Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3rd stage larvae (L3s) in the intermediate host (molluscs) act as the source of infection for accidental hosts through ingestion. Larvae can spontaneously emerge from dead gastropods (slugs and snails) in water, which are experimentally infective to rats. We sought to identify the time when infective A. cantonensis larvae can autonomously leave dead experimentally infected Bullastra lessoni snails. The proportion of A. cantonensis larvae that emerge from crushed and submerged B. lessoni is higher in snails 62 days post-infection (DPI) (30.3%). The total larval burden of snails increases at 91 DPI, indicating that emerged larvae subsequently get recycled by the population. There appears to be a window of opportunity between 1 and 3 months for infective larvae to autonomously escape dead snails. From a human and veterinary medicine viewpoint, the mode of infection needs to be considered; whether that be through ingestion of an infected gastropod, or via drinking water contaminated with escaped larvae.

Pseudosuccinea columella is considered invasive and has become an important intermediate host of both Fasciola species in many regions of the world. This systematic review assessed the geographical distribution of P. columella, and its implications in the transmission of Fasciola hepatica and Fasciola gigantica, globally. A literature search was conducted on Google Scholar, JSTOR and PubMed databases using Boolean operators in combination with predetermined search terms for thematic analysis. Results show that P. columella has been documented in 22 countries from Europe (3), Africa (8), Oceania (2), North America (3) and South America (6). Furthermore, this snail species has shown to adapt to and inhabit a vast array of freshwater bodies including thermal lakes and ditches with acidic soils. Studies showed that P. columella transmits F. hepatica, with natural and experimental infections documented in sub-Saharan Africa, Europe, South America and North America. Experimental infection studies in Cuba showed the presence of P. columella populations resistant to F. hepatica infection. Furthermore, some populations of this invasive snail collected from F. hepatica endemic locations in Brazil, Venezuela, Australia, South Africa, Colombia and Argentina were found without Fasciola infection. As a result, the role played by this snail in the transmission of Fasciola spp. in these endemic areas is still uncertain. Therefore, further studies to detect natural infections are needed in regions/countries where the snail is deemed invasive to better understand the veterinary and public health importance of this snail species in Fasciola-endemic areas and determine the global dispersion of resistant populations of P. columella.

Chagas disease is a serious parasitic infection caused by Trypanosoma cruzi. Unfortunately, the current chemotherapeutic tools are not enough to combat the infection. The aim of this study was to evaluate the trypanocidal activity of benznidazole-loaded microparticles during the acute phase of Chagas infection in an experimental murine model. Microparticles were prepared by spray-drying using copolymers derived from esters of acrylic and methacrylic acids as carriers. Dissolution efficiency of the formulations was up to 3.80-fold greater than that of raw benznidazole. Stability assay showed no significant difference (P > 0.05) in the loading capacity of microparticles for 3 years. Cell cultures showed no visible morphological changes or destabilization of the cell membrane nor haemolysis was observed in defibrinated human blood after microparticles treatment. Mice with acute lethal infection survived 100% after 30 days of treatment with benznidazole microparticles (50 mg kg−1 day−1). Furthermore, no detectable parasite load measured by quantitative polymerase chain reaction and lower levels of T. cruzi-specific antibodies by enzyme-linked immunosorbent assay were found in those mice. A significant decrease in the inflammation of heart tissue after treatment with these microparticles was observed, in comparison with the inflammatory damage observed in both infected mice treated with raw benznidazole and untreated infected mice. Therefore, these polymeric formulations are an attractive approach to treat Chagas disease.

In order to elucidate the infection pathways of third stage larvae (L3) of Angiostrongylus cantonensis, we performed experiments to assess: (i) the shedding of L3 from two species of experimental veronicellid slugs drowned in water and the ratio of emerged larvae, (ii) the transmission of viable L3 from drowned terrestrial gastropods to aquatic snails, and (iii) the transmission of viable L3 between terrestrial snails. Molluscs were experimentally infected by first stage larvae (L1) of A. cantonensis. Significantly more L3 larvae were released from Veronicella cubensis than from Veronicella sloanei. Numerous L3 were observed in the muscular foot, and also in the connective tissue between internal organs. Experimental exposure of P. maculata to L3 of A. cantonensis liberated from other gastropod species led to their infection and the infectivity of larvae after intermediesis was demonstrated by infection of laboratory rats (Rattus norvegicus). The transmission of L3 was observed in three out of four experiment replications and L3 were retrieved from 6 out of 24 Subulina octona snails. The infected synanthropic molluscs represent a key component in the epidemiology of human infections by A. cantonensis. Escape of L3 larvae from bodies of dead snails or slugs and their ability to infect further gastropod hosts (intermediesis) represents a public health risk. Thus, control of molluscs living in peri-domestic environment is an essential part of prevention of human infections.

We have learned a lot about infections of the mammary gland of dairy cows from experimental investigations of the pathogenesis of the various diseases. The understanding gained has contributed to huge successes in reducing the prevalence of infection in properly managed dairy herds. Now descriptive studies using DNA technologies reject previous concepts of mammary gland sterility by default. Bacteria, at least markers of genes, of many genera are reported even from absolutely healthy mammary glands. This may be a technological artefact. No direct evidence exists because experimental studies of infection are no longer fashionable. A regeneration of the lost arts in the pathogenesis of infection is essential to separate truth from conjecture and deal with coming challenges from rapidly changing farm systems and the reduction in access to antimicrobial drugs. In this Opinion Paper I argue for a return to experimental approaches that construct hypotheses, and then test them, in intramammary disease research.

Fecal samples from wild-caught common voles (n = 328) from 16 locations in the Czech Republic were screened for Cryptosporidium by microscopy and PCR/sequencing at loci coding small-subunit rRNA, Cryptosporidium oocyst wall protein, actin and 70 kDa heat shock protein. Cryptosporidium infections were detected in 74 voles (22.6%). Rates of infection did not differ between males and females nor between juveniles and adults. Phylogenetic analysis revealed the presence of eight Cryptosporidium species/genotypes including two new species, C. alticolis and C. microti. These species from wild-caught common voles were able to infect common and meadow voles under experimental conditions, with a prepatent period of 3–5 days post-infection (DPI), but they were not infectious for various other rodents or chickens. Meadow voles lost infection earlier than common voles (11–14 vs 13–16 DPI) and had significantly lower infection intensity. Cryptosporidium alticolis infects the anterior small intestine and has larger oocysts (5.4 × 4.9 µm), whereas C. microti infects the large intestine and has smaller oocysts (4.3 × 4.1 µm). None of the rodents developed clinical signs of infection. Genetic and biological data support the establishment of C. alticolis and C. microti as separate species of the genus Cryptosporidium.

In South America, visceral leishmaniasis is frequently caused by Leishmania infantum and, at an unknown frequency, by Leishmania amazonensis. Therefore, mixed infections with these organisms are possible. Mixed infections might affect the clinical course, immune response, diagnosis, treatment and epidemiology of the disease. Here we describe the clinical course of mixed infections with L. amazonensis and L. infantum in a hamster model. We show that mixed infections are associated with more severe clinical disease than infection with L. amazonensis or L. infantum alone. In spleens with mixed infections, L. infantum outcompeted L. amazonensis in the tissue, but not in culture from tissue. We found increased levels of IgG in animals infected with L. infantum. Although more than 30 bands were revealed in a Western blot, the highest immunogenicity was observed with proteins having molecular masses of 95 and 90 kDa, whereas proteins with molecular masses of lower than 50 kDa were reactive frequently with serum from hamsters infected with L. amazonensis, and proteins with molecular masses of 80 and 70 kDa were reactive only with serum from hamsters infected with L. infantum. This finding has important implications regarding the biology of Leishmania and humoral immune responses to infections with these organisms.

Aelurostrongylus abstrusus is a worldwide occurring lungworm affecting felids. This metastrongyloid nematode has an indirect lifecycle relying on slugs and snails as intermediate hosts. In the present study the development of first-stage (L1) to third-stage larvae (L3) in the tropical freshwater pulmonate snail Biomphalaria glabrata was assessed. A total of 306 snails were individually exposed to 300 A. abstrusus L1, which were obtained from a naturally infected stray cat. The species was confirmed by biomolecular analysis. Second stage larvae (L2) and L3 were first isolated by artificial digestion of snails in the second and fourth week post exposure (wpe), respectively. From 8 wpe onwards, all larvae had developed into L3. Snails remained infected for up to 26 wpe. Only 0.4% of the L1 had pursued their development into L3, indicating low suitability of this artificial intermediate host for production of infective A. abstrusus L3.

Introduction. Bacterial canker,caused by P. syringae pv. syringae,is an important disease of stone fruit worldwide. The possibilityof P. syringae pv. syringae infectionthrough leaf scars and lenticels was evaluated in cherry, peachand prune. Materials and methods. Laboratory and fieldinoculations were performed using cherry, peach and prune stemsto evaluate leaf scar age, chilling and freezing-thawing on bacterialinfection through leaf scars and lenticels. Results and discussion. Increasingleaf scar age was associated with significant decreases in diseaseincidence and length of lesions resulting from leaf scar inoculationwith Pseudomonas syringae pv. syringae incherry, peach and prune. A significant reduction in incidence andlesion length was observed after 4 h of air exposure, and both measuresof infection were reduced to essentially 0 by 2 days of exposure. Prolongedchilling temperature (2.2 °C) prior to leaf removal had no cleareffect on disease incidence of leaf scar infection, but significantlydecreased lesion length due to leaf scar infection. Cherry was more susceptibleto P. syringae pv. syringae infectionthrough leaf scars than peach and ‘French’ prune. The leaf scar inoculationresults were consistent with the previous studies. The disease incidenceof lenticel infection caused by bacterial inoculation in ‘French’prune was very low, but significantly higher than the water control.Freezing-thawing significantly increased both the disease incidence andthe lesion size via lenticel infection. The lenticel inoculationdata suggest that P. syringae pv. syringae infection throughlenticels is possible under field conditions.

Introduction. Bacterial canker, caused by Pseudomonassyringae pv. syringae, is a damaging disease of stone fruitworldwide. The effects of rootstock, temperature and incubation duration on bacterialcanker in peach were assessed using both field and laboratory inoculation assays.Materials and methods. Both field and laboratory experiments were conductedto study the effects of rootstock, temperature and incubation duration on disease severityin peach. All inoculations were achieved with P. syringae pv.syringae strain B3A. Bacterial inoculations were applied to 1-year-oldshoots of peach trees [Prunus persica (L.) Batsch]. After inoculation,the inoculated shoots were allowed to incubate either under field conditions or in a coldroom at different temperatures [constantly at 0 °C, constantly at 14.4 °C, and in afluctuating temperature regime of 12 h at 0 °C (night) and 12 h at 14.4 °C (day)] forexcised shoots. The lesions were determined 1 to 6 weeks after inoculation to determinethe effect of incubation duration. Results and discussion. The fieldexperiment using peach grafted on three rootstocks (Nemaguard, K119-50 and P30-135) showedthat shoots on Nemaguard developed the longest lesions and shoots on K119-50 the shortestamong all three rootstocks. Shoots on Nemaguard had significantly lower bark calcium andhigher nitrogen concentrations than those on K119-50 and P30-135. A negative correlationwas found between lesion length and bark calcium concentration and the [calcium/ nitrogen] ratio. Laboratory experiments with excised shoots on Nemaguard, K119-50,P30-135, Lovell and Guardian rootstocks growing in a second orchard showed inconsistentresults. Shoots from Nemaguard developed significantly smaller lesions than those onK119-50 and P30-135. Shoots on Guardian and Lovell also developed significantly smallerlesions than those of shoots on K119-50 and P30-135. Temperature fluctuation duringincubation (0 °C to 14.4 °C) had no effect on shoot lesion length compared with thoseincubated constantly at 14.4 °C, but produced significantly longer lesions than shootsincubated constantly at 0 °C. These inconsistent results suggest that, in the absence ofmajor predisposing factors (i.e., ring nematodes or low soil pH),rootstocks may play a minor role in peach susceptibility to bacterial canker even underfavorable disease development conditions.

Cryptosporidium is a zoonotic protozoan parasite with public health importance worldwide. The objectives of this study were to (1) conduct a meta-analysis of published literature for oocyst shedding and diarrhoea outcomes, and (2) develop recommendations for standardization of experimental dose–response studies. Results showed that for the outcome of oocyst shedding in faeces, the covariates ‘experimental species’, ‘immunosuppression’, ‘oocyst dose’ and ‘oocyst dose’ × ‘age’ were all significant (P ⩽ 0·05). This study suggests that exposing mice, piglets, or ruminants, and using immunosuppressed experimental hosts, is more likely to result in oocyst shedding. For the outcome of diarrhoea in experimentally infected animal species, the key covariates ‘experimental species’, ‘age’ and ‘immunosuppression’ were significant (P ⩽ 0·2). Therefore, based on the results of this meta-analysis, these variables should be carefully reported and considered when designing experimental dose–response studies. Additionally, detection of possible publication bias highlights the need to publish additional studies that convey statistically non-significant as well as significant results in the future.

The consumption of a high-fat diet modifies both the morphology of the small intestine and experimentally tested effects of schistosomiasis mansoni. However, whether a schistosomiasis infection associated with a high-fat diet causes injury to the small intestine has never been investigated. Mice were fed either a high-fat or a standard-fat diet for 6 months and were then infected with Schistosoma mansoni cercariae. Physical characteristics of the intestinal tissue (mucosal thickness, small intestinal villi length and height, and abundance of goblet cells and enterocytes on the villous surface) and the distribution of granulomas along the intestinal segments and their developmental stage were measured at the time of sacrifice (9 or 17 weeks post-infection). The group fed a high-fat diet exhibited different granuloma stages, whereas the control group possessed only exudative granulomas. The chronically infected mice fed a high-fat diet exhibited higher granuloma and egg numbers than the acutely infected group. Exudative, exudative/exudative-productive and exudative-productive granulomas were present irrespective of diet. Computer-aided morphometric analysis confirmed that villus length, villus width, muscular height and submucosal height of the duodenal and jejunal segments were affected by diet and infection. In conclusion, a high-fat diet and infection had a significant impact on the small intestine morphology and morphometry among the animals tested.

A study was conducted of the biological, morphological and molecular characters of 3 strains of Trypanosoma cruzi (SI5, SI8 and SIGR3) isolated from specimens of Triatoma sordida collected in Santo Inácio and a domestic cat. In order to carry out the study, the following parameters were evaluated: pre-patent period, parasitaemia curves, morphology of the parasites, mortality rates, histopathological lesions and molecular typing. The strains presented variable pre-patent periods, low parasitaemia and no animal mortality. The morphological study of trypomastigotes showed a predominance of intermediate-width and short-length forms, as well as low nuclear index. Epimastigotes presented a low nuclear index, intermediate-width forms in strains SI5 and SI8, and large-width forms in SIGR3. A shorter length could be noted in strains SI8 and SIGR3, whereas SI5 displayed an intermediate length. The histopathological study did not detect amastigote nests in tissues. The amplification of the divergent domain of 24Sα rRNA, HSP60 and GPI genes of strains SI5, SI8 and SIGR3 classified the 3 strains into Group II. Biological parameters made it possible to classify the strains isolated in Santo Inácio (BA) into Biodeme III, Zymodeme 1 and Group II of T. cruzi.

Trypanosoma cruzi (Tc), the causative agent of Chagas disease, is a diverse species with 2 primary genotypes, TcI and TcII, with TcII further subdivided into 5 subtypes (IIa–e). This study evaluated infection dynamics of 4 genetically and geographically diverse T. cruzi strains in 2 South American reservoirs, degus (Octodon degus) and grey short-tailed opossums (Monodelphis domestica). Based on prior suggestions of a genotype-host association, we hypothesized that degus (placental) would more readily become infected with TcII strains while short-tailed opossums (marsupial) would be a more competent reservoir for a TcI strain. Individuals (n=3) of each species were intraperitoneally inoculated with T. cruzi trypomastigotes of TcIIa [North America (NA)-raccoon (Procyon lotor) origin], TcI [NA-Virginia opossum (Didelphis virginiana)], TcIIb [South America (SA)-human], TcIIe (SA-Triatoma infestans), or both TcI and TcIIa. Parasitaemias in experimentally infected degus peaked earlier (7–14 days post-inoculation (p.i.)) compared with short-tailed opossums (21–84 days p.i.). Additionally, peak parasitaemias were higher in degus; however, the duration of detectable parasitaemias for all strains, except TcIIa, was greater in short-tailed opossums. Infections established in both host species with all genotypes, except for TcIIa, which did not establish a detectable infection in short-tailed opossums. These results indicate that both South American reservoirs support infections with these isolates from North and South America; however, infection dynamics differed with host and parasite strain.

The evolution of host specificity and the potential trade-off between being a generalist and a specialist are central issues in the evolutionary ecology of parasites. Different species of parasites or even different populations of the same species often show different degrees of host specificity. However, less is known about intraspecific variation in host specificity within a population. We investigated intraspecific variation by experimentally exposing cercariae from different clones of the trematode Curtuteria australis to two species of second intermediate hosts, the New Zealand cockle Austrovenus stutchburyi and the wedge shell Macomona liliana. We found an overall difference in infection success between the two bivalve species, with A. stutchburyi being the more heavily infected host. However, the cercariae showed a consistent preference for encysting at the tip of the bivalve's foot, regardless of host species. Importantly, there were no significant differences among parasite clones in either relative infection success in the two hosts or preference for the host foot tip. This lack of intraspecific variation may be due to the life-history traits of both parasite and hosts in our system, which may limit opportunities for variation in performance and exploitation strategies in different hosts to evolve within the population.

A cysticercus of a possibly undescribed species of Taenia which occurs commonly in Taiwan aborigines was used to establish an experimental infection in a human volunteer. Symptomatic effects attributed to the infection included diarrhoea, upper abdominal pain, and increase or loss of appetite over a four-month period. After an expelled proglottid was observed 122 days post-exposure, eggs and proglottids were found continuously until the patient was treated with anthelmintics. Antibody titres measured by enzyme-linked immunosorbent assay and levels of eosinophilia seemed to correlate with symptoms. Haematological analyses revealed an abnormal lipid metabolism during the entire symptomatic period.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.

Experimental completion of the life-cycle of Paragonimus westermani (Trematoda: Troglotrematidae) (diploid type) from egg to adult has been achieved in the laboratory for the first time. A dog was fed metacercariae of P. westermani from naturally infected freshwater crabs, Geothelphusa dehaani. The adult flukes were recovered and their eggs were incubated in water at 28°C, hatching after 20 days. Laboratory reared marsh snails, Semisulcospira libertina, were exposed to miracidia from embryonated eggs. One of 47 snails became infected with a large number of 2nd generation rediae containing fully mature cercariae 245 days after exposure. Paragonimus-free crabs (G. dehaani) were then infected with the intramolluscan larvae. Of the 40 crabs examined 34–70 days after infection, 17 were infected with a total of 84 metacercariae. A cat and 4 dogs were fed with 55 metacercariae obtained. Eighty to 100 days later, a total of 40 flukes was recovered; 39 flukes were adults with eggs in their Uteri.

The possibility of coproantigen detection in the diagnosis of strongyloidiasis was investigated in rats experimentally infected with Strongyloides ratti. A capture ELISA was developed using affinity-purified polyclonal antibodies raised in rabbits against adult and filariform larval somatic antigens. The first detection of coproantigen coincided with patency and the coproantigen level remained elevated even when the parasite count was low. Following challenge infection, when examination of stool for larvae or ova was negative, coproantigen was detected by anti-adult somatic capture ELISA. Despite the use of antisera raised against crude worm extracts, the test was specific showing only low cross-reactivity with Necator americanus and Syphacia muris.

Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence of B. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. Gorging B. divergens infected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.