Thomas Schnieder

Release of Hepatic Plasmodium yoelii Merozoites into the Pulmonary Microvasculature

Plasmodium undergoes one round of multiplication in the liver prior to invading erythrocytes and initiating the symptomatic blood phase of the malaria infection. Productive hepatocyte infection by sporozoites leads to the generation of thousands of merozoites capable of erythrocyte invasion. Merozoites are released from infected hepatocytes as merosomes, packets of hundreds of parasites surrounded by host cell membrane. Intravital microscopy of green fluorescent protein–expressing P. yoelii parasites showed that the majority of merosomes exit the liver intact, adapt a relatively uniform size of 12–18 μm, and contain 100–200 merozoites. Merosomes survived the subsequent passage through the right heart undamaged and accumulated in the lungs. Merosomes were absent from blood harvested from the left ventricle and from tail vein blood, indicating that the lungs effectively cleared the blood from all large parasite aggregates. Accordingly, merosomes were not detectable in major organs such as brain, kidney, and spleen. The failure of annexin V to label merosomes collected from hepatic effluent indicates that phosphatidylserine is not exposed on the surface of the merosome membrane suggesting the infected hepatocyte did not undergo apoptosis prior to merosome release. Merosomal merozoites continued to express green fluorescent protein and did not incorporate propidium iodide or YO-PRO-1 indicating parasite viability and an intact merosome membrane. Evidence of merosomal merozoite infectivity was provided by hepatic effluent containing merosomes being significantly more infective than blood with an identical low-level parasitemia. Ex vivo analysis showed that merosomes eventually disintegrate inside pulmonary capillaries, thus liberating merozoites into the bloodstream. We conclude that merosome packaging protects hepatic merozoites from phagocytic attack by sinusoidal Kupffer cells, and that release into the lung microvasculature enhances the chance of successful erythrocyte invasion. We believe this previously unknown part of the plasmodial life cycle ensures an effective transition from the liver to the blood phase of the malaria infection.

Kupffer cells are obligatory for Plasmodium yoelii sporozoite infection of the liver

Previous studies suggested Plasmodium sporozoites infect hepatocytes after passing through Kupffer cells, but proof has been elusive. Here we present new information strengthening that hypothesis. We used homozygous op/op mice known to have few Kupffer cells because they lack macrophage colony stimulating factor 1 required for macrophage maturation due to a deactivating point mutation in the osteopetrosis gene. We found these mice to have 77% fewer Kupffer cells and to exhibit reduced clearance of colloidal carbon particles compared with heterozygous phenotypically normal littermates. Using a novel quantitative reverse transcription polymerase chain reaction assay for P. yoelii 18S rRNA, we found liver infection of op/op mice to be decreased by 84% compared with controls. However, using another way of limiting Kupffer cells, treatment with liposome‐encapsulated clodronate, infection of normal mice was enhanced seven‐ to 15‐fold. This was explained by electron microscopy showing temporary gaps in the sinusoidal cell layer caused by this treatment. Thus, Kupffer cell deficiency in op/op mice decreases sporozoite infection by reducing the number of portals to the liver parenchyma, whereas clodronate increases sporozoite infection by opening portals and providing direct access to hepatocytes. Together these data provide strong support for the hypothesis that Kupffer cells are the portal for sporozoites to hepatocytes and critical for the onset of a malaria infection.

Prevalence of benzimidazole resistance on horse farms in Germany

Faecal egg counts (FECS) were made on samples from 1383 horses on 64 farms in northern Germany between August 2000 and November 2001. There were significant differences between the mean FECS in the two years; in 2000, 59.6 per cent of 369 samples were positive and in 2001,32.6 per cent of 1014 samples were positive for strongyle eggs. The results of a FEC reduction test indicated that resistance to fenbendazole was present on all 10 farms where it had been used, including in 33 of 60 horses tested. In contrast, treatment with ivermectin resulted in the complete elimination of nematode eggs in all the 77 horses tested. The mean LD50 values of the egg hatch test for thiabendazole indicated resistance on all 20 farms investigated and in 94 of 134 samples (70 per cent).

Larval development of Toxocara canis in dogs.

The parasitic roundworm Toxocara canis is present in dog populations all over the world. Due to its zoonotic potential, this roundworm is of special interest not only for veterinarians, but also for medical practitioners. In the present review, current knowledge of infection routes and the subsequent development of larvae within the canine host is summarised. Furthermore, information about the clinical, pathological, enzymatic, haematological and histopathological changes was collected, giving a broad overview of current knowledge of the infection. Although the data collected over the years give an idea of what happens during the larval development of T. canis, many questions remain open. Nevertheless, it is important that we continue our efforts to further understand the biology of this versatile and compelling parasite and try to improve and optimise strategies to prevent the infection in dogs and thereby to protect humans from this infection.

Quantitative analysis of ITS2 sequences in trichostrongyle parasites

Real-time PCR assays were developed to identify and quantify common gastrointestinal nematodes of ruminants. The assays were based on genus-specific primer and probe combinations derived from the second internal transcribed spacer (ITS2) ribosomal DNA transcription unit of Haemonchus contortus, Ostertagia leptospicularis, Trichostrongylus colubriformis and Cooperia curticei. The TaqMan probes for the first two species were labelled with 6-carboxyfluorescein and those for the latter two with VIC and all were synthesised as dihydrocyclopyrroloindole minor groove binder conjugates. Cloned ITS2 DNA or genomic DNA isolated from first stage larvae, derived from overnight cultures, were used as template. The real-time PCRs reproducibly allowed the identification of at least 100 copies of cloned ITS2 DNA or one hundredth part of a single larval genomic DNA equivalent. The assays proved to be genus-specific since the addition of DNA from heterologous trichostrongyle genera did not change the cycle threshold values obtained with only homologous DNA. Furthermore, the use of genomic DNA of several other ruminant nematode parasite genera gave negative results. In duplex experiments, 6-carboxyfluorescein-labelled H. contortus or O. leptospicularis probes were used together with the VIC-labelled T. colubriformis and C. curticei probes, respectively, confirming the specificity and sensitivity of the probes found in the simplex experiments. The primer and probe combinations gave comparable results when applied with core reagents from different suppliers and with both the M x 4000 and the ABI 7700 instruments. This technique provides means for a rapid, reliable and quantitative detection and differentiation of the most important parasitic nematodes of sheep and cattle.

Antibodies to major pasture borne helminth infections in bulk-tank milk samples from organic and nearby conventional dairy herds in south-central Sweden

The objective of this randomised pairwise survey was to compare the regional distribution of antibody levels against the three most important helminth infections in organic and conventional dairy herds in Sweden. Bulk-tank milk from 105 organic farms and 105 neighbouring conventional dairy farms with access to pasture in south-central Sweden were collected in September 2008. Samples were also collected from 8 organic and 8 conventional herds located in a much more restricted area, on the same as well as 3 additional occasions during the grazing season, to reveal evidence for seasonal patterns against cattle stomach worm (Ostertagia ostertagi). Antibody levels to the stomach worm (O. ostertagi), liver fluke (Fasciola hepatica) and lungworm (Dictyocaulus viviparus) were then determined by detection of specific antibodies using three different enzyme-linked immunosorbent assays (ELISAs). According to the Svanovir Ostertagia ELISA, the mean optical density ratio (ODR) was significantly higher in the milk from organic compared to conventional herds, i.e. 0.82 (95% CL=0.78-0.86) versus 0.66 (0.61-0.71). However, no significant differences were observed in the samples collected at different time points from the same 16 herds (F(3,39)=1.18, P=0.32). Antibodies to D. viviparus infection were diagnosed with an ELISA based on recombinant major sperm protein (MSP), and seropositivity was found in 21 (18%) of the 113 organic herds and 11 (9%) of the 113 conventional herds. The seroprevalence of D. viviparus was somewhat higher in the organic herds (Chi-square=3.65, P=0.056), but with the positive conventional herds were located in the vicinity of infected organic herds. Of the 16 herds that were sampled on repeated occasions, as many as 10 (63%), were seropositive on at least one sampling occasion. Many of these turned positive towards the end of the grazing season. Only one herd was positive in all 4 samples and 3 were positive only at turn-out. Considering F. hepatica there was no difference in seroprevalence between organic and conventional herds according to the Institute Pourquier ELISA. In general, liver fluke infection was low and it was only diagnosed in 8 (7%) organic and 7 (6%) conventional herds.

Comparative use of faecal egg count reduction test, egg hatch assay and beta-tubulin codon 200 genotyping in small strongyles (cyathostominae) before and after benzimidazole treatment.

A survey on benzimidazole (BZ) resistance in small strongyles was performed on three farms in the tenth region in Chile. Samples from a total of 100 horses were tested using the faecal egg count reduction test (FECRT), the egg hatch assay (EHA) and an allele-specific PCR for the detection of beta-tubulin isotype 1 genes coding for phenylalanine (phe) or tyrosine (tyr) at codon 200. In the past, BZ-type drugs have been used within anthelmintic campaigns on all the three farms. This has predictably led to a high degree of BZ resistance at the Valdivia and Riñihue farms and to a lesser degree at the Frutillar farm, as demonstrated by all the three tests. The FECRT indicated resistance in every farm by faecal egg count reductions (FECR) of 27% (S.D. +/- 33), 26.5% (S.D. +/- 26.9) and 83.9% (S.D. +/- 22.8) for the Valdivia, Riñihue and Frutillar farms, respectively. With the EHA, the following mean LD(50) values were found before and after treatment with fenbendazole (FBZ): 0.093, 0.141 and 0.066 microg TBZ/ml and 0.149, 0.158 and 0.091 microg TBZ/ml, respectively, for the Valdivia, Riñihue and Frutillar samples. The corresponding LD(96) values were 0.222, 0.263 and 0.188 microg TBZ/ml before treatment and 0.316, 0.322 and 0.221 microg TBZ/ml after treatment, indicating BZ resistance in all the cases. Genotyping was performed on more than 1700 single larvae, at least 10 per faecal sample, for 98 pre- and 66 post-treatment samples. Despite a general trend toward higher percentages of phe/tyr and tyr/tyr individuals following treatment, no statistically significant difference was found between these two and the phe/phe genotype percentages. However, a significantly negative correlation was detected between the LD(50) values and the phe/phe percentages and there was a positive correlation between the FECRT results and the phe/phe percentages. Thus, there seems to be a difference in the significance of the codon 200 polymorphism in the mechanisms of BZ resistance in small strongyles of the horse and sheep trichostrongyles.

Endoparasite control management on horse farms - lessons from worm prevalence and questionnaire data.

REASONS FOR PERFORMING STUDY Increasing prevalence of anthelmintic resistance in equine nematodes calls for a reexamination of current parasite control programmes to identify factors influencing control efficacy and development of resistance. OBJECTIVES To investigate if associations occur between prevalence of parasitic nematodes and management practices. METHODS German horse farms (n = 76) were investigated in 2003 and 2004. Information on farm and pasture management with respect to endoparasite control measures obtained using a questionnaire survey. Faecal examinations were performed in parallel. RESULTS Horses (n = 2000) were examined by faecal nematode egg counts, grouped into foals, yearlings and mature individuals for statistical analyses. Farms were categorised into 3 types, riding, stud farms and small holdings. Count regression models were used to analyse strongyle faecal egg count data. Following dichotomisation of faecal egg count (FEC) data, prevalence of strongyle and Parascaris equorum infections were assessed by logistic regression models as a function from various management factors. Yearlings on stud farms showed a 2-fold higher risk of being positive for strongyle FEC, higher (i.e. > or =3 per year) anthelmintic drug treatment frequencies were associated with reduced strongyle infection rates only in mature individuals but not in foals or yearlings, foals on farms fertilising pastures with horse manure had a significantly higher risk of being P. equorum FEC positive and yearlings on stud farms were more often showing incomplete FECR following anthelmintic treatment compared to yearlings on other farm types. The mean yearly treatment frequencies per age group were: foals 4.52, yearlings 3.26 and mature horses 2.72 times, respectively. CONCLUSION AND POTENTIAL RELEVANCE To delay the development of anthelmintic, resistance management should include additional nonchemotherapeutic parasite control strategies, FEC-monitoring, controlled quarantine treatment of new arrivals and control of efficacy by the faecal egg count reduction test on a regular basis.

Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus

Quantitative real-time PCR (qPCR) is the most sensitive technique for transcript quantification provided that gene transcription patterns are normalized to an evaluated reference gene. For Dictyocaulus viviparus, the housekeeping genes beta-tubulin, beta-actin, elongation factor 1alpha (ef-1alpha), glyceraldehyde-3-phosphatase dehydrogenase (gapdh), and 60S ribosomal protein L37a (60S rpL37a) were characterized and evaluated. Evaluation using the geNorm software revealed ef-1alpha and beta-tubulin as the most suitable reference genes, whereas the coefficient of variation approach resulted in ef-1alpha and 60S rpL37a as transcripts with the least variation among 12 developmental lungworm stages. The critical influence of reference genes on qPCR data analysis, with the possible consequence of erroneous, misleading results due to inappropriate reference genes used for data normalization, is shown for protein disulfide isomerase 2 (pdi-2) transcription patterns. Proper normalization of pdi-2 transcription using ef-1alpha and beta-tubulin as reference genes resulted in a more than 7-fold enriched pdi-2 transcription in L1 compared to that in eggs, and a dramatic decrease in L3. Following an increase in the L5 stage there is again a decrease of pdi-2 transcription in adult lungworms. These fluctuations in the transcription levels reflect the requirement of cuticule collagen during bovine lungworm development.

Analysis of the beta-tubulin codon 200 genotype distribution in a benzimidazole-susceptible and -resistant cyathostome population

To study the prevalence of the polymorphism in position 200 of the beta-tubulin gene in the mechanism of benzimidazole (BZ) resistance in cyathostomes of horses, an allele-specific PCR was used to detect the genotype of individuals of BZ-susceptible and BZ-resistant populations. The molecular analysis of 100 adults recovered from an anthelmintic-naïve horse revealed 80% homozygous TTC/TTC individuals, 17% heterozygous TTC/TAC and 3% homozygous TAC/TAC. A naturally infected horse was treated with increasing fenbendazole (FBZ) dosages to select a BZ-resistant population of cyathostomes. The PCR based analysis of 3rd-stage larvae (L3) during the experiment revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals. After treatment 42.3% of the adults (n=104) were homozygous TTC/TTC, 55.8% were heterozygous TTC/TAC and only 1.9% showed the homozygous genotype TAC/TAC. The results of the molecular analysis lead to the proposal that polymorphism within codon 200 is not the only reason for the development of BZ resistance in small strongyles.