Ascaris lumbricoides eggs or artefacts? A diagnostic conundrum.

Due to the presence of artefacts in stool samples, the copromicroscopic diagnosis of Ascaris lumbricoides is not always straightforward, particularly in the case of fertilized decorticated eggs. A total of 286 stool samples from 115 schoolchildren in India and 171 adult immigrants in Italy were screened for the presence of A. lumbricoides eggs by both Kato-Katz thick smear and Mini-FLOTAC. If the outer layer of A. lumbricoides eggs was absent, two aliquots of each stool sample were preserved: one for coproculture to identify larvae after development and one to compose a pool of stool for molecular analysis. A total of 64 stool samples (22.4%) were positive for A. lumbricoides using the Kato-Katz thick smear; 36 (56.3%) of these showed mammillated A. lumbricoides eggs, 25 (39.1%) showed elements resembling fertilized decorticated eggs, while three samples (4.7%) showed both mammillated and decorticated eggs. By Mini-FLOTAC, 39 stool samples (13.6%) were positive, while decorticated A. lumbricoides-like eggs were identified as artefacts. These results were confirmed by negative coprocultures and quantitative polymerase chain reaction. Mini-FLOTAC can be used for a reliable diagnosis of A. lumbricoides, thanks to the flotation and translation features which allow a clearer view, resulting in the correct identification of A. lumbricoides eggs.

Evaluation of the therapeutic efficacy of praziquantel against schistosomes in seven countries with ongoing large-scale deworming programs.

The World Health Organization (WHO) recommends periodic assessment of the therapeutic efficacy of praziquantel (PZQ) to detect reduced efficacy that may arise from drug resistance in schistosomes. In this multi-country study (2014), we assessed the therapeutic efficacy of a single oral dose of PZQ (40 mg/kg) against Schistosoma mansoni (Brazil, Cameroon, Ethiopia, Mali, Madagascar and Tanzania), S. haematobium (Cameroon, Ethiopia, Mali, Tanzania and Zanzibar) and S. japonicum (the Philippines) infections in school-aged children, across a total of 12 different trials. Each trial was performed according to the standardized methodology for evaluating PZQ efficacy as described by the WHO. Overall, therapeutic efficacy, measured as the reduction in arithmetic mean of schistosome egg counts following drug administration (egg reduction rate; ERR), was high for all three schistosome species (S. mansoni: 93.4% (95%CI: 88.8-96.8); S. haematobium: 97.7% (95%CI: 96.5-98.7) and S. japonicum: 90.0% (95%CI: 68.4-99.3). At the trial level, therapeutic efficacy was satisfactory (point estimate ERR ≥90%) for all three Schistosoma species with the exception of S. mansoni in Cameroon where the ERR was 88.5% (95%CI: 79.0-95.1). Furthermore, we observed that in some trials individual drug response could vary significantly (wide 95%CI) and that few non-responsive individuals could significantly impact ERR point estimates. In conclusion, these results do not suggest any established reduced efficacy of the standard PZQ treatment to any of the three schistosome species within these countries. Nevertheless, the substantial degree of variation in individual responses to treatment in some countries underpins the need for future monitoring. The reported ERR values serve as reference values to compare with outcomes of future PZQ efficacy studies to ensure early detection of reduced efficacies that could occur as drug pressure continues increase. Finally, this study highlights that 95%CI should be considered in WHO guidelines to classify the therapeutic efficacy of PZQ.

Measuring larval nematode contamination on cattle pastures: Comparing two herbage sampling methods.

Assessing levels of pasture larval contamination is frequently used to study the population dynamics of the free-living stages of parasitic nematodes of livestock. Direct quantification of infective larvae (L3) on herbage is the most applied method to measure pasture larval contamination. However, herbage collection remains labour intensive and there is a lack of studies addressing the variation induced by the sampling method and the required sample size. The aim of this study was (1) to compare two different sampling methods in terms of pasture larval count results and time required to sample, (2) to assess the amount of variation in larval counts at the level of sample plot, pasture and season, respectively and (3) to calculate the required sample size to assess pasture larval contamination with a predefined precision using random plots across pasture. Eight young stock pastures of different commercial dairy herds were sampled in three consecutive seasons during the grazing season (spring, summer and autumn). On each pasture, herbage samples were collected through both a double-crossed W-transect with samples taken every 10 steps (method 1) and four random located plots of 0.16 m(2) with collection of all herbage within the plot (method 2). The average (± standard deviation (SD)) pasture larval contamination using sampling methods 1 and 2 was 325 (± 479) and 305 (± 444)L3/kg dry herbage (DH), respectively. Large discrepancies in pasture larval counts of the same pasture and season were often seen between methods, but no significant difference (P = 0.38) in larval counts between methods was found. Less time was required to collect samples with method 2. This difference in collection time between methods was most pronounced for pastures with a surface area larger than 1 ha. The variation in pasture larval counts from samples generated by random plot sampling was mainly due to the repeated measurements on the same pasture in the same season (residual variance component = 6.2), rather than due to pasture (variance component = 0.55) or season (variance component = 0.15). Using the observed distribution of L3, the required sample size (i.e. number of plots per pasture) for sampling a pasture through random plots with a particular precision was simulated. A higher relative precision was acquired when estimating PLC on pastures with a high larval contamination and a low level of aggregation compared to pastures with a low larval contamination when the same sample size was applied. In the future, herbage sampling through random plots across pasture (method 2) seems a promising method to develop further as no significant difference in counts between the methods was found and this method was less time consuming.

Monitoring the efficacy of drugs for neglected tropical diseases controlled by preventive chemotherapy.

In the last decade, pharmaceutical companies, governments and global health organisations under the leadership of the World Health Organization (WHO) have pledged large-scale donations of anthelmintic drugs, including ivermectin (IVM), praziquantel (PZQ), albendazole (ALB) and mebendazole (MEB). This worldwide scale-up in drug donations calls for strong monitoring systems to detect any changes in anthelmintic drug efficacy. This review reports on the outcome of the WHO Global Working Group on Monitoring of Neglected Tropical Diseases Drug Efficacy, which consists of three subgroups: (i) soil-transmitted helminthiases (ALB and MEB); (ii) onchocerciasis and lymphatic filariasis (IVM); and (iii) schistosomiasis (PZQ). Progress of ongoing work, challenges and research needs for each of the four main drugs used in helminthic preventive chemotherapy (PC) are reported, laying the ground for appropriate implementation of drug efficacy monitoring programmes under the co-ordination and guidelines of the WHO. Best practices for monitoring drug efficacy should be made available and capacity built as an integral part of neglected tropical disease (NTD) programme monitoring. Development of a disease-specific model to predict the impact of PC programmes, to detect outliers and to solicit responses is essential. Research studies on genetic polymorphisms in relation to low-efficacy phenotypes should be carried out to identify markers of putative resistance against all NTD drugs and ultimately to develop diagnostic assays. Development of combination and co-administration of NTD drugs as well as of new drug entities to boost the armamentarium of the few drugs available for NTD control and elimination should be pursued in parallel.

Efficacy of different albendazole and mebendazole regimens against heavy-intensity Trichuris trichiura infections in school children, Jimma Town, Ethiopia.

Recent studies have shown that the efficacy of benzimidazole drugs is influenced by the intensity of trichuriasis. Therefore, the objective of this study was to determine the efficacy of albendazole (ALB) and mebendazole (MBZ) administered randomly for 1 (ALB×1 and MBZ×1) or 2 days (ALB×2 and MBZ×2) to 385 school children with heavy-intensity trichuriasis (mean faecal egg counts (FEC) >1000 eggs per gram of stool (epg)) in Jimma Town, Ethiopia. The efficacies (95% confidence intervals) by means of reduction in faecal egg counts (FECs) were 29·3% (-9·9-56·2), 60·0% (48·5-70·9), 73·5% (64·2-81·3), and 87·1% (81·4-91·2) for ALB×1, MBZ×1, ALB×2, and MBZ×2, respectively. These observations highlight that assessment of the anthelmintic efficacy of existing or new compounds against Trichuris trichiura should be assessed under varying levels of infection intensity.

Risk factors for Psoroptes ovis mange on Belgian Blue farms in Northern Belgium.

The parasitic mite Psoroptes ovis causes important economic losses in Belgian Blue cattle. Because mange problems vary significantly between farms, a cross-sectional questionnaire survey and subsequent farm visits were performed to identify potential risk factors for P. ovis infections on Belgian Blue farms in Flanders, Belgium. The questionnaire was sent to 1800 beef farms to evaluate the presence and severity of psoroptic mange in the herd and to assess farm management practices, including antiparasitic treatments. Subsequently, about 10% of the farms with a completed questionnaire were visited to validate the questionnaire and to retrieve supplementary information on additional management parameters, such as barn infrastructure and climate. Associations between parasitism and putative risk factors were assessed by logistic regression. Out of 1800 contacted farms, 680 (38%) completed questionnaires were received. Data were collected from 238 barns during 66 farm visits. The questionnaire results demonstrated a high farm prevalence of mange (74%; 95% CI (70.7-77.3)) and half of the farmers declared that the problem was difficult to control. Nevertheless, in only 14% of the barns a high scratching index was recorded and in most of the sampled animals (80%) the affected body surface was less than 10%. This indicates that despite the high prevalence and the difficulty to control the infection, clinical signs were often quite moderate. Logistic regression analyses of the questionnaire and the farm visit data suggested that heavily infested farms treat more intensively against mange. On most farms mange occurred the whole year round and more problems with mange were found on farms where a higher number of animals were purchased per year. In addition, the disease was more prevalent when the animals had a lower hygiene score. This score was strongly correlated with environmental hygiene, indicating that transmission of mites from the environment to the animals should not be underestimated. Conflicting results were obtained on the effect of supplementing minerals on the occurrence of mange. In this study, temperature, light intensity and relative humidity in the barns, ventilation systems, barn infrastructure, animal stocking rate and blood mineral levels were not indicated as risk factors for mange. In conclusion, maintaining a good animal hygiene and if possible, avoiding introduction of cattle may help to control psoroptic mange in Belgian Blue cattle.

The economic effects of whole-herd versus selective anthelmintic treatment strategies in dairy cows.

Current control practices against gastrointestinal nematodes in dairy cows rely strongly on anthelmintic use. To reduce the development of anthelmintic resistance or disposition of drug residues in the environment, novel control approaches are currently proposed that target anthelmintic treatment to individual animals instead of the whole herd. However, such selective treatment strategies come with additional costs for labor and diagnostics and, so far, no studies have addressed whether they could be economically sustainable. The objectives of this study were to (1) investigate the economic effects at farm level of whole-herd versus more selective anthelmintic treatment strategies in adult dairy cows, and (2) determine how these economic effects depend on level of infection and herd size. A Monte Carlo simulation, fed by current epidemiological and economical knowledge, was used to estimate the expected economic effects and possible variation of different control strategies under Belgian conditions. Four treatment strategies were compared with a baseline situation in which no treatments were applied: whole herd at calving (S1), selective at calving with (S2) or without (S3) treatment of the first-calf cows, and whole-herd when animals are moved from grazing to the barn in the fall (housing treatment, S4). The benefit per lactation for an average dairy herd varied between -$2 and $131 (average $64) for S1, between -$2 and $127 (average $62) for S2, between -$17 and $104 (average $43) for S3, and between -$41 and $72 (average $15) for S4. The farmer's risk associated with any treatment strategy, as indicated by the width of the 95% credible intervals of economic benefit of anthelmintic treatment, decreased with increasing level of exposure, as assessed by bulk tank milk ELISA. The order of the different strategies when sorted by expected benefit was robust to changes in economic input parameters. We conclude that, on average, strategies applying anthelmintic treatment at calving outperform a strategy applying treatment at housing. Within the strategies that applied treatment at calving, more selective treatment strategies can be economically sustainable. However, given the large variation in possible benefits within each treatment strategy, decision support systems are needed to account for the multitude of cow, epidemiological, and economic factors that determine the economics of nematode control and select the optimal treatment strategy for a specific farm.

The bias, accuracy and precision of faecal egg count reduction test results in cattle using McMaster, Cornell-Wisconsin and FLOTAC egg counting methods.

The faecal egg count reduction test (FECRT) is the recommended method to monitor anthelmintic drug efficacy in cattle. There is a large variation in faecal egg count (FEC) methods applied to determine FECRT. However, it remains unclear whether FEC methods with an equal analytic sensitivity, but with different methodologies, result in equal FECRT results. We therefore, compared the bias, accuracy and precision of FECRT results for Cornell-Wisconsin (analytic sensitivity = 1 egg per gram faeces (EPG)), FLOTAC (analytic sensitivity = 1 EPG) and McMaster method (analytic sensitivity = 10 EPG) across four levels of egg excretion (1-49 EPG; 50-149 EPG; 150-299 EPG; 300-600 EPG). Finally, we assessed the sensitivity of the FEC methods to detect a truly reduced efficacy. To this end, two different criteria were used to define reduced efficacy based on FECR, including those described in the WAAVP guidelines (FECRT <95% and lower limit of 95%CI <90%) (Coles et al., 1992) and those proposed by El-Abdellati et al. (2010) (upper limit of 95%CI <95%). There was no significant difference in bias and accuracy of FECRT results across the three methods. FLOTAC provided the most precise FECRT results. Cornell-Wisconsin and McMaster gave similar imprecise results. FECRT were significantly underestimated when baseline FEC were low and drugs were more efficacious. For all FEC methods, precision and accuracy of the FECRT improved as egg excretion increased, this effect was greatest for McMaster and least for Cornell-Wisconsin. The sensitivity of the three methods to detect a truly reduced efficacy was high (>90%). Yet, the sensitivity of McMaster and Cornell-Wisconsin may drop when drugs only show sub-optimal efficacy. Overall, the study indicates that the precision of FECRT is affected by the methodology of FEC, and that the level of egg excretion should be considered in the final interpretation of the FECRT. However, more comprehensive studies are required to provide more insights into the complex interplay of factors inherent to study design (sample size and FEC method) and host-parasite interactions (level of egg excretion and aggregation across the host population).

Novel insights in the faecal egg count reduction test for monitoring drug efficacy against gastrointestinal nematodes of veterinary importance.

The faecal egg count reduction test (FECRT) is the method of choice to monitor anthelmintic efficacy against gastro-intestinal nematodes in livestock. Guidelines on how to conduct a FECRT are made available by the World Association for the Advancement of Veterinary Parasitology (WAAVP). Since the publication of these guidelines in the early 1990 s, some limitations have been noted, including (i) the ignorance of host-parasite interactions that depend on animal and parasite species, (ii) their feasibility under field conditions, (iii) appropriateness of study design, and (iv) the high detection limit of the recommended faecal egg count (FEC) method. Therefore, the objective of the present study was to empirically assess the impact of the level of excretion and aggregation of FEC, sample size and detection limit of the FEC method on the sensitivity and specificity of the FECRT to detect reduced efficacy (<90% or <95%) and to develop recommendations for surveys on anthelmintic resistance. A simulation study was performed in which the FECRT (based on the arithmetic mean of grouped FEC of the same animals before and after drug administration) was conducted under varying conditions of mean FEC, aggregation of FEC (inversely correlated with k), sample size, detection limit and 'true' drug efficacies. Classification trees were built to explore the impact of the above factors on the sensitivity and specificity of detecting a truly reduced efficacy. For a reduced-efficacy threshold of 90%, most combinations resulted in a reliable detection of reduced and normal efficacy. For the reduced-efficacy threshold of 95% however, unreliable FECRT results were found when sample sizes <15 were combined with highly aggregated FEC (k=0.25) and detection limits ≥ 5 EPG or when combined with detection limits ≥ 15 EPG. Overall, an increase in sample size and mean preDA FEC, and a decrease in detection limit improved the diagnostic accuracy. FECRT remained inconclusive under any evaluated condition for drug efficacies ranging from 87.5% to 92.5% for a reduced-efficacy-threshold of 90% and from 92.5% to 97.5% for a threshold of 95%. The results highlight that (i) the interpretation of this FECRT is affected by a complex interplay of factors, including the level of excretion and aggregation of FEC and (ii) the diagnostic value of FECRT to detect small reductions in efficacy is limited. This study, therefore, provides a framework allowing researchers to adapt their study design according to a wide range of field conditions, while ensuring a good diagnostic performance of the FECRT.

Comparison of the Kato-Katz thick smear and McMaster egg counting techniques for monitoring drug efficacy against soil-transmitted helminths in schoolchildren on Pemba Island, Tanzania.

The Kato-Katz thick smear technique is widely used to assess prevalence and intensity in soil-transmitted helminth (STH) control programmes, but its usefulness in monitoring anthelminthic drug efficacy needs to be validated and compared with other methods. A promising alternative is the McMaster egg counting technique. In the present study, the efficacy of single-dose albendazole against STH infections in 430 schoolchildren on Pemba Island was assessed using both the Kato-Katz and McMaster techniques. The study revealed comparable drug efficacy results for both methods and confirmed the potency of the McMaster technique as an alternative method for monitoring large-scale deworming programmes.

Doramectin resistance in Haemonchus contortus on an alpaca farm in Belgium.

Parasitism by gastrointestinal nematodes is a health concern in New World Camelids (NWC) worldwide, and anthelmintic treatment is often needed for parasite control. Although anthelmintic resistance has been reported in ruminants worldwide, data in NWC are only scarce. In the present study, a case of suspected doramectin resistance in alpacas was examined. A field efficacy study was conducted for the evaluation of two different dosages of doramectin using a faecal egg count reduction test. A group of 8 alpacas was treated with a subcutaneous injection of doramectin at 0.2mg/kg bodyweight. Individual faecal samples were collected before treatment and 7 days after treatment. The faecal egg counts indicated a treatment efficacy of only 68%. To determine whether the treatment failure was caused by true anthelmintic resistance or suboptimal dosage in this animal species, a group of 4 alpacas was subsequently treated at 0.3mg/kg bodyweight. Faecal egg counts 7 days post treatment were reduced by only 41%, indicating that the treatment failure was more likely to be caused by the presence of resistant parasites on this farm. Coprocultures of faecal samples collected after treatment indicated the presence of 98.5% Haemonchus contortus and a small percentage of Cooperia oncophora (<1.5%). A controlled efficacy trial in sheep, for which the optimal dosage of doramectin is known, was conducted to ensure that this truly was a case of resistant parasites. Infective larvae collected from the faeces of these alpacas were used to infect eight nematode-free lambs. These lambs were assigned to one of two groups based on faecal egg counts post infection. One group was treated with doramectin injectable at 0.2mg/kg bodyweight, the other group served as a non treated control group. Pharmacokinetics indicated that the doramectin treatment was adequate, yet an efficacy of only 16% was determined on day 7 after treatment. Identification of the larvae after treatment revealed 100% H. contortus. On day 7 after treatment, H. contortus worm counts were only reduced by 8% in the treated lambs. The results of the present study report for the first time a case of doramectin resistance in alpacas, mainly in H. contortus.

The impact of baseline faecal egg counts on the efficacy of single-dose albendazole against Trichuris trichiura.

There is considerable variation in the efficacy of single-dose albendazole (400mg) against Trichuris trichiura across human trials. Factors contributing to this variation have not yet been identified. We assessed the impact of mean baseline faecal egg counts (FEC) on the efficacy of single-dose albendazole against T. trichiura in five previously conducted trials. Our results suggest that efficacy measured by reduction in mean FECs decreased significantly (p<0.0001) when mean baseline FECs increased, highlighting that this parameter should be considered as an important confounding factor for drug efficacy.

Molecular differentiation of Entamoeba spp. in a rural community of Loja province, South Ecuador.

Although previous epidemiological surveys in Ecuador indicate the presence of Entamoeba histolytica, prevalence data of this parasite remain scarce. Most of the studies were based on microscopic examination, which does not allow a morphological differentiation from the non-pathogenic Ent. dispar and Ent. moshkovskii. In the present study, 674 stool samples from a South Ecuadorian rural community were screened for Entamoeba spp. Subsequently, molecular identification was performed on 101 samples containing Ent. histolytica/Ent. dispar/Ent. moshkovskii cysts. The study indicated the absence of Ent. histolytica in this South Ecuadorian community and confirmed the difficulty of differentiating Entamoeba spp. based on morphological features.

Mixed Giardia duodenalis assemblage A, B, C and E infections in pet chinchillas (Chinchilla lanigera) in Flanders (Belgium).

Worldwide there is an increasing trend to keep exotic animals as pets. In contrast to domestic animals, few studies have addressed the importance of infectious diseases of these exotic animals harbor. Chinchillas are host to Giardia, but prevalence studies are scarce. Moreover, little is known about their role as a zoonotic reservoir for Giardia. Therefore, the objective of the present study was (1) to study the occurrence of Giardia in pet chinchillas, (2) to identify risk factors and (3) to determine the role of these animals as potential zoonotic reservoir. To this end, pet chinchillas (Chinchilla lanigera) from both pet owners and breeders in Flanders (Belgium) were screened for the presence of Giardia spp. using a sedimentation flotation technique. A questionnaire was distributed among the pet owners to identify putative risk factors. A subset of the Giardiaisolates was characterized by direct sequencing of the ß-giardin gene. In addition, assemblage specific PCRs targeting the triose phosphate isomerase (tpi) gene were performed. Of the 80 chinchillas screened, 53 (66.3%) excreted cysts of Giardia. Youngsters and animals participating in shows were significantly more at risk for infection. A total of 22 isolates were characterized. Direct sequencing of the ß-giardin gene solely revealed the presence of G. duodenalis assemblage B. The assemblage specific PCRs confirmed these findings, but also revealed the presence of assemblage A (11 samples), C (15 samples) and E (2 samples). This study indicates that multiple Giardia spp. are highly prevalent in pet chinchillas and that these animals are a potential reservoir for zoonotic transmission. In addition, the results highlight the benefit of using an assemblage specific PCR in molecular studies as mixed infections are likely to be missed using conventional PCR approaches.

Molecular identification of Entamoeba spp. in captive nonhuman primates.

This study describes the molecular identification of 520 Entamoeba-positive fecal samples from a large and diverse population of captive nonhuman primates (NHP). The results revealed the presence of Entamoeba histolytica (NHP variant only), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms.

Use of hydrophobically modified inulin for the preparation of polymethyl methacrylate/polybutyl acrylate latex particles using a semicontinuous reactor.

The seeded semicontinuous emulsion copolymerization of methyl methacrylate (MMA) and butyl acrylate (BuA) stabilized with a graft polymeric surfactant based on inulin, INUTEC SP1, as well as its mixture with sodium lauryl sulfate (SLS) is described. The mixture of SLS and Brij58 (alcohol ethoxylated) and the mixture of SLS and Pluronic P85 (block copolymer PEO-PPO-PEO) are also used as surfactant systems. The addition of methacrylic acid (MAA) or acrylic acid (AA) as comonomers is also studied. Previous results proved this inulin-derivative surfactant, INUTEC SP1, to be very effective on synthesizing latexes using a very low surfactant concentration. The kinetic features of the emulsion polymerization (instantaneous conversion and total conversion) were gravimetrically determined along the reactions. Latex dispersions were characterized by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) to obtain the average particle size, the particle size distributions (PSDs) as well as the polydispersity index (PdI). The stability was determined by turbidimetry measurements and expressed in terms of critical coagulation concentration. The results showed that the use of the graft polymeric surfactant allowed obtaining highly stable nanoparticles, at low surfactant concentrations and high solid contents (up to 37 wt %). This is an improvement with respect to previous works, in which a mixture of the graft polymeric surfactant with another surfactant was required to obtain stable nanoparticles with low polydispersity, at high solid content. In the present work, low polydispersity was achieved using INUTEC as the only emulsifier, which was related to the absence of secondary nucleations. When a mixture of INUTEC SP1 and SLS is used, a wider PSD is obtained due to secondary nucleations. Replacing INUTEC SP1 by other nonionic surfactants such as Brij58 or Pluronic P85 leads to an increase of average particle size and wider PSD.

The use of a simplified faecal egg count reduction test for assessing anthelmintic efficacy on Belgian and German cattle farms.

Anthelmintic resistant cattle nematodes have been reported in different regions around the world. However, in Western Europe the assessment of the problem relies largely on case reports and no prevalence data based on wide-scale surveys are available. Therefore, we performed a survey to (1) screen for reduced anthelmintic efficacy in Belgian and German cattle farms; (2) evaluate the usefulness of a simplified faecal egg count reduction test (FECRT), where efficacies are based on the mean FECs of 10 at random collected faecal samples pre- and post-treatment per farm and (3) identify possible risk factors for reduced anthelmintic efficacy. Of 88 farms included in this study, 84 farms used macrocyclic lactones (MLs). A FECR <95% was observed on 39% of these 84 farms. However, using a Markov chain Monte Carlo simulation analysis, to correct for the used McMaster FEC technique with a detection limit of 50 epg, reduced efficacy could only be confirmed in 25% of the farms (21/84). Only Cooperia spp. were found in significant numbers in the coprocultures post-treatment. Reduced efficacy was significantly associated with farm type and with a lower efficacy in beef herds compared to dairy herds. Four farms were revisited and a standardized FECRT was performed to confirm anthelmintic resistance (AR). Surprisingly, macrocyclic lactone resistance against Cooperia oncophora was only confirmed in one of four farms. In conclusion, our results show that a reduced efficacy observed in a FECRT are not only caused by AR but that the detection limit of the FEC technique used and the (in)correct administration of the anthelmintic drugs are confounding factors of major importance.

Multilocus genotyping of Cryptosporidium and Giardia in non-outbreak related cases of diarrhoea in human patients in Belgium.

Stool samples from Belgian patients suffering from abdominal pain and/or diarrhoea were examined for Cryptosporidium and Giardia. Cryptosporidium-positive samples were genotyped using the 70 kDa heat shock protein and the 60 kDa glycoprotein (GP60) genes: C. hominis was identified in 54.2% and C. parvum in 45.8% of the samples. Sequencing at the GP60 locus indicated that subgenotype IbA10G2 of C. hominis and subgenotype IIaA15G2R1 of C. parvum were the most prevalent, although several other subgenotypes were identified. For Giardia, sequencing at the beta-giardin, triose phosphate isomerase (TPI) and glutamate dehydrogenase (GDH) genes revealed assemblage B as the most prevalent (74.4%) in human patients. A high degree of heterogeneity was found, especially on the beta-giardin gene, and to a lesser extent on the GDH gene. Furthermore, using a novel species-specific PCR based on the TPI gene, mixed infections with both assemblage A and B were detected in a large number (32.4%) of human patients, which might have important epidemiological implications.

Giardia and other intestinal parasites in different dog populations in Northern Belgium.

The objectives of this study were to obtain data on the prevalence of intestinal parasites in different dog populations in northern Belgium, to estimate the zoonotic risk associated with these infections and to identify potential risk factors. Between 2004 and 2007 a total of 1159 faecal samples were collected from 451 household dogs, 357 dogs from breeding kennels and 351 dogs with gastrointestinal disorders. The samples from dogs with gastrointestinal disorders were sent to the diagnostic Laboratory for Parasitology at Ghent University by veterinary practitioners. In household dogs the prevalence of intestinal parasites was relatively low. Giardia was the most commonly found parasite (9.3%, CI 5.5-13.1), followed by Toxocara canis (4.4%, CI 2.7-6.8). Much higher infection rates were observed in kennel dogs, especially for Giardia spp. (43.9%, CI 37.8-50.0); T. canis (26.3%, CI 21.8-31.2) and Cystoisospora spp. (26.3%, CI 21.8-31.2). Also in dogs with gastrointestinal problems, Giardia spp. (18.1%, CI 13.1-23.1), Cystoisospora spp. (8.8%, CI 6.1-12.3) and T. canis (7.4%, CI 4.9-10.7) were the most frequently detected parasites. In all dog populations pups were more frequently infected with Cystoisospora (P<0.0001 to P<0.05), Giardia (P<0.001 to P<0.05), and T. canis (P<0.0001 to P<0.001) than adult dogs, except for T. canis in household dogs, where this correlation was not significant. A significant association of anthelmintic treatment with T. canis infections was only observed within the household population. Household dogs with a higher number of treatments per year were more frequently infected with T. canis (P<0.05). There was a significant difference between the different breeding kennels for the occurrence of Cystoisospora, Giardia and T. canis (P<0.0001) and large kennels harboured relatively more infected animals than smaller breeding facilities (P<0.05). However, this was not significant for Giardia spp. Breed and gender did not affect the risk of an infection in any of the study populations. Toxocara and Giardia present a zoonotic risk, especially in household dogs, where the majority of Giardia positive samples (80%) belonged to the zoonotic assemblage A. In kennel dogs and clinically affected dogs the host-specific Giardia assemblages C and D were most prevalent (94% and 80%, respectively).

Mixed Giardia duodenalis assemblage A and E infections in calves.

A molecular epidemiological study was conducted on 100 dairy (499 calves) and 50 beef (333 calves) farms in Belgium to estimate the prevalence of different Giardia duodenalis assemblages in calves younger than 10 weeks of age. Positive samples from the epidemiological study and from a previous clinical study were selected and genotyped based on the amplification of the beta-giardin gene. To investigate the occurence of mixed assemblage A and E infections in calves, a novel assemblage-specific PCR was developed based on the triose-phosphate isomerase gene. The prevalence was 22% (95% Probability Interval (PI): 12-34%) in dairy calves and 45% (95% PI: 30-64%) in beef calves. In total, 120 Giardia-positive samples from dairy and beef calves collected in the epidemiological study and from clinically affected calves were identified based on the amplification of the beta-giardin gene. Overall G. duodenalis assemblage E was more prevalent (in 64% of the samples), although the majority (59%) of the dairy calves were infected with G. duodenalis assemblage A. Furthermore, mixed G. duodenalis assemblage A and E infections were identified in 31% of the calf samples (n=101) using the assemblage-specific PCR. We believe this is the first report of mixed infections in calves, and the results of the present study indicate that calves, although mainly infected with the host-specific G. duodenalis assemblage E, are frequently infected with the zoonotic assemblage A, either as a mixed or mono-infection, suggesting that calves might be underestimated as a potential zoonotic reservoir for human infections.