Rapid environmental effects on gut nematode susceptibility in rewilded mice.

Genetic and environmental factors shape host susceptibility to infection, but how and how rapidly environmental variation might alter the susceptibility of mammalian genotypes remains unknown. Here, we investigate the impacts of seminatural environments upon the nematode susceptibility profiles of inbred C57BL/6 mice. We hypothesized that natural exposure to microbes might directly (e.g., via trophic interactions) or indirectly (e.g., via microbe-induced immune responses) alter the hatching, growth, and survival of nematodes in mice housed outdoors. We found that while C57BL/6 mice are resistant to high doses of nematode (Trichuris muris) eggs under clean laboratory conditions, exposure to outdoor environments significantly increased their susceptibility to infection, as evidenced by increased worm burdens and worm biomass. Indeed, mice kept outdoors harbored as many worms as signal transducer and activator of transcription 6 (STAT6) knockout mice, which are genetically deficient in the type 2 immune response essential for clearing nematodes. Using 16S ribosomal RNA sequencing of fecal samples, we discovered enhanced microbial diversity and specific bacterial taxa predictive of nematode burden in outdoor mice. We also observed decreased type 2 and increased type 1 immune responses in lamina propria and mesenteric lymph node (MLN) cells from infected mice residing outdoors. Importantly, in our experimental design, different groups of mice received nematode eggs either before or after moving outdoors. This contrasting timing of rewilding revealed that enhanced hatching of worms was not sufficient to explain the increased worm burdens; instead, microbial enhancement and type 1 immune facilitation of worm growth and survival, as hypothesized, were also necessary to explain our results. These findings demonstrate that environment can rapidly and significantly shape gut microbial communities and mucosal responses to nematode infections, leading to variation in parasite expulsion rates among genetically similar hosts.

Intensity of infection with intracellular Eimeria spp. and pinworms is reduced in hybrid mice compared to parental subspecies.

Genetic diversity in animal immune systems is usually beneficial. In hybrid recombinants, this is less clear, as the immune system could also be impacted by genetic conflicts. In the European house mouse hybrid zone, the long-standing impression that hybrid mice are more highly parasitized and less fit than parentals persists despite the findings of recent studies. Working across a novel transect, we assessed infections by intracellular protozoans, Eimeria spp., and infections by extracellular macroparasites, pinworms. For Eimeria, we found lower intensities in hybrid hosts than in parental mice but no evidence of lowered probability of infection or increased mortality in the centre of the hybrid zone. This means ecological factors are very unlikely to be responsible for the reduced load of infected hybrids. Focusing on parasite intensity (load in infected hosts), we also corroborated reduced pinworm loads reported for hybrid mice in previous studies. We conclude that intensity of diverse parasites, including the previously unstudied Eimeria, is reduced in hybrid mice compared to parental subspecies. We suggest caution in extrapolating this to differences in hybrid host fitness in the absence of, for example, evidence for a link between parasitemia and health.

Holobiont suture zones: Parasite evidence across the European house mouse hybrid zone.

Parasite hybrid zones resulting from host secondary contact have never been described in nature although parasite hybridization is well known and secondary contact should affect them similarly to free-living organisms. When host populations are isolated, diverge and recontact, intimate parasites (host specific, direct life cycle) carried during isolation will also meet and so may form parasite hybrid zones. If so, we hypothesize these should be narrower than the host's hybrid zone as shorter parasite generation time allows potentially higher divergence. We investigate multilocus genetics of two parasites across the European house mouse hybrid zone. We find each host taxon harbours its own parasite taxa. These also hybridize: Parasite hybrid zones are significantly narrower than the host's. Here, we show a host hybrid zone is a suture zone for a subset of its parasite community and highlight the potential of such systems as windows on the evolutionary processes of host-parasite interactions and recombinant pathogen emergence.

Localization and phosphorylation of Plasmodium falciparum nicotinamide/nicotinate mononucleotide adenylyltransferase (PfNMNAT) in intraerythrocytic stages.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) is an essential molecule in the energy metabolism of living beings, and it has various cellular functions. The main enzyme in the biosynthesis of this nucleotide is nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) because it is the convergence point for all known biosynthetic pathways. NMNATs have divergences in both the number of isoforms detected and their distribution, depending on the organism. METHODS: In the laboratory of basic research in biochemistry (LIBBIQ: acronym in Spanish) the NMNATs of protozoan parasites (Leishmania braziliensis, Plasmodium falciparum, Trypanosoma cruzi, and Giardia duodenalis) have been studied, analysing their catalytic properties through the use of proteins. Recombinants and their cellular distribution essentially. In 2014, O'Hara et al. determined the cytoplasmic localization of NMNAT of P. falciparum, using a transgene coupled to GFP, however, the addition of labels to the study protein can modify several of its characteristics, including its sub-cellular localization. RESULTS: This study confirms the cytoplasmic localization of this protein in the parasite through recognition of the endogenous protein in the different stages of the asexual life cycle. Additionally, the study found that PfNMNAT could be a phosphorylation target at serine, tyrosine and threonine residues, and it shows variations during the asexual life cycle. CONCLUSIONS: These experiments confirmed that the parasite is situated in the cytoplasm, fulfilling the required functions of NAD+ in this compartment, the PfNMNAT is regulated in post-transcription processes, and can be regulated by phosphorylation in its residues.

Isobolographic analysis of co-administration of two plant-derived antiplasmodial drug candidates, cryptolepine and xylopic acid, in Plasmodium berghei.

BACKGROUND: Increasing resistance to current anti-malarial therapies requires a renewed effort in searching for alternative therapies to combat this challenge, and combination therapy is the preferred approach to address this. The present study confirms the anti-plasmodial effects of two compounds, cryptolepine and xylopic acid and the relationship that exists in their combined administration determined. METHODS: Anti-plasmodial effect of cryptolepine (CYP) (3, 10, 30 mg kg-1) and xylopic acid (XA) (3, 10, 30 mg kg-1) was evaluated in Plasmodium berghei-infected male mice after a 6-day drug treatment. The respective doses which produced 50% chemosuppression (ED50) was determined by iterative fitting of the log-dose responses of both drugs. CYP and XA were then co-administered in a fixed dose combination of their ED50s (1:1) as well as different fractions of these combinations (1/2, 1/4, 1/8, 1/16 and 1/32) to find the experimental ED50 (Zexp). The nature of interaction between cryptolepine and xylopic acid was determined by constructing an isobologram to compare the Zexp with the theoretical ED50 (Zadd). Additionally, the effect of cryptolepine/xylopic acid co-administration on vital organs associated with malarial parasiticidal action was assessed. RESULTS: The Zadd and Zexp were determined to be 12.75 ± 0.33 and 2.60 ± 0.41, respectively, with an interaction index of 0.2041. The Zexp was significantly (P < 0.001) below the additive isobole indicating that co-administration of cryptolepine and xylopic acid yielded a synergistic anti-plasmodial effect. This observed synergistic antiplasmodial effect did not have any significant deleterious effect on the kidney, liver and spleen. However, the testis were affected at high doses. CONCLUSION: The co-administration of cryptolepine and xylopic acid produces synergistic anti-malarial effect with minimal toxicity.

Anti-plasmodial effect of plant extracts from Picrolemma huberi and Picramnia latifolia.

BACKGROUND: Malaria is an infectious disease caused by parasites of the genus Plasmodium, of which Plasmodium vivax and Plasmodium falciparum are the major species that cause the disease in humans. As there are relatively few alternatives for malaria treatment, it is necessary to search for new chemotherapeutic options. Colombia possesses a great diversity of plants, which are potential sources of new compounds of medical interest. Thus, in this study the antiplasmodial effect of extracts from two species of plants from the families Simaroubaceae and Picramniaceae (Picramnia latifolia and Picrolemma huberi) was evaluated in vitro and in vivo. These plants were chosen because they contain secondary metabolites with interesting medicinal effects. RESULTS: The ethanolic extracts of both species were highly active with IC50: 1.2 ± 0.19 µg/mL for P. latifolia and IC50: 0.05 ± 0.005 µg/mL for P. huberi. The P. latifolia extract had a stage specific effect on trophozoites and inhibited parasite growth in vivo by 52.1 ± 3.4%, evaluated at 1000 mg/kg in Balb/c mice infected with Plasmodium berghei. On the other hand, evaluated at 150 mg/kg body weight in the same murine model, the ethanolic extract from P. huberi had an antiplasmodial effect in all the asexual intraerythrocytic stages of P. falciparum FCR3 and inhibited the parasitic growth in 93 ± 32.9%. CONCLUSIONS: This is the first report of anti-malarial activity for these two species of plants. Thus, P. latifolia and P. huberi are potential candidates for the development of new drugs for treating malaria.

Differential activity of methylene blue against erythrocytic and hepatic stages of Plasmodium.

BACKGROUND: In the context of malaria elimination/eradication, drugs that are effective against the different developmental stages of the parasite are highly desirable. The oldest synthetic anti-malarial drug, the thiazine dye methylene blue (MB), is known for its activity against Plasmodium blood stages, including gametocytes. The aim of the present study was to investigate a possible effect of MB against malaria parasite liver stages. METHODS: MB activity was investigated using both in vitro and in vivo models. In vitro assays consisted of testing MB activity on Plasmodium falciparum, Plasmodium cynomolgi and Plasmodium yoelii parasites in human, simian or murine primary hepatocytes, respectively. MB in vivo activity was evaluated using intravital imaging in BALB/c mice infected with a transgenic bioluminescent P. yoelii parasite line. The transmission-blocking activity of MB was also addressed using mosquitoes fed on MB-treated mice. RESULTS: MB shows no activity on Plasmodium liver stages, including hypnozoites, in vitro in primary hepatocytes. In BALB/c mice, MB has moderate effect on P. yoelii hepatic development but is highly effective against blood stage growth. MB is active against gametocytes and abrogates parasite transmission from mice to mosquitoes. CONCLUSION: While confirming activity of MB against both sexual and asexual blood stages, the results indicate that MB has only little activity on the development of the hepatic stages of malaria parasites.

Parasitic nematodes of the genus Syphacia Seurat, 1916 infecting Muridae in the British Isles, and the peculiar case of Syphacia frederici.

Syphacia stroma (von Linstow, 1884) Morgan, 1932 and Syphacia frederici Roman, 1945 are oxyurid nematodes that parasitize two murid rodents, Apodemus sylvaticus and Apodemus flavicollis, on the European mainland. Only S. stroma has been recorded previously in Apodemus spp. from the British Isles. Despite the paucity of earlier reports, we identified S. frederici in four disparate British sites, two in Nottinghamshire, one each in Berkshire and Anglesey, Wales. Identification was based on their site in the host (caecum and not small intestine), on key morphological criteria that differentiate this species from S. stroma (in particular the tail of female worms) and by sequencing two genetic loci (cytochrome C oxidase 1 gene and a section of ribosomal DNA). Sequences derived from both genetic loci of putative British S. frederici isolates formed a tight clade with sequences from continental worms known to be S. frederici, clearly distinguishing these isolates from S. stroma which formed a tight clade of its own, distinct from clades representative of Syphacia obvelata from Mus and S. muris from Rattus. The data in this paper therefore constitute the first record of S. frederici from British wood mice, and confirm the status of this species as distinct from both S. obvelata and S. stroma.

Cryptosporidium infecting wild cricetid rodents from the subfamilies Arvicolinae and Neotominae.

We undertook a study on Cryptosporidium spp. in wild cricetid rodents. Fecal samples were collected from meadow voles (Microtus pennsylvanicus), southern red-backed voles (Myodes gapperi), woodland voles (Microtus pinetorum), muskrats (Ondatra zibethicus) and Peromyscus spp. mice in North America, and from bank voles (Myodes glareolus) and common voles (Microtus arvalis) in Europe. Isolates were characterized by sequence and phylogenetic analyses of the small subunit ribosomal RNA (SSU) and actin genes. Overall, 33·2% (362/1089) of cricetids tested positive for Cryptosporidium, with a greater prevalence in cricetids from North America (50·7%; 302/596) than Europe (12·1%; 60/493). Principal Coordinate analysis separated SSU sequences into three major groups (G1-G3), each represented by sequences from North American and European cricetids. A maximum likelihood tree of SSU sequences had low bootstrap support and showed G1 to be more heterogeneous than G2 or G3. Actin and concatenated actin-SSU trees, which were better resolved and had higher bootstrap support than the SSU phylogeny, showed that closely related cricetid hosts in Europe and North America are infected with closely related Cryptosporidium genotypes. Cricetids were not major reservoirs of human pathogenic Cryptosporidium spp.

Endoparasites of Small Mammals in Edo State, Nigeria: Public Health Implications.

Some small mammals occur as household pests and harbour a number of parasites that could be of public health importance. This study profiled the helminth and protozoan parasites in trapped small mammals within and around human dwelling places (houses) located across 4 major towns (Auchi, Benin, Ekpoma, and Uromi) and environs in Edo state, Nigeria. Six genera (Apodemus sp., Crocidura sp., Mastomys natalensis, Mus musculus, Rattus sp., and Sorex sp.) were identified from 502 trapped small mammals. Overall, M. musculus (71.9%) and Rattus rattus (20.1%) were the most frequently trapped. In total, on examination of blood, gastrointestinal contents, and brain tissues, 12 helminth taxa (Angiostrongylus sp., Aspicularis sp., Capillaria sp., Gongylonema sp., Heterakis spumosa, Hymenolepis diminuta, Hymenolepis nana, Mastophorus muris, Moniliformis moniliformis, Nippostrongylus brasiliensis, Strongyloides sp., Trichosomoides sp., and Trichuris sp.), and 6 protozoan parasites (Babesia sp., Trypanosoma lewisi, Plasmodium sp., Eimeria sp., Isospora sp., and Toxoplasma gondii) were isolated. Most prevalent helminths with relatively heavy mean intensity were Strongyloides sp. and Heterakis spumosa, while Plasmodium, Eimeria, and Isospora were the most prevalent protozoan parasites. Generally, intrinsic factors like sex and age had marginal influence on the rate and burden of infection in M. musculus and R. rattus. Although the infection rate and prevalence of zoonotic parasites were low, they were largely recovered in rodents from Ekpoma. This study elucidates the public health implication of the presence of zoonotic parasites in these small mammals.

Pattern of tick aggregation on mice: larger than expected distribution tail enhances the spread of tick-borne pathogens.

The spread of tick-borne pathogens represents an important threat to human and animal health in many parts of Eurasia. Here, we analysed a 9-year time series of Ixodes ricinus ticks feeding on Apodemus flavicollis mice (main reservoir-competent host for tick-borne encephalitis, TBE) sampled in Trentino (Northern Italy). The tail of the distribution of the number of ticks per host was fitted by three theoretical distributions: Negative Binomial (NB), Poisson-LogNormal (PoiLN), and Power-Law (PL). The fit with theoretical distributions indicated that the tail of the tick infestation pattern on mice is better described by the PL distribution. Moreover, we found that the tail of the distribution significantly changes with seasonal variations in host abundance. In order to investigate the effect of different tails of tick distribution on the invasion of a non-systemically transmitted pathogen, we simulated the transmission of a TBE-like virus between susceptible and infective ticks using a stochastic model. Model simulations indicated different outcomes of disease spreading when considering different distribution laws of ticks among hosts. Specifically, we found that the epidemic threshold and the prevalence equilibria obtained in epidemiological simulations with PL distribution are a good approximation of those observed in simulations feed by the empirical distribution. Moreover, we also found that the epidemic threshold for disease invasion was lower when considering the seasonal variation of tick aggregation.

Sex-specific effects of parasitism on survival and reproduction of a rodent host in a subtropical montane region.

Parasites can generate complex life history trade-offs in a host. In this study, we experimentally reduced the infection level of intestinal helminth parasites in the Taiwan field mouse (Apodemus semotus) to test (1) whether parasite richness and load are biased towards male or female mice (sex-biased parasitism) and (2) whether the effects of parasitism on the host's survival and reproduction are different between the sexes (sex-specific effects of parasitism). Our findings indicate that neither parasite richness (number of helminth taxa found in a fecal sample) nor parasite load (number of helminth eggs per gram of fecal material) was sexually biased in our A. semotus study population. These results are in agreement with those of previous studies on endoparasites in Apodemus spp., but are in contrast to those on ectoparasites in Apodemus spp. Parasite removal reduced the survival rate of reproducing females, possibly by allowing reproducing females to increase maternal investment in their current litters at the cost of their own future survival. Single-litter mothers with reduced parasitism had a higher body mass than the untreated single-litter mothers, suggesting an increased maternal investment. In addition, the reproductively more active A. semotus, particularly the females, carried higher parasite loads, suggesting a trade-off between reproduction and parasite defense. By demonstrating that parasites can affect life history trade-offs in A. semotus, our results highlight the importance of maintaining variation in life history traits under parasitism risks and illustrate the subtle demographic processes (e.g. reduced future survival among healthy reproducing females) that might be driven by parasitism.

Two New Species of Demodex (Acari: Demodecidae) with a Redescription of Demodex musculi and Data on Parasitism in Mus musculus (Rodentia: Muridae).

This article describes two new skin mite species found on the house mouse Mus musculus L., 1758. Demodex marculus sp. nov. is a very small demodecid mite (adult stages, on average, 99 µm in length) found in mouse skin in the abdomen, back, limbs, and anal area. It is characterized by relatively large bossing hammer-shaped supracoxal spines, embedded in the trapezoidal gnathosoma. Demodex fusiformis sp. nov., in turn, is a little larger (adult stages on average 111 µm in length), with a small oval gnathosoma equipped with fine, knob-like supracoxal spines. It was found in the skin of abdomen, back, and limbs. Moreover, Demodex musculi (Oudemans, 1897) was redescribed, which is small demodecid mite (adult stages on average 142 µm in length) and characterized by relatively large morphological variation and considerable sexual dimorphism. The characteristic feature of this species is the strongly elongated and rectangular gnathosoma equipped with very large wedge-shaped supracoxal spines. D. musculi was found in the skin of various, haired regions of the mice body (head, neck, abdomen, back, limbs, genital-anal region, and tail). Moreover, one more demodecid mite was found in the skin of the examined mice, it was Demodex flagellurus Bukva, 1985, which was found only in the genital area. Overall infection of Mus musculus L. by all species of Demodex was with the prevalence of 100%, mean intensity of 24.0, and range of intensity of 1-109. Despite high infection levels, no symptoms of parasitosis were observed in the hosts.

Survey of Hymenolepis spp. in pet rodents in Italy.

We carried out the first survey of Hymenolepis spp. infection in pet rodents in Italy. Fresh fecal samples were collected from 172 pet rodents as follows: guinea pigs (Cavia porcellus; n = 60), squirrels (Callosciurus finlaysonii, Callosciurus prevosti, Tamias striatus, Tamias sibiricus, Sciurus calorinensis; n = 52), hamsters (Phodopus campbelli, Mesocricetus auratus; n = 30), chinchillas (Chinchilla lanigera; n = 13), rats (Rattus norvegicus; n = 10), and mice (Mus minutoides; n = 7). These animals were housed either in pet shops or in private houses. All fecal samples were processed using the FLOTAC pellet technique to assess the number of eggs per gram (EPG) of feces. Eggs of Hymenolepis nana were found in 24 out of 172 (13.9 %; 95 % confidence interval = 9.3-20.2 %) pet rodents. Of those rodents, 41.6 % (10/24) were rats (mean EPG = 55.7; range = 2-200), 29.2 % (7/24) mice (mean EPG = 64.5; range = 32-120), 25.0 % (6/24) were chinchillas (mean EPG = 25.5; range = 10-50), and 4.2 % (1/24) hamsters (P. campbelli) (EPG = 86.0). In addition, Hymenolepis diminuta eggs were found in 2 out of 172 (1.2 %; 95 % confidence interval = 0.2-4.6 %) rodents examined, both of which (100 %; 2/2) were pet squirrels (C. prevosti) (mean EPG = 10; range = 4-16). To the authors' knowledge, this is the first report of a natural infection of H. diminuta in pet squirrels.

Behavioural changes in the flour beetle Tribolium confusum infected with the spirurid nematode Protospirura muricola.

We examined changes to the behaviour of flour beetles, Tribolium confusum, infected with the rodent stomach worm, the spirurid Protospirura muricola, in the context of the 'Behavioural Manipulation Hypothesis'. Trobolium confusum infected with the third-stage infective larvae of P. muricola showed consistently altered patterns of behaviour. Relative to uninfected beetles, over a measured time period, beetles infected with P. muricola were likely to move over a shorter distance, when moving their speed of movement was slower, they were more likely to stay in the illuminated area of their environment, more likely to emerge from darkened areas into the illuminated areas, and their longevity was significantly shortened. The changes in behaviour, as reflected in effects on speed of movement, were only evident among beetles that actually harboured infective cysts and not among those carrying younger infections when the larvae within their haemocoels would have been at an earlier stage of development and not yet capable of infecting the definitive murine hosts. We discuss whether these changes would have made the beetles more susceptible to predation by rodents, and specifically by the omnivorous eastern spiny mouse, Acomys dimidiatus, the natural definitive host of this parasite in Egypt, from where the P. muricola isolate originated, and whether they support the Behavioural Manipulation Hypothesis or reflect parasite-induced pathology.

The role of the immunological background of mice in the genetic variability of Schistosoma mansoni as detected by random amplification of polymorphic DNA.

Schistosomiasis is a parasitic disease caused by flatworms of the genus Schistosoma. Among the Schistosoma species known to infect humans, S. mansoni is the most frequent cause of intestinal schistosomiasis in sub-Saharan Africa and South America: the World Health Organization estimates that about 200,000 deaths per year result from schistosomiasis in sub-Saharan Africa alone. The Schistosoma life cycle requires two different hosts: a snail as intermediate host and a mammal as definitive host. People become infected when they come into contact with water contaminated with free-living larvae (e.g. when swimming, fishing, washing). Although S. mansoni has mechanisms for escaping the host immune system, only a minority of infecting larvae develop into adults, suggesting that strain selection occurs at the host level. To test this hypothesis, we compared the Belo Horizonte (BH) strain of S. mansoni recovered from definitive hosts with different immunological backgrounds using random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR). Schistosoma mansoni DNA profiles of worms obtained from wild-type (CD1 and C57BL/6J) and mutant (Jα18- / - and TGFßRIIdn) mice were analysed. Four primers produced polymorphic profiles, which can therefore potentially be used as reference biomarkers. All male worms were genetically distinct from females isolated from the same host, with female worms showing more specific fragments than males. Of the four host-derived schistosome populations, female and male adults recovered from TGFßRIIdn mice showed RAPD-PCR profiles that were most similar to each other. Altogether, these data indicate that host immunological backgrounds can influence the genetic diversity of parasite populations.

A new species of the genus Demodex Owen, 1843 (Acari: Demodecidae) from the ear canals of the house mouse Mus musculus L. (Rodentia: Muridae).

A new species Demodex conicus n. sp. is described based on adult and juvenile stages from the ear canals of the house mouse Mus musculus L. in Poland. The new species is most similar to D. auricularis Izdebska, Rolbiecki & Fryderyk, 2014 from the ear canals of the wood mouse Apodemus sylvaticus (L.), but differs in the following features: the gnathosoma is triangular, the supracoxal spines (setae elc.p) are conical, the spines on the terminal segment of palp are four, the striation on opisthosoma is fine but dense, the vulva is located at a distance of c.17 µm from posterior level of legs IV, and the male genital opening is located at the level of legs I. The differences also relate to body size and proportions, female D. conicus n. sp. being, on average slightly larger, and male significantly larger than D. auricularis. Males of the new species also have longer and more massive opisthosoma than males of D. auricularis. Demodex conicus n. sp. was found in 17.5% of the mice studied from different locations in Poland.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.

Survival of host blood proteins in Ixodes scapularis (Acari: Ixodidae) ticks: a time course study.

Ixodes scapularis Say, 1821 larvae were fed on mice and allowed to molt under laboratory conditions. A liquid chromatography-tandem mass spectrometry-based proteomic study was conducted to identify the type of mammalian proteins present in the derived nymphal ticks at different time intervals after molting. Albumin was present for 85 d; transferrin was present for 29 d; and, more importantly, hemoglobin remained detectable for up to 309 d postmolting. Peptides of actin, keratin, and tubulin are highly similar between mouse and tick, and therefore, unambiguous assignment of these proteins to different species was not possible. Establishing a time line for the persistence of hemoglobin, one of the most abundant blood proteins, at detectable levels in ticks after the bloodmeal and molting advances our efforts to use this protein to identify the host species.

A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents.

Cryptosporidium spp. are cosmopolitan protozoa that infect fishes, reptiles, amphibians, birds and mammals. More than 20 species are recognized within this genus. Rodents are a group of abundant and ubiquitous organisms that have been considered reservoirs of Cryptosporidium for humans and livestock. The aim of this study was to design specific primers for the gene encoding 18S rRNA, potentially capable of amplifying any species or genotype of Cryptosporidium spp. and evaluate the diagnostic attributes of the nested-PCR based on such probes. The primers were designed to amplify the shortest segment as possible to maximize the sensitivity of the test, but preserving the discriminatory potential of the amplified sequences for phylogenetic inferences. The nested-PCR standardized in this study (nPCR-SH) was compared in terms of sensitivity with another similar assay (nPCR-XIAO) that has been largely used for the detection and identification of Cryptosporidium spp. worldwide. We also aimed to molecularly characterize samples of Cryptosporidum spp. isolated from synanthropic rodents using these probes. Forty-five rodents were captured in urban areas of the municipality of Umuarama, Paraná State, Brazil. Fecal samples were submitted to three molecular tests (nested-PCRs), two of them targeted to the 18S rDNA gene (nPCR-SH and nPCR-XIAO) and the third targeted to the gene encoding actin (nPCR-actin). The nPCR-SH was tested positive on samples of Cryptosporidum parvum, Cryptosporidum andersoni, Cryptosporidum meleagridis, Cryptosporidum hominis, Cryptosporidum canis, and Cryptosporidum serpentis. Sixteen samples of rodents were positive by nPCR-SH, six by nPCR-XIAO and five by nPCR-actin. Sequencing of amplified fragments allowed the identification of Cryptosporidum muris in three samples of Rattus rattus, and two genotypes of Cryptosporidium, the genotypes mouse II and III. Cryptosporidium genotype mouse II was found in one sample of Mus musculus and genotype mouse III, in twelve samples, being five from R. rattus and seven from M. musculus. The results of this study demonstrated that the primers designed for detection of Cryptosporidium spp. were more efficient than those used in the nPCR-XIAO. Genotypes or species of Cryptosporidium that can be usually transmitted for human beings and livestock were not found in synanthropic rodents, suggesting that the importance of these animals in zoonotic transmission of cryptosporidiosis should be revisited.