiasis caused by this fly, is a grave problem in terms of both the animal welfare and economic loss. This review highlights important aspects of the biology, pest status, epidemiology, population genetic structure, economics and control of W. magnifica and wohlfahrtiosis, with an emphasis on recent outbreaks in Greece and Morocco and fly population dynamics in the Mediterranean Basin. Potential areas for future studies on genetics, host tolerance, in vitro rearing, field behaviour and range expansion of the species are also discussed.

Clinical and laboratory study of rosacea in northern Greece.

BACKGROUND: Numerous factors have been implicated in the pathogenesis of rosacea, which remains obscure. OBJECTIVES: To examine the epidemiological characteristics of rosacea patients, the histopathological alterations, the prevalence of gastric Helicobacter pylori infection and the role of ultraviolet radiation, to detect the presence of Demodex folliculorum on affected skin and to elucidate the immunological nature of this disorder. METHODS: The study included 100 patients with rosacea. Each patient was assessed with a clinical, haematological, biochemical and histological examination; serology test for the detection of antibodies against H. pylori; direct immunofluorescence on perilesional, sun exposed skin and indirect immunofluorescence with monkey oesophagus as a substrate; antinuclear antibody titre and a skin surface biopsy to search for Demodex folliculorum. RESULTS: Women were more frequently affected. Half of our patients were 51-70 years old. About two-thirds were photo-types I and II and 73% complained of worsening of conditions after sun exposure. An almost permanent histopathological feature was solar elastosis. Higher prevalence of H. pylori was not established. Prevalence and mean density of Demodex folliculorum were significantly increased in rosacea patients. Direct and indirect immunofluorescence tests were positive in 6.4% and 6.7% respectively. Antinuclear antibody titres were found in 21.1%. CONCLUSIONS: Our results suggest the pivotal role of chronic sun exposure in the pathogenesis of rosacea. Demodex folliculorum represents a significant cofactor that may contribute to the transition of the disease from a vascular to an inflammatory stage. The low positive results of direct and indirect immunofluorescence do not support a potential autoimmune role in the development of rosacea.

Anthelmintic activity of some Mediterranean browse plants against parasitic nematodes.

The anthelmintic properties of tannin-rich plants are being explored as an alternative to chemical drugs. Most data have been acquired on legume forages, but only few on browse plants. The present study aimed to (i) screen the in vitro effects of extracts from 7 Mediterranean plants on Haemonchus contortus, (ii) verify the role of tannins using an inhibitor, polyvinyl polypyrrolidone (PVPP) and (iii) verify the in vivo effects of extracts from 4 plants. Significant inhibition was shown in vitro using a larval migration inhibition (LMI) assay for all extracts except that from Olea europaea var. koroneiki. After adding PVPP, the LMI values were restored to control levels for all plants except Pistacia lentiscus and Ceratonia siliqua, confirming a role for tannins in the activity. In the in vivo experiment, 48 lambs composed 6 groups, depending on diet. On Day 0, groups G1-G5 received H. contortus and Trichostrongylus colubriformis larvae and G6 remained uninfected. The various diets were distributed from Days 14 to 45: P. lentiscus (G1), Quercus coccifera (G2), C. siliqua (G3), Onobrychis viciifolia (G4), or Medicago sativa for the 2 control groups (G5, G6). Egg excretion, packed cell volumes (PCVs) and inorganic phosphate were measured weekly throughout the entire experimental period. At slaughter, the worms were enumerated and their fecundity assessed. Consumption of the 4 browser plants did not provoke differences in pathophysiological measurements but there were significant decreases in egg excretion, mainly explained by significant decreases in worm fecundity for both species, without any statistical difference in worm numbers.

Molecular genetic analysis of populations of Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica, in outbreak populations from Greece and Morocco.

Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae), is the most important cause of traumatic myiasis in the southern Palaearctic region. Larval stages are obligate parasites and the wounds caused by infestations are very similar to those caused by Old and New World screwworm flies. During the last decade, W. magnifica appears to have expanded its range to parts of northern and central Morocco, and to Crete, Greece. Specimens of W. magnifica were collected in Morocco and Crete either as larvae (preserved in 80% ethanol) or as adults (dry-pinned). Comparison specimens were collected in Spain, Hungary and mainland Greece. A DNA fragment containing the 3' 715 base pairs of the mitochondrial cytochrome b gene was amplified by polymerase chain reaction from each of 132 larvae or adults of W. magnifica and the amplicons were directly sequenced and analysed phylogeographically. Twelve cytochrome b haplotypes were detected. All haplotypes from Morocco belonged to a lineage that included specimens from the Iberian peninsula, and restricted mixing of central and northern populations in Morocco was demonstrated. Cytochrome b haplotyping combined with an analysis of larval size provided clear evidence of multiple infestations of hosts in all geographical areas, with one quarter of wounds containing larvae from two to at least four females. More than 80% of specimens from Crete contained a haplotype predominating in mainland Greece and Hungary. Our survey indicated that wohlfahrtiosis was more widespread in northern and central Morocco than previously recorded by government veterinarians. However, the prevalence of wohlfahrtiosis was low (< 1%). The high genetic diversity of Moroccan populations is consistent with longterm endemicity, rather than recent introduction. Crete showed a higher prevalence of wohlfahrtiosis (< or = 15%) and less genetic diversity of W. magnifica, which is consistent with a recent introduction. The western and eastern Mediterranean lineages may have been isolated in different Pleistocene ice-age refugia, from which there has been limited post-glacial dispersal.