We examined the parasite-induced cataract formation in rainbow trout (Oncorhynchus mykiss) using slit-lamp microscopy to determine the relationship between cataract intensity and number of Diplostomum spathaceum parasites that were established in the lens. Cataract intensity increased significantly with parasite burden, but was also affected by the pattern of exposure to the parasite cercariae. The slit-lamp methodology proved useful in scoring the cataracts since it provides a 3-dimensional view into the lens and gives an actual picture of the location and intensity of the cataracts, which allows detailed investigations of mechanisms underlying cataract formation in various fish species. Potential applications of the method in fish farming and parasitological studies are discussed.

Two superoxide dismutases (SODI and SODII) have been purified by differential centrifugation, fractionation with ammonium sulphate followed by chromatographic separation (ionic exchange and affinity), from a plant trypanosomatid isolated from Euphorbia characias, and then characterized for several biochemical properties. Both enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing superoxide dismutase. SODI had a molecular mass of approximately 66 kDa, whereas the molecular mass of SODII was approximately 22 kDa, both enzymes showing single bands. The isoelectric points of SODI and SODII were 6·8 and 3·6, respectively. The enzymatic stability persisted at least for 6 months when the sample was lyophilized and preserved at −80 °C. Digitonin titration and subcellular fractionation showed that both enzymes were in the cytoplasmic fraction, although part of SODII isoenzyme was also associated with glycosomes. We assayed these activities (SOD) in 18 trypanosomatid isolates on isoelectric focusing gels, and have demonstrated that the SOD is a biochemical marker sufficient to identify a trypanosomatid isolated from a plant as belonging to the genus Phytomonas and to distinguish between a true Phytomonas and other trypanosomatids that are capable of causing transient infections in plants.

Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n=46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n=19) or H. muscae (n=4), and others (n=7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3·3%) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100% and a sensitivity of ~97% (being able to specifically detect as little as ~0·02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.

We studied the pattern of infection and the inter-annual variation and individual factors affecting the infection of 2 species of nasopharyngeal bot flies, Cephenemyia auribarbis and Pharyngomyia picta (Diptera: Oestridae), in a population of Iberian red deer (Cervus elaphus hispanicus) from south central Spain (10 annual periods between 1990 and 2003). Mean prevalence±S.E.95%CI of infection was 35·19±4·24% (n=486). The frequency distribution of the parasites was markedly aggregated (K: 0·213, mean abundance±S.D.: 5·49±12·12). Parasite load of Oestridae peaked at calf and subadult age groups and declined thereafter, which suggests that acquired immunity may be acting. In common with other host–parasite relationships, male hosts were found to have higher prevalence and abundance levels than females. The prevalence of P. picta was positively affected by the presence of C. auribarbis whereas the intensity of infection of P. picta was negatively affected by the presence of C. auribarbis. Intensity of P. picta in concomitant infections with C. auribarbis was lower than in pure P. picta infections, whilst the intensity of C. auribarbis infections did not change. This provides good evidence of interspecific competence, which could be dealt with by parasites by means of asynchronous life-cycles and different maturation periods. Weather also affects the dynamics and transmission rates of these parasites. Previous annual rainfalls positively affected the level of infection with oestrids. Yearly autumn rainfalls affected positively P. picta, possibly due to an effect on the pupal stage survival. Infection of Oestridae affected body condition in calves and subadults, suggesting that oestrids could have sublethal effects on Iberian red deer. Future research is needed to investigate the effect of parasites on the dynamics of the Iberian red deer.

Major sperm protein (msp) genes were isolated from complementary (cDNA) and genomic DNA libraries prepared from the parasitic nematode, Oesophagostomum dentatum, characterized at the nucleotide and amino acid (aa) levels, and their expression was investigated. Three different msp cDNA and 2 genomic sequences were determinedThe nucleotide sequences reported in this article have been deposited in the EMBL, GenBank and DDJB databases under the Accession numbers AJ627869–AJ627873., each with an open reading frame (ORF) of 381 nucleotides. Nucleotide variation was detected at 30 positions in the ORF among all 5 sequences. Conceptual translation of the full-length msp sequences inferred 4 different MSPs each of 126 aa. These predicted MSPs differed at aa positions 15 (serine<->threonine), 101 (alanine<->glycine), 103 (glutamine<->leucine) and 126 (proline<->leucine). Southern blot analysis of O. dentatum genomic DNA, digested separately with various restriction endonucleases, displayed multiple (n=7–13) bands for each enzyme, providing support for a multigene family. Also, at the genomic level, sequence tracts consistent with a ‘substitute’ TATA box sequence motif were identified within a region (−1 to −123 nt) preceding the 2 msp genes. In contrast to other species of nematode investigated to date, no GATA transcription factor binding motif was detected immediately upstream of the msp coding region. Real-time PCR analysis demonstrated that msp mRNA was expressed exclusively in the males of both fourth-stage larvae (L4s) and adults of O. dentatum (raised in pigs after intragastric inoculation). The magnitude of expression in male O. dentatum raised in pigs in the presence of female worms was the same as in males in the absence of females. Comparative analyses showed aa sequence conservation among MSPs from various nematodes, suggesting similar functional roles for these proteins.

The glutamate-gated chloride channels (GluCls) are members of the ligand-gated ion channel superfamily that are thought to be involved in the mode of action of ivermectin and mechanism of resistance. Using reverse-transcriptase PCR techniques, 2 full-length GluCl cDNAs, encoding GluClα3 and GluClβ subunits, were cloned from Cooperia oncophora, a nematode parasite of cattle. The two sequences show a high degree of identity to similar subunits from other nematodes. The C. oncophora GluClα3 subunit is most closely related to the Haemonchus contortus GluClα3B subunit, while C. oncophora GluClβ subunit shares high sequence identity with the H. contortus GluClβ subunit. Using single-strand conformation polymorphism, the genetic variability of these two genes was analysed in an ivermectin-susceptible isolate and an ivermectin-resistant field isolate of C. oncophora. Statistical analysis suggested an association between the C. oncophora GluClα3 gene and ivermectin resistance. No such association was seen with the GluClβ gene.

This paper describes the structure of the microfilarial reservoir of Loa loa in an endemic population of central Cameroon. The possible effects of age and sex on the prevalence and intensity of microfilaraemia have been explored. Logistic analysis showed that the prevalence of microfilaraemia increased significantly with age, reaching 60% in the oldest males. This result suggests that the figure commonly reported, according to which only one third of the infected individuals were microfilaraemic, should be reconsidered; in addition, as part of surveys of loiasis, crude microfilaraemia prevalence values should be replaced by adjusted ones. The intensity of infection did not show any age-specific change. As a result, even if the oldest members of the male population are clearly the most at risk of developing post-ivermectin serious adverse reactions, especially Loa-encephalopathy, the other members of the population are not risk-free. Therefore, in those areas where the African Programme for Onchocerciasis Control is undertaking regular mass distributions of ivermectin for onchocerciasis control, and where loiasis is co-endemic, no subpopulation should be excluded from surveillance and monitoring during community directed treatments with ivermectin.

This paper reports a longitudinal study, conducted in 1989/90, of 1893 children aged 5 to 14 years in 9 poor urban areas of the city of Salvador (population 2·44 million), capital of Bahia State in northeast Brazil. Stool examinations were performed to measure nematode infection and reinfection 9 months after treatment, and an extensive questionnaire was applied to collect information on each child and on the conditions of the household. Comparison of areas with different levels of infrastructure showed the following trends as the level of community sanitation improved: clustering of cases by household became more significant, predisposition of individuals to reinfection and to heavy infection became more marked, and infections with different species were increasingly aggregated in the same individuals. These results suggest that sewerage and drainage can significantly reduce transmission of intestinal nematode infections in the public domain, but that other measures are required to control transmission within the household.

In this article I begin with a few current ideas on some physiological factors that influence mating choice in insects. Emphasis is placed on those proteins produced by the male reproductive accessory glands which increase female fecundity and reduce her receptivity to subsequent males. Strategies used by late-arriving males to favour their paternity are also mentioned. With a number of insect models as background, I then review what is currently known about several male factors in ticks (a capacitation factor, a male factor, an engorgement factor and a vitellogenesis stimulating factor) and suggest where we might focus our experimental activities in the future.

In the production of the infective cercariae of trematodes, the terminal investment hypothesis of life-history theory predicts that the rate of host exploitation and cercarial production should increase during the period of cercarial shedding since the reproductive value of the parasite decreases during this period. In contrast, a bet hedging hypothesis that focuses on the success of transmission when host contact rate is variable predicts that cercarial production should decrease in an attempt to keep the host alive for longer and thus would increase the probability of successful transmission. We examined these two hypotheses under laboratory conditions and recorded the production of Diplostomum spathaceum cercariae from naturally infected snail hosts, Lymnaea stagnalis. The average number of cercariae produced per day decreased as the snail host approached death counter to the terminal investment hypothesis. The finding supports the prediction of the bet hedging hypothesis and implies that the pattern of cercarial production may be explained by reduced virulence of the parasite within the snails to ensure extended total production time of cercariae. Nevertheless, survival of infected snails was still lower than uninfected snails suggesting that ultimately the infection still increased snail mortality rate. Cercarial production varied between days but was not cyclic, probably because of the physiology of the sporocysts within snails. Fewer cercariae were released at night, which may increase transmission efficiency to diurnally-active fish hosts. The mechanisms associated with daily cercarial production are discussed.

The swim-bladder nematode Anguillicola crassus originates from the Far East where it is a parasite of the Japanese eel (Anguilla japonica). After A. crassus was introduced to Europe, it became a predominant parasite of the European eel (Anguilla anguilla). A study performed with experimentally infected eels (98 days, 23 °C) revealed significant differences in the susceptibility of the two eel species to this parasite. The recovery rate of 30 administered infective A. crassus larvae (L3) from A. japonica was less than half of that from A. anguilla (33·2% and 13·8%, respectively). Almost 60% of the worms recovered from A. japonica were found as dead, encapsulated and necrotic larvae in the swimbladder wall. In contrast, no dead larvae were found in A. anguilla. Additionally, the development of the worms was shown to be significantly slower in A. japonica compared with A. anguilla. The lower survival rate of the worms, together with their slower development, resulted in a significantly lower adult worm burden (11 and 428 mg wet weight, respectively) and in a decreased reproductive success in A. japonica compared with A. anguilla. These results demonstrate that the original host, A. japonica, possesses more effective defence mechanisms against A. crassus than does the non-adapted host, A. anguilla.

The parasitic nematode Strongyloides stercoralis, has several alternative developmental pathways. Upon exiting the host (humans, other primates and dogs) in faeces, 1st-stage larvae (L1) can enter the direct pathway, in which they moult twice to reach the infective 3rd-stage. Alternatively, if they enter the indirect pathway, they moult 4 times and become free-living adults. The choice of route depends, in part, on environmental cues. In this investigation it was shown that at temperatures below 34 °C the larvae enter the indirect pathway and develop to free-living adulthood. Conversely, at temperatures approaching body temperature (34 °C and above), that are unfavorable for the survival of free-living stages, larvae develop directly to infectivity. The time-period within the L1's development during which temperature influenced the choice of the pathway depended on the temperature, but, at any given temperature, occurred approximately in the middle of the time-span spent in the L1 stage, which varied inversely with temperature. This critical period was associated with the time-interval in which the number of cells in the genital primordium began to increase, thus providing a morphological marker for the pathway decision in individual worms. Sensing the environment is the function of the amphidial neurons, and therefore we examined the role of individual amphidial neurons in controlling entry into the direct pathway to infectivity. The temperature-sensitive developmental switch is controlled by the neuron pair ALD (which also controls thermotaxis), as seen by the loss of control when these neurons are ablated. Thus, in S. stercoralis a single amphidial neuron pair controls both developmental and behavioural functions.

The fox tapeworm Echinococcus multilocularis is typically perpetuated in a cycle with red foxes as definitive hosts and various rodent species as intermediate hosts. In this study, foxes were baited with a highly efficient drug against cestodes (praziquantel) in 5 blocks of 1 km2. Voles, Arvicola terrestris, the most abundant intermediate host species, were trapped in the 5 baited blocks and in 5 non-baited control blocks. Baiting the foxes reduced the prevalence of E. multilocularis in fox faecal samples in the baited blocks, but voles trapped in the two blocks did not differ in their infection rates. However, voles from the baited blocks had significantly smaller spleen masses and were more likely to be infested with mites than those from the control blocks, possibly reflecting different immunological activities. Our study suggests that the environmental contamination with E. multilocularis eggs, and perhaps those of other tapeworms, influences the immune system of the intermediate host species A. terrestris in the wild.

Ticks are of vast medical and veterinary public health importance due to direct damage caused by feeding and their roles in transmitting well known and emerging infectious agents. Ticks and tick-borne pathogens stimulate the immune system of the host. Those immune interactions are of importance in tick biology, pathogen transmission and control of ticks and tick-borne diseases. Both innate and specific acquired immune defenses are involved in the responses of vertebrate hosts to infestation. Ticks have evolved countermeasures to circumvent host immune defenses. This review addresses the immunobiology of the tick–host interface from the perspectives of the pharmacology of tick saliva; relationship of tick saliva to pathogen transmission; host immune responses to infestation; tick modulation of host immune defences; and genomic/proteomic strategies for studying tick salivary gland molecules.

Homologues of c-fos and c-jun from total DNA of Taenia crassiceps and Taenia solium were cloned and sequenced. The amino acid alignment analysis revealed that c-fos DNAs from T. crassiceps and T. solium were highly homologous (96%), and both have high homology compared to several mammalian c-fos proteins (93% to mouse, 96% to rat and 86% to human). The c-jun protein alignment showed higher homology (T. crassiceps and T. solium have 98%), when compared with mouse, rat and human, being 92%, 98% and 93% respectively. RT–PCR amplification of the parasite's total RNA, showed that T. crassiceps expressed both AP-1 complex genes, while T. solium only expressed c-fos. Southern blot hybridization analysis confirmed the true origin of each amplified gene. AP-1 transcription gene expression is regulated by oestradiol in the same fashion as their mammalian counterparts only in T. crassiceps. To study if AP-1 genes are involved in a physiological function of the cyst, reproduction was studied in vitro. Oestradiol treatment stimulated reproduction in T. crassiceps but not in T. solium cysticerci. This is the first report of the detection and functionality of AP-1 transcription factor genes in any species of helminth parasite.

Thelazia callipaeda (Spirurida, Thelaziidae) eyeworm causes ocular infection in carnivores and humans in the Far East; this infection has been recently reported also in Europe – northern and southern Italy – in dogs, cats and foxes. The natural vector/s of T. callipaeda is/are unknown and the development of the nematode in its definitive hosts is limited to an experimental trial on dogs. To contribute new insights into the development of T. callipaeda in the definitive host in field conditions, eyeworms were collected from naturally infected dogs from an area with a high prevalence of infection (up to 60·14%) in the Basilicata region of southern Italy, from January 2002 to December 2003. Conjunctival secretions were also collected and examined for the presence of immature stages. The presence of blastomerized eggs throughout the period – except for the months from May to November – indicates a seasonality in the reproductive activity of T. callipaeda, coinciding with the presence/absence of the vector. In fact, 1st-stage larvae were found in the lachrymal secretions of dogs in summer (June–July 2002 and 2003), ready to be ingested by flies feeding about the eyes. The evidence of 4th-stage larvae in March 2002 and April, July and October 2003 may be accounted for by the presence of flies that act as intermediate hosts of T. callipaeda from early spring to early autumn. The presence of immature stages in October indicates an overlapping generation of nematodes and a 2nd cycle of vector infection. This basic knowledge of the development of T. callipaeda will hopefully help future epidemiological studies to identify the intermediate hosts and define the likely risk for vectors in field conditions.

Parasite community ecology has recently focused on understanding the forces structuring these communities. There are few surveys, however, designed to study the spatial repeatability and predictability of parasite communities at the local scale in one host. The purpose of our study was to address the relationship between infracommunity and component community richness, and to describe spatial variations on the local scale, of helminth parasite communities in an avian host, the red-legged partridge (Alectoris rufa). We sampled 235 wild partridges from 8 separate localities, with different partridge population densities, in the Ciudad Real and Toledo provinces of central Spain, and we determined their overall and intestinal helminth species. We found that habitat variables (mean temperature and land use) were not significantly associated with any component community. The partridge population abundance index was directly correlated with the prevalence and mean intensity of infection but not with component community species richness. There was a curvilinear relationship between infracommunity and component community species richness, as well as negative interspecific associations, for the helminth species assemblage parasitizing the intestine. A nestedness/anti-nestedness pattern, considered as part of a continuum, was associated with prevalence, mean intensity and partridge population abundance index, but not with component community richness. Increases in the partridge population abundance index and the prevalence and mean intensity of infection were associated with increases in helminth community nestedness. Although negative interactions between helminth species could not be ruled out as forces structuring helminth communities, our results suggest that parasite community structure in the red-legged partridge was primarily determined by the extrinsic influence of parasite habitat heterogeneity and its amplification of the differing probabilities of colonization of parasite species.

Surgical treatment of human hydatidsosis involves the use of various scolicidal agents to kill infective Echinococcus granulosus protoscoleces that may disseminate into the peritoneal cavity during surgery and potentially re-infect the patient. Currently, no scolicidal agent is completely effective in killing intracystic protoscoleces in humans. Cyclosporin A (CsA) has previously been found to be lethal for E. granulosus protoscoleces in vitro. In this study, we further assessed the effectiveness of CsA as a scolicidal agent by testing the toxic effect of CsA at higher doses over various time-periods. Experiments were performed on activated and unactivated protoscoleces cultured in nutrient medium or sheep hydatid cyst fluid. All activated protoscoleces were killed following culture in 100 μg/ml of CsA for 3 days and 50 or 20 μg/ml for 5 days. The lethal effect of CsA on unactivated protoscoleces varied but reached 100% over 15 days in culture with 100 or 50 μg/ml of CsA. Pulse treatment of protoscoleces with 50, 20 or 10 μg/ml of CsA for 5 min or 72 h killed all parasites by day 10 and day 5 respectively. Untreated protoscoleces remained greater than 95% viable for the duration of experiments. Changes in protoscolex ultrastructure induced by treatment with 10 μg/ml of CsA over 10 days in in vitro culture was assessed by TEM. Protoscolex alterations observed in treated parasites included an increase in cellular vacuolization, swelling of mitochondria, rounding of cells, damage to the tegument, decrease in glycogen, a breakdown of the extracellular matrix and an increase in lipid globules. The untreated protoscoleces, by comparison, had few changes during the 10-day culture period with the exception of large amounts of extracellular glycogen observed in the protoscoleces at culture days 7 and 10. From these results, CsA is clearly an effective scolicidal agent in vitro that may have potential application as a new therapeutic agent in the treatment of human hydatid disease.

Gastrointestinal helminth infections remain a major constraint to livestock production globally. This study evaluated anthelmintic efficacy of 7 plants used as dewormers by farmers and pastoralists in Kenya. Thus 3 commercial anthelmintics and 7 plant preparations were tested in lambs infected with 5000 or 3000 L3 Haemonchus contortus in 4 experiments. In the first experiment, ivermectin, levamisole and albendazole were tested in 46 lambs. Seven plant preparations of Hagenia abyssinica, Olea europaea var. africana, Annona squamosa, Ananas comosus, Dodonea angustifolia, Hildebrandtia sepalosa and Azadirachta indica were tested in 151 lambs in 3 experiments. All 3 anthelminitics were highly effective in reducing faecal egg counts (FEC) and total worm counts (TWC) in lambs. Plant preparations had varying levels of crude proteins from 2·6% for O. europaea to 18·4% for A. indica. Compared with controls, no significant reductions in FEC were observed for any of the treated groups either 2 or 3 weeks post-treatment. Lambs treated with A. squamosa and A. comosus were slaughtered 4 weeks post-treatment. No significant differences were observed in mean TWC or number of eggs per female worm between treated animals and the controls. No significant improvements in weight gain were observed in treated lambs.