The culture method developed by Geiman and co-workers has been modified to give average multiplication rates for Plasmodium knowlesi of at least six-fold in 24 h.

Individual modifications have been assessed on the basis of parasite multiplication and [3H]leucine incorporation into parasite protein. The second cycle of in vitro development which is particularly influenced by the conditions of culture was improved by: (i) culture of washed parasitized red cells in a medium containing dialysed homologous serum screened for its ability to support parasite growth; (ii) addition of ATP, Co-enzyme A (or pantothenate) and glutamine.

We thank Mr E. D. Dennis and Miss Susan Wanstall for technical assistance. This work was supported by grants from the Medical Research Council and the World Health Organization.

An enzyme analysis of diploid and triploid Paragonimus westermani was conducted using starch gel electrophoresis. In total, 16 enzymes, probably encoded by 18 loci, were studied for 3 populations of the diploid form sampled from 2 localities, and 4 populations of the triploid form from 4 localities. Comparison of the enzymes of the triploid and the diploid digeneans showed 5 different patterns; diaphorase (EC 1.6.2.2), glutamic-oxaloacetic transaminase (EC 2.6.1.1), hexokinase (EC 2.7.1.1), leucylglycylglycine aminopeptidase (EC 3.4.1.3), and phosphoglucomutase (EC 2.7.5.1). On the basis of the numbers of bands and their patterns, all individuals of the triploid are probably heterozygous at each of these 5 loci and homozygous at the remaining 13 loci. The occurrence of fixed heterozygotes found in triploid populations cannot be easily explained by only a single mutation. It is suggested that the variability may have been introduced by hybridization with a different sub-species or a closely related species and may, thus, have been maintained since the time of the origin of triploids.

We examined the relative importance of abiotic and biotic factors on the ability of adult Dermacentor variabilis ticks to attach and engorge with blood across 10 populations of free-ranging raccoons (Procyon lotor). We developed a priori models that represented explicit hypotheses based on the literature and tested the ability of these models to explain non-replete and replete (fully engorged with blood) tick infestation using generalized linear models and Akaike's Information Criterion. Abiotic models that included month and site of collection clearly provided a better fit for non-replete tick abundance data, while biotic models with host age and sex covariates best fit the replete tick data. Abiotic models of non-replete abundance were superior to biotic models because of large seasonal and site fluctuations in non-replete abundance that masked differences due to host characteristics. Conversely, best-fitting models of replete tick abundance included only age and sex and suggest that once a tick has reached a host, host-parasite interactions are the primary determinant of engorgement by female ticks. Host population structure may have a large influence on potential cohort size of ticks by reducing or increasing the total number and proportion that can become engorged and moult or lay eggs.

Echinococcus multilocularis is a cestode parasite that predominantly infects red and arctic foxes as definitive hosts. Ingestion of E. multilocularis eggs and subsequent post-oncospheral infection with the larval stage (metacestode) of the parasite results in alveolar echinococcosis (AE), a life-threatening hepatic disease concerning humans and other intermediate hosts such as small rodents. The primary fluid-filled vesicles of the asexually proliferating metacestode are comprised of an inner germinal layer, a syncytial tegument, and an outer, acellular, so-called laminated layer. This laminated layer may play an important role in protecting the developing E. multilocularis metacestode from host immune reactions, and laminated layer-associated components represent potential targets for intervention during the course of AE. We have used an in vitro cultivation technique for the long-term maintenance and proliferation of E. multilocularis metacestodes in order to generate premature (protoscolex-free) parasite vesicles. A polyclonal antiserum was raised against this host-free parasite tissue. Subsequent immunoblot analysis of parasite fractions obtained by Triton X-114 extraction led to the identification of a 116 kDa protein (named EmP2) within the Triton-insoluble fraction. The characterization of EmP2 by SDS–PAGE, Western blotting, and by immunofluorescence revealed that EmP2 is a laminated layer-associated protein.

A single specimen of a blood fluke was obtained from the gills of an amberjack or coronado, Seriola lalandi Cuv. and Val., taken by the writer on November 30th, 1929, from the Atlantic Ocean off Miami, Florida. This fluke belongs to the family Aporocotylidae Odhner 1912, and is regarded as the type of a new genus.

The epidemiology of trematode infections in cattle was investigated within highland and lowland areas of Iringa Rural District, in southern Tanzania. Fecal samples were collected from 450 cattle in 15 villages at altitudes ranging from 696 to 1800 m above the sea level. Freshwater snails were collected from selected water bodies and screened for emergence of cercariae. The infection rates in cattle were Fasciola gigantica 28·2%, paramphistomes 62·8% and Schistosoma bovis 4·8%. Notably, prevalence of trematode infections in cattle was much higher in highland (altitude > 1500 m) as compared with lowland (altitude < 1500 m) areas and was statistically significant (P-value = 0·000) for F. gigantica and paramphistomes but not for S. bovis. The snails collected included Lymnaea natalensis, Bulinus africanus, Bulinus tropicus, Bulinus forskali, Biomphalaria pfeifferi, Melanoides tuberculata and Bellamya constricta with a greater proportion of highland (75%) than lowland (36%) water bodies harbouring snails. Altitude is a major factor shaping the epidemiology of F. gigantica and paramphistomes infections in cattle in Iringa Rural District with greater emphasis upon control needed in highland areas.

Ivermectin (IVE), one of the most important anthelmintics, is often used in the treatment of haemonchosis in ruminants. The objective of our work was (1) to find and identify phase I and II metabolites of IVE formed by the Barber's pole worm (Haemonchus contortus), and (2) to compare IVE metabolites in helminths with IVE biotransformation in sheep (Ovis aries) as host species. Ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, microsomes (from adult worms or from ovine liver) and a primary culture of ovine hepatocytes were incubated with IVE. In the ex vivo study, living H. contortus adults were incubated in the presence of 1 μM IVE for 24 h. The results showed that the H. contortus enzymatic system is not able to metabolize IVE. On the other hand, 7 different phase I as well as 9 phase II IVE metabolites were detected in ovine samples using UHPLC/MS/MS analyses. Most of these metabolites have not been described before. Haemonchus contortus is not able to deactivate IVE through biotransformation; therefore, biotransformation does not contribute to the development of IVE-resistance in the Barber's pole worm.

Cerebral angiostrongyliasis is a central nervous system disease caused by Angiostrongylus cantonensis and can produce eosinophilic meningitis or meningoencephalitis in humans. Sero-immunological techniques, such as the enzyme-linked immunosorbent assay (ELISA) and immunoblotting, are most commonly used for the diagnosis of human angiostrongyliasis. However, diagnosis in remote areas remains problematic because sophisticated equipment and specialized skills are required. To overcome, we have developed the immunochromatographic test (ICT) kit for rapid serological diagnosis of angiostrongyliasis through the detection of anti-A. cantonensis-specific antibodies in human serum. A recombinant A. cantonensis galectin-2 (rAcGal2) from young adult female worms was used as an antigen for the ICT kit development. Diagnostic values were evaluated and compared using the ELISA. The sensitivity, specificity and positive and negative predictive values of the ICT kit were 87.0, 96.5, 94.6 and 91.4%, respectively, and those of the ELISA were 91.0, 97.2, 95.8 and 94.0%, respectively. The concordance of the ICT kit was 93.9%. We, thus, determined that the ICT kit is sensitive and specific and provides reliable diagnostic results. It is rapid and simple to perform and can be utilized for both point-of-care diagnosis in the bedside laboratory and epidemiological surveys in endemic regions where access to diagnostic equipment is limited.

Feral swine are known reservoirs of various pathogens, including Toxoplasma gondii. Here, we report the first national survey of viable T. gondii in feral swine in the USA. We paired serological surveys with parasite isolation and bioassay to evaluate the prevalence and genetic diversity of these parasites. From 2012–2017, sera and tissues from 1517 feral swine across the USA were collected for the isolation of viable T. gondii. Serum samples were initially screened for antibodies to T. gondii, and then the tissues of seropositive feral swine were bioassayed in mice. Antibodies were detected in 27.7% of feral swine tested by the modified agglutination test (1:25 or higher). Antibody positive rates increased significantly with age, with 10.1% of juveniles, 16.0% of sub-adults and 38.4% of adults testing seropositive. Myocardium (50 g) from 232 seropositive feral swine was digested in pepsin and bioassayed in mice. Viable T. gondii was isolated from 78 feral swine from 21 states. Twelve of the 78 isolates were pathogenic to outbred Swiss Webster mice and 76 of the 78 isolates could be propagated further in cell culture and were genotyped. For genotyping, deoxyribonucleic acid extracted from cell culture-derived tachyzoites was characterized by polymerase chain reaction restriction fragment length polymorphism using the genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genotyping revealed 15 ToxoDB genotypes, including 43 isolates for genotype #5 (haplogroup 12), 11 isolates for #24, four isolates for #2 (haplogroup 3), two isolates for each of genotypes #3 (haplogroup 2), #4 (haplogroup 12), #216, #221, #289 and #297 and one isolate for each of genotypes #1 (haplogroup 2), #39, #66, #260, #261 and #299. Genotype #5 was the most frequently isolated, accounted for 57% (43/76) of the isolates, followed by #24, accounted for 14% (11/76). Genotypes #260, #289, #297 and #299 are new types. Genotype #289 was highly virulent to mice and originated from feral swine collected in Louisiana on the same day at the same location. Genotype #216 was previously demonstrated to be highly virulent to mice. Our results indicate moderate genetic diversity of T. gondii in feral swine in the USA, with the genotype #5 (haplogroup 12) dominant in the continental USA, whereas genotype #24 (10/14) was dominant in Hawaii, suggesting different population structures of the parasites among the two distinct geographical locations.

Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0–3 h (84·3% for banana-shaped and 49·2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

The rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruzi antibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected with T. cruzi VI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected with T. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence of T. cruzi circulating lineages in the region.

Phylogenetic analyses of SSU rDNA sequences have previously revealed the existence of 2 Ichthyobodo species able to infect Atlantic salmon (Salmo salar L.). Ichthyobodo necator sensu stricto (s.s.) is assumed to be a freshwater parasite, while a genetically distinct but undescribed species, Ichthyobodo sp. II sensu Todal et al. (2004) have been detected on Atlantic salmon in both fresh- and seawater. In the present study a morphological description of Ichthyobodo sp. II from the gills of salmon reared in fresh-, brackish- and seawater is presented, using both light- and electron microscopy. Comparative morphometry show that Ichthyobodo sp. II from both freshwater and seawater displays a different cell shape, and is significantly smaller than I. necator s.s. Also, ultrastructural characteristics distinguish these two species, notably differences in the attachment region and the presence of spine-like surface projections in Ichthyobodo sp. II. Based on both unique SSU rDNA sequences and morphological characteristics, we conclude that Ichthyobodo sp. II. represents a novel species for which we propose the name Ichthyobodo salmonis sp. n.

Previous studies using monoclonal antibody (mAb) 4C9 specific for a 32 kDa antigen (p32) of Theileria parva demonstrated expression of the antigen on the surface of the sporozoite, making it a potential antigen for sporozoite neutralization. A full-length cDNA encoding the major merozoite/piroplasm surface antigen (mMPSA) of T. parva was cloned and expressed in bacteria. The expressed product reacted strongly with mAb 4C9, demonstrating identity between the p32 and mMPSA of T. parva. Using immunoblot analysis and immunoelectron microscopy with mAb 4C9 it was shown that the mMPSA is a major antigen of the merozoite and piroplasm at the cell surface, while lower levels of antigen are expressed in the sporozoite and schizont stages. Upregulation of the mMPSA occurs at merogony and can be induced by culturing schizont-infected lymphocytes at 42 °C. Recombinant mMPSA of T. parva induced high titres of specific antibodies in cattle but failed to confer protection against a T. parva sporozoite stabilate challenge. The pre-challenge sera also failed to neutralize infectivity of sporozoites in an in vitro assay. Possible reasons for the lack of parasite neutralization in vivo and in vitro are discussed.

In this study we evaluated the effects of the anti-microsporidial exospore monoclonal antibody 3B6, recognizing 3 Encephalitozoon species, Encephalitozoon intestinalis (Syn. Septata intestinalis), Encephalitozoon cuniculi, and Encephalitozoon hellem on microsporidial growth in vitro. Pre-treatment of spores for 24 h with mAb 3B6 resulted in 21–29% fewer infected host cells 4 days after inoculation of the cultures compared to cultures pre-treated with medium or an irrelevant isotype control mAb (P<0·001). Fewer intracellular spores (1·2±0·2) in infected cells were found when mAb 3B6 was present in cultures compared to cultures with medium alone (4·3±0·8) or an irrelevant isotype control mAb (4·2±0·9; P<0·001). This decrease appeared not to be dependent on time of exposure, mAb concentration, or presence of complement. It is concluded that antibodies, particularly those directed to potential neutralizing-sensitive epitopes on spores, may have a role in the control of microsporidial growth in vitro.

Antisera were produced by immunizing rabbits with either a trichloroacetic acid-soluble fraction, or a high molecular weight (Mr) fraction of Schistosoma mansoni SEA (a salinesoluble fraction of homogenized egg). Both of these sera reacted monospecifically in immunoelectrophoresis against unfractionated SEA, recognizing a cathodally migrating antigen. This antigen had been identified previously as being responded to by S. mansoni-infected mouse sera, and has been designated K3 (Kappa 3). The rabbit antisera were used to partially characterize antigen K3 as having Mr in the range > 750 – 70 K, and being resistant to boiling, resistant to the action of proteases, but sensitive to periodate. It partially binds to Concanavalin A. In addition to SEA, the antigen was present in homogenized cercariae and schistosomula, but not adult worms, and it was also present in detergent extracts of intact cercariae and schistosomula. Using an antibody-dependent cell adherence assay, anti-K3 serum was found to react with the surface of live cercariae and with the surface of schistosomula recovered from the skin of mice infected up to 48 h previously. Anti-K3 serum also reacted with the surface of S. bovis, S. haematobium and to a lesser extent S. japonicum schistosomula.

The relationship between infection success, the density of Schistosoma mansoni miracidia and the density of the snail host Biomphalaria glabrata was examined experimentally. Within laboratory infection arenas, the distribution of miracidial infections/snail was approximately uniform and the net rate of snail infection was directly positive binomial to miracidial density. In contrast, however, the rate of infection declined exponentially as snail density increased. Mathematical models are developed to aid in the estimation of infection rates and experimental results are discussed in the context of the transmission dynamics of schistosomiasis.

The present report compares the macrophage function in rodent hosts susceptible and resistant to the human lymphatic filariid Brugia malayi. Macrophages from both mastomys (resistant) and gerbil (susceptible) infected intraperitoneally (i.p.) with the infective larvae (L3) of B. malayi were isolated from peritoneal lavage at different time-intervals and formation rate of NO, H2O2, O2−, TNF-α, glutathione peroxidase and reductase was assayed. NO release was found to be significantly increased in resistant mastomys as compared to gerbils and the release was markedly suppressed by i.p. administration of the NOS inhibitor aminoguanidine (AG). The AG-treated mastomys also demonstrated significantly greater establishment of larvae which correlated well with suppressed formation of NO. Nitric oxide synergizes with superoxide to form peroxynitrite radical (potent oxidant), which is known to be more toxic per se than NO. Results indicate the possible involvement of peroxynitrite in the rapid killing of larvae in the peritoneal cavity of mastomys. In contrast, the production of H2O2 was found to be enhanced in both species indicating that B. malayi L3 could withstand the toxic effects of H2O2. The higher level of glutathione peroxidase and reductase, as observed in mastomys compared with the gerbil after larval introduction, possibly protects the cell against the injurious effect of H2O2. The TNF-α level remained virtually unchanged in both the hosts, suggesting an insignificant role for this cytokine in parasite establishment.

The patch clamp technique was used to investigate the action of the anthelmintic drug, oxantel, on nicotinic acetylcholine receptor (nAChR) currents recorded from vesicles of the somatic muscle cells of the nematode parasite Ascaris suum. The amplitudes of the currents were analysed at different membrane potentials to determine the single channel conductance. Also the open and closed durations were measured to determine the kinetic properties of the activated channel. Oxantel activated single nAChR currents throughout a concentration range 10–100 μM, these currents were not observed with oxantel-free pipette solutions. The mean open time of the activated channels at a membrane potential of –75 mV and a concentration of 10 μM was 1·34 ms. At higher concentrations the open times were shorter and voltage sensitive, decreasing in duration on hyperpolarization, thus suggesting open channel block. The kinetics were analysed using a simple channel block model. The forward block rate, K + B, increased with increasing oxantel concentration but showed little increase as the membrane was hyperpolarized. K + B was 2·41×107 M−1s−1 – 50 mV and 2·64 × 107 M−1s−1 at – 100mV. The unblocking rate constant, K – B, did exhibit voltage sensitivity being 443·6 s−1 at – 50 mV and 86·8 s−1 at –100 mV. Thus the blocking dissociation constant KB (= K – B/K + B) was 18·5 μM at –50 mV and 3·3 μM at –100 mV. The simple channel block scheme was found to be insufficient to explain fully the observations made; reasons for this are discussed.

Students of the history of Protozoology are generally agreed that the earliest recorded observations on the Coccidia are those of Hake (1839). Bütschli (1882, p. 490) mentions his work, though he did not see it; and it has been cited by Leuckart (1879), Labbé (1896), and several other writers on the Sporozoa—including myself (1919)—as the first publication in which coccidia are recognizably described.

Changes in the specialization of parasite–host interactions will be influenced by variations in host species composition. We evaluated this hypothesis by comparing the composition of bats and bat flies within a roost cave over one annual. Five bat and five bat fly species occupied the cave over the course of the study. Bat species composition was 40% different in the rainy season compared with the dry–cold and dry–warm seasons. Despite the incorporation of three new bat species into the cave during the rainy season, bat fly species composition was not affected by seasonality, since the bats that arrived in the rainy season only contributed one new bat fly species at a low prevalence. Bat–bat fly ecological networks were less specialized in the rainy season compared with the dry–cold and dry–warm seasons because of the increase of host overlap among bat fly species during this season. This study suggests that seasonality promote: (1) differences in host species composition, and (2) a reduction in the specialization of host–parasite ecological networks.