Henrik O. Bøgh

A survey of anthelmintic resistance in nematode parasites of goats in Denmark.

The occurrence of anthelmintic resistance in 15 goat herds in Denmark was investigated using the faecal egg count reduction test (FECRT), egg hatch assay (EHA) for thiabendazole (TBZ) and a microlarval development assay (LDA) for TBZ, levamisole (LEV) and ivermectin (IVM). Resistance to both benzimidazole (BZ) and levamisole (LEV) anthelmintics was detected on six farms, and to both BZs and IVM on one farm. Resistance to BZs was also detected on two farms and to LEV on two farms. Ivermectin resistance was present on one farm. Trichostrongylus and Ostertagia spp. were the predominant nematode species in both pre-treatment and post-treatment faecal cultures. In all nine instances where BZ resistance was detected in the FECRT, the LD50 values for TBZ in the EHA were higher than 0.5 microM TBZ (0.1 microgram TBZ/ml), indicating resistance. LD50 values for TBZ in the LDA were also higher than 0.5 microM where resistance to BZs was detected in the FECRT, except two farms where LD50 values of 0.21 and 0.29 microM TBZ were recorded. LD50 values in the LDA for isolates declared resistant to LEV ranged from 0.78 to 5.62 microM LEV. For IVM, the two resistant isolates had LD50 values of 25 and 68 nM IVM. There were disagreements in the declaration of resistance between methods of calculating faecal egg count reduction percentage (FECR%) based on the arithmetic mean and those where geometric mean EPG is used. Similarly, inclusion of pre-treatment EPG or control group EPG in the calculation of FECR% influenced declaration of resistance.

The prevalence and intensity of helminth and coccidial infections in dairy cattle in central Kenya.

A survey of gastrointestinal parasite infections of young (<6 months old), immature (6–12 months old) and adult (>12 months old) dairy cattle on 16 farms in Kiambu District, Kenya was conducted during a dry season (September 1991 to January 1992) and during a wet season (March to July 1992). The survey was based on monthly coproparasitological examination of cohorts and worm counts in tracer calves. The effects of age, sex, farm and season on the prevalence and intensity of helminth and coccidial infections were determined. Faecal egg and oocyst counts revealed that the overall prevalences were: strongyles (including trichostrongyles) (85.5%), liver flukes (Fasciola gigantica) (34.0%), coccidia (30.9%) and tapeworms (9.6%). Eight species of the protozoan Eimeria were identified, the most prevalent species being E. bovis and E. zuernii. The most prevalent nematode genera were Haemonchus, Cooperia, Oesophagostomum and Trichostrongylus. Season, farm and age of the animals had a significant (p<0.05) influence on the intensity of infection with strongyles, liver flukes and coccidia, whereas the sex of the animals had no significant (p>0.05) effect on the prevalence or intensity of infections. A higher intensity of infection with strongyles and coccidia was found in the wet season than in the dry season (p<0.05). The age-specific intensity was in the following order: for strongyles, immature animals of 6–12 months of age had the highest egg counts, followed by young calves and adults. Calves had significantly (p<0.05) higher oocyst counts than immatures or adults. Liver fluke egg counts did not differ significant (p>0.05) between immatures and adult cattle.

Schistosoma japonicum infection in the pig as a model for human schistosomiasis japonica

Valuable information on human schistosomiasis japonica has been provided using primates and experimental rodent hosts. However, major drawbacks such as high costs and ethical concerns for the primate models and large biological deviations for the rodent models have led to the search for more appropriate models. Recent data on the pig indicate that this natural host for Schistosoma japonicum might be a realistic alternative. As only very few research groups have investigated the S. japonicum/pig model, the present review mainly deals with the experimental methods and the major host/parasite findings obtained from the authors own research group. With emphasis on a critical evaluation of the work, the results are compared to the scarce information existing on human schistosomiasis japonica. Like in humans, S. japonicum establishes mainly in the large intestinal veins, with high faecal egg counts during the acute phase of infection, which varies greatly within and between days. Concomitant resistance is another shared feature, but studies in pigs have indicated that the phenomenon is more complex than generally thought. Clinical signs as eosinophilia and diarrhoea with mucus and blood in the acute phase of infection and hepatomegaly, increased portal diameter, periportal fibrosis and ascites in chronic infections are common findings in both humans and pigs. Low protein diet aggravates the disease in pigs by increasing the establishment rates, the faecal egg excretion and the morbidity. A 100% cure rate is achieved when treating S. japonicum infected pigs with praziquantel at 40 mg/kg, and 4 weeks post treatment pigs remain resistant to reinfection. Lastly, human congenital S. japonicum infection has been confirmed in pigs but the implications of such infections for the pathogenesis of schistosomiasis japonica remain to be investigated.

PCR-based identification of individuals of Schistosoma japonicum representing different subpopulations using a genetic marker in mitochondrial DNA

A mitochondrial NADH dehydrogenase I gene fragment (NDI) was sequenced for three laboratory maintained isolates of Schistosoma japonicum. Comparison of sequences representing the isolates (originally obtained from the Anhui and Zhejiang provinces of the Peoples Republic of China, and from the Philippines) revealed inter-isolate sequence variations of 0.2-0.6% and no intra-isolate variation was found. The sequences also indicated that while the amplification products of the Zhejiang and Philippine isolates contained a recognition site for the endonuclease RsaI, there was no such site in the Anhui isolate. This was tested by digesting amplification products from a number of individual worms with RsaI. Then an infection experiment was designed to test the value of this genetic marker for studies of the population biology of S. japonicum in the final host. For this, the two Chinese isolates were used. Three groups of mice (A-C) were exposed firstly to a primary infection and then challenge-infected at weeks 4 and 7 of the experiment. In group A the first infection was done with the Anhui isolate, and the two others with the Zhejiang isolate, thereby providing a specific, detectable cohort. In groups B and C the Anhui isolate was used for the second and third infection. All mice were perfused 5 weeks after the last challenge infection, and the NDI was subsequently amplified from DNA of the perfused worms and digested with RsaI. The digestion revealed that while infection groups A and B contained mixed populations of the Anhui and Zhejiang isolates, only Zhejiang worms were present in group C. We concluded that the absence/presence of the RsaI site in the NDI provides a useful marker for the delineation of cohorts of S. japonicum.

Scanning for nucleotide variations in mitochondrial DNA fragments of Schistosoma japonicum by single-strand conformation polymorphism

In this study, we employed a mutation scanning approach for the direct visual display of genetic variability in mitochondrial DNA (mtDNA) fragments within and among populations of Schistosoma japonicum from the Peoples Republic of China. Fragments of the NADH dehydrogenase 1 gene (ND1) and the cytochrome c oxidase subunit I (COI) were individually amplified from parasite DNA by polymerase chain reaction (PCR), denatured and subjected to single-strand conformation polymorphism (SSCP) analysis. Using ND1 and COI fragments, individuals representing different genotypes could be readily identified based on characteristic and reproducible SSCP profiles. The results demonstrated the usefulness of SSCP for the direct visual display of low-level sequence variation in mtDNA of S. japonicum prior to DNA sequence analysis. This approach has important implications for studying the genetic structure and biology of S. japonicum populations, and for analysing the inheritance of mitochondrial DNA.

Worm establishment and egg production of Schistosoma japonicum in pigs infected by percutaneous methods or intramuscular injection.

Worm establishment and egg production were compared in Landrace/Yorkshire crossbred pigs infected with Schistosoma japonicum using 3 different infection techniques. S. japonicum worm establishment and overall egg production following intramuscular injection of cercariae suspended in Iscoves medium exceeded that in pigs infected by the leg immersion or the coverslip percutaneous technique. However, the egg production per mature female was similar for all three infection methods. The results support the use of intramuscular injection of medium-suspended cercariae as an effective, safe, rapid and convenient infection method for studies on the population biology of S. japonicum in pigs when dermal cercarial penetration is not required.

Variation in the sequence of a mitochondrial NADH dehydrogenase I gene fragment among six natural populations of Schistosoma japonicum from China

The present study investigated the level of genetic variation among Schistosoma japonicum populations of different geographical origins from mainland China. Polymerase chain reaction-based methods were employed to determine the sequence for a subunit of the mitochondrial NADH dehydrogenase I gene for populations from Zhejiang, Anhui, Jiangxi, Hunan, Hubei and Sichuan. No variation was detectable in the NADH dehydrogenase I sequence within populations from Zhejiang and Hubei, whereas sequence variation of 0.2% was detected within populations from Anhui, Jiangxi, Hunan and Sichuan. Pairwise comparison of the sequences representing the six different populations revealed genetic differences ranging from 0 to 0.6%.

Comparison of the vaccine efficacy of γ-irradiated Schistosoma japonicum cercariae with the defined antigen Sj62(IrV-5) in pigs

Development of a vaccine against Schistosoma japonicum which can protect both man and the domestic animal zoonotic reservoirs of infection would be an invaluable tool in attempts to control this infection in those areas in which conventional control methods have failed to break transmission. The pig is a natural host of S. japonicum and because of its anatomical and immunological similarities to humans, it is a potentially valuable host for studies on S. japonicum in particular and schistosomes in general. Radiation-attenuated cercariae are highly effective in inducing immunity in experimental schistosomosis and there are promising reports of partial protection against schistosomes with recombinant-derived individual antigens. In the present study we have set out to establish a protocol for inducing protection with gamma-irradiated cercariae in pigs and to assess the protective capacity of recombinant and naked DNA formulations of Sj62, a 62kDa region of S. japonicum myosin. The corresponding S. mansoni version or Sj62, recombinant IrV-5, has previously been implicated in irradiated vaccine immunity in S. mansoni infections and has been shown to induce high levels of immunity in a variety of hosts. Groups of pigs were immunised three times at 2-week intervals with 2000 cercariae irradiated at 20krad, with Sj62 as a recombinant (rSj62) incorporated in Freunds adjuvant, a micellar preparation, or as a naked DNA construct. Vaccination with irradiated cercariae did not induce significant anti-Sj62 antibody but following intramuscular challenge with 2000 cercariae, the vaccinated pigs showed >95% resistance as assessed by reduced faecal egg output, worm tissue egg burdens and also reduced septal fibrosis. Immunisation with each of the Sj62 formulations induced significant anti-Sj62 antibody responses, the highest titre (>12,800) being with the Freunds preparation but none of the Sj62-immunised groups showed significant resistance to challenge. The data suggest that Sj62 shows little promise as a vaccine candidate for schistosomosis.

Distribution of Taenia saginata cysts by muscle group in naturally infected cattle in Tanzania

The distribution of Taenia saginata cysts among organs and muscle groups was determined by slicing the heart, masseters, liver and muscles of the half carcass of 21 naturally infected Zebu cattle from Morogoro abattoir, Tanzania. The carcasses harboured from 1 to 310 cysts. In the muscle groups, normally considered predilection sites, the heart and Musculus triceps brachii harboured the highest proportions of cysts (mean 17% and 12%, respectively, of the total cysts in the carcass) while lower proportions were found in the tongue, masseters and psoas muscles (3%, 2% and 5%, respectively). A relatively high proportion was found in the liver (10%). Relative cyst density was calculated for the different muscle groups by dividing the mean proportion of total weight of the muscle group into the mean proportion of cysts located in that site. The cysticerci in the examined tissues were found in the following order of relative density: heart (10.4), tongue (2.1), masseters (1.9), M. triceps brachii (1.7), liver (1.4), M. psoas (1.3), hindlimb muscles (0.71), trunk muscles (0.67), and other forelimb muscles (0.32). The proportion of cysts expected to be found at the surfaces exposed by visual examination or incision at meat inspection was calculated using an indirect method, which incorporated the area revealed by incision and visual inspection of an organ and the proportion of cysts located in the particular organ. It was estimated that 17% of the cysts would be located at an inspected site if the regulations were followed carefully. Based on cyst density, we recommend that the heart should be paid particular attention during meat inspection.

Vaccination against Taenia taeniaeformis infection in rats using a recombinant protein and preliminary analysis of the induced antibody response

Primary screening of a cDNA expression library of Taenia taeniaeformis oncospheres in lambda gt11 bacteriophage was carried out using rabbit anti-T, taeniaeformis oncosphere serum affinity-purified from oncosphere pellets. From approximately 1.6 x 10(5) plaques, 21 single clones that were positive with the affinity-purified antibodies were isolated. Sibling analysis revealed that 17 clones out of the 21 could be assigned to five different antigen families. Only family 1 was strongly recognized by a serum prepared in a rabbit against a partially purified host-protective oncosphere antigen fraction. The fragments of lambda DNA were inserted into a pGEX plasmid vector that encodes glutathione S-transferase (GST) of Schistosoma japonicum. Clones designated TtO-18, -49.53 (family 1), 46 (family 2), 15 (family 3), 40 (family 4) and 66 (family 5) were established as subclones in pGEX-1 plasmid vectors which produced GST fusion proteins. All GST fusion proteins were soluble and recognized by anti-GST and anti-TtO sera. Three vaccination experiments with these fusion proteins using specific-pathogen-free Wistar rats revealed that all three fusion proteins of family 1 were exclusively effective against T. taeniaeformis oncosphere challenge with approximately 95% and 91% reductions in cystic metacestode and total metacestode recoveries, respectively. Rats vaccinated with fusion proteins of family 1 produced antibodies which reacted with a 21-kDa oncosphere antigen component which appeared to be a major oncosphere stage-specific antigen.