New insights about cross-reactive epitopes of six trypanosomatid genera revealed that Crithidia and Leptomonas have antigenic similarity to L. (L.) chagasi

We investigated whether ELISA using crude antigens from insect and plant trypanosomatids, which are non-pathogenic and easily cultivated in large scale, has the same positivity data as Leishmania (Leishmania) chagasi, the etiological agent of human visceral leishmaniasis (VL) or canine leishmaniasis (CanL), or as Trypanosoma cruzi, the etiological agent of Chagas disease (CD). The antigens from Crithidia fasciculata, Crithidia luciliae, and Leptomonas seymouri showed 100% cross-reactivity with VL and CanL samples, with no statistically titers differences from L. (L.) chagasi, however, 34% (17/50) of VL samples revealed higher titers using the insect trypanosomatids than the homologous antigen. On the other hand, antigens from Strigomonas culicis, Angomonas deanei, and Phytomonas serpens showed low cross-reactivity with VL and CanL samples. The sera from patients with American tegumentary leishmaniasis showed low levels of cross-reactivity with all trypanosomatids investigated, even with L. (L) chagasi, without titers dissimilarity among them. These parasites were also worthless as antigen source for detection of CD cases, which required homologous antigens to reach 100% positivity. This study showed, by ELISA, that crude extract of Crithidia and Leptomonas have epitopes similar to L. (L.) chagasi, which supports the idea of using them as antigens source for the serodiagnosis of visceral leishmaniasis.     

Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand

The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002-2004. The pfcrt 76T allele was identified in 109 isolates (99.1%) while pfcrt 76K was found in a single (0.9%) isolate. The pfmdr 86N, 86Y, and the combination (86N + 86Y) alleles were identified in 83 (75.5%), 22 (20%), and 5 (4.5%) isolates, respectively. The pfmdr1 1042N, 1042D alleles and a mixture (1042N + 1042D) of the alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphisms (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates; 61.2%), Y-N-D (22 isolates; 19.8%), N-D-D (11 isolates; 9.9%), N-D-Y (1 isolate; 0.9%), N/Y-N-D (4 isolates; 3.6%), N-N/D-D (3 isolates; 2.7%), and N/Y-N/D-D (1 isolate; 0.9%). Eight different combinations of pfcrt-pfmdr1 genotypes were observed. Only one CQ-, MQ- and QN-sensitive isolate was found at the Thai-Laos border and no cases of QN resistance were found in this study.     

Anisakis/Ascaris IgE ratio improves specificity for the diagnosis of Anisakis simplex sensitization in travellers and immigrants

Anisakis simplex is a fish parasite responsible for human infection and is able to induce IgE-mediated reactions with several clinical manifestations. Laboratory diagnosis of Anisakis allergy is based on the detection of specific IgE using parasite whole antigen. Unfortunately, these diagnostic tools detect cross-reactivities with other nematodes and micro-organisms leading to low specificity of the diagnostic tests. The aim of this retrospective study was to assess the diagnostic value of specific IgE to Anisakis for diagnosis of A. simplex-sensitization in native Spanish residents (IMM, n = 766) and subjects coming from tropical and sub-tropical geographic areas (TRO, n = 233). Since Ascaris is the human parasite most closely related to Anisakis, specific IgE to Ascaris was also determined to assess Anisakis cross-reaction with other nematodes and the diagnostic value of Anisakis/Ascaris IgE ratio for Anisakis allergy was examined. IMM and TRO groups showed similar specific IgE to Anisakis levels, while TRO had higher levels of specific IgE to Ascaris than IMM group (p = 0.001). ROC curve analysis determined that an Anisakis specific IgE threshold of 0.71 kU/L yielded 93% and 82% specificities in IMM and TRO groups, respectively. A cut-off value ≥4.4 for Anisakis/Ascaris IgE ratio increased specificity to 95% for samples having IgE to Ascaris ≥0.35. In conclusion, the ratio of specific IgE to Anisakis and Ascaris improved remarkably the specificity and this parameter easily obtained from the commercially available system could be useful in the diagnosis of hypersensitivity to A. simplex.     

Laboratory and field evaluation of neem (Azadirachta indica A. Juss) and Chinaberry (Melia azedarach L.) oils as repellents against Phlebotomus orientalis and P. bergeroti (Diptera: Psychodidae) in Ethiopia

The study evaluated the efficacy of neem (Azadirachta indica A. Juss.) and Chinaberry (Melia azedarach L.) seed oils as repellents against laboratory and field populations of some sandflies in Ethiopia. In the laboratory, concentrations of 2% and 5% neem oil in coconut oil tested against Phlebotomus orientalis (vector of visceral leishmaniasis) provided 96.28% (95% CI = 95.60-96.97) protection up to a mean time of 7 h and 20 min and 98.26% (95% CI = 93.46-104. 07) protection up to 9 h, respectively. Similarly, M. azedarach oil at 2% concentration produced 95.13% (95% CI = 90.74-99.52) protection for the same duration (7 h and 20 min), while the 5% oil gave 96.20 (95% CI = 86.98-105.41) protection for 8 h and 20 min against the same species with no significant difference in percentage protection between the two oils at 2% and 5% concentrations. In the field tests with only neem oil (A. indica) against field populations of P. orientalis and P. bergeroti, similar high level of repellencies were recorded with about the same duration of protection. Application of both neem and Chinaberry oils can be safe and low-cost means of personal protection against sandfly bites in endemic areas of Ethiopia, if the community is advised and encouraged to grow the plants abundantly.     

Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis

Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research.     

dSir2 and longevity in Drosophila

The silent information regulator 2 (Sir2 or Sirtuin) family of proteins is highly conserved and has been implicated in the extension of longevity for several species. Mammalian Sirtuins have been shown to affect various aspects of physiology including metabolism, the stress response, cell survival, replicative senescence, inflammation, the circadian rhythm, neurodegeneration, and even cancer. Evidence in Drosophila implicates Sir2 in at least some of the beneficial effects of caloric restriction (CR). CR delays age-related pathology and extends life span in a wide variety of species. Here we will review the evidence linking Drosophila Sir2 (dSir2) to longevity regulation and the pathway associated with CR in Drosophila, as well as the effects of the Sir2 activator resveratrol and potential interactions between dSir2 and p53.     

Anthelmintic activity of benzimidazole derivatives against Toxocara canis second-stage larvae and Hymenolepis nana adults

The anthelmintic activity of 11 benzimidazole derivatives (A1-A11) and 2 thioureides N,N′-disubstituted (B1-B2) was determined. Each compound and albendazole was tested in vitro against Toxocara canis larvae and in vivo against Hymenolepis nana adult. Compounds A1-A6 and B1-B2 were designed as albendazole prodrugs. Compounds A8-A11 were designed as direct analogues of A7, which had previously proved to be an effective agent against Fasciola hepatica. Results of the in vitro screening showed that A6 was more active than albendazole at 0.18 μM (relative mobility 40% and 80%, respectively). Whereas that the in vivo evaluation against H. nana, compounds A7-A11 demonstrated significant activity in terms of removing cestode adults in the range of 88-97%, displaying better efficacy than albendazole (83%).     

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayesian phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes. Within the Albimanus Section the monophyly of the Strodei Subgroup was strongly supported and within the Myzorhynchella Section Anopheles antunesi and An. lutzii formed a strongly supported monophyletic group. The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An. lanei-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An. goeldii were recovered as sister species. Finally, there was evidence of complexes in several species, including An. antunesi, An. deaneorum, and An. strodei.     

First human cases of Leishmania (Viannia) naiffi infection in Ecuador and identification of its suspected vector species

Epidemiological surveillance of leishmaniasis was conducted in a northern Amazonian region of Ecuador, in which cutaneous leishmaniasis cases were recently reported. Sand flies were captured in the military training camp, and the natural infection of sand flies by Leishmania species was examined. Out of 334 female sand flies dissected, the natural infection by flagellates was microscopically detected in 3.9% of Lutzomyia yuilli yuilli and 3.7% of Lutzomyia tortura, and the parasite species were identified as Endotrypanum and Leishmania (Viannia) naiffi, respectively. After the sand fly surveillance, specimens from cutaneous leishmaniasis (CL) patients considered to have acquired the infection in the training camp area were obtained, and the infected parasite species were identified as L. (V.) naiffi. The present study reported first cases of CL caused by L. (V.) naiffi infection in Ecuador. In addition, a high ratio of infection of Lu. tortura by L. (V.) naiffi in the same area strongly suggested that Lu. tortura is responsible for the transmission of L. (V.) naiffi in this area.     

Antifilarial activity in vitro and in vivo of some flavonoids tested against Brugia malayi

We evaluated the antifilarial activity of 6 flavonoids against the human lymphatic filarial parasite Brugia malayi using an in vitro motility assay with adult worms and microfilariae, a biochemical test for viability (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-reduction assay), and two animal models, Meriones unguiculatus (implanted adult worms) and Mastomys coucha (natural infections). In vitro, naringenin and hesperetin killed the adult worms and inhibited (>60%) MTT-reduction at 7.8 and 31.2 μg/ml concentration, respectively. Microfilariae (mf) were killed at 250-500 μg/ml. The half maximal inhibitory concentration (IC₅₀) of naringenin for motility of adult females was 2.5 μg/ml. Flavone immobilized female adult worms at 31.2 μg/ml (MTT > 80%) and microfilariae at 62.5 μg/ml. Rutin killed microfilariae at 125 μg/ml and inhibited MTT-reduction in female worms for >65% at 500 μg/ml. Naringin had adulticidal effects at 125 μg/ml while chrysin killed microfilariae at 250 μg/ml. In vivo, 50 mg/kg of naringenin elimiated 73% of transplanted adult worms in the Meriones model, but had no effect on the microfilariae in their peritoneal cavity. In Mastomys, the same drug was less effective, killing only 31% of the naturally acquired adult worms, but 51%, when the dose was doubled. Still, effects on the microfilariae in the blood were hardly detectable, even at the highest dose. In summary, all 6 flavonoids showed antifilarial activity in vitro, which can be classed, in a decreasing order: naringenin > flavone = hesperetin > rutin > naringin > chrysin. In jirds, naringenin and flavone killed or sterilized adult worms at 50 mg/kg dose, but in Mastomys, where the parasite produces a patent infection, only naringenin was filaricidal. Thus naringenin and flavone may provide a lead for design and development of new antifilarial agent(s). This is the first report on antifilarial efficacy of flavonoids.     

Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity

A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63 μg/ml, IC50: 6.00 μg/ml) than macrofilaricidal (LC100: >250; IC50: 88 μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25 μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250–500 μg/ml, IC50: 44.16 μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100–200 mg/kg; fractions: 100 mg/kg, i.p. × 5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38–40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63 μg/ml, IC50: 6.57–10.31 μg/ml) and microfilaricidal (LC100: 31.25–62.5 μg/ml, IC50: 11.05–22.10 μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).     

Taxonomic assessment of Anopheles crawfordi and An. dangi of the Hyrcanus Group of subgenus Anopheles in Vietnam

Anopheles dangi, introduced as a new species of the Hyrcanus Group of subgenus Anopheles in an illustrated dichotomous key for the identification of the Anopheles mosquitoes of Vietnam published in 1987, was distinguished from Anopheles crawfordi based on the presence of a humeral pale spot on the base of the costal vein of the wing. However, this character has been known to occur occasionally in An. crawfordi. To determine whether An. dangi is distinct from An. crawfordi, we analyzed nucleotide sequences of the COI, COII and Cyt-b genes of mtDNA and the D3 gene of rDNA obtained from specimens collected in south-central Vietnam that were identified as An. dangi and An. crawfordi based on the presence or absence, respectively, of a humeral pale spot. Maximum Likelihood and Bayesian analyses of the sequences showed a low mean genetic distance of 0.004 for specimens identified as An. crawfordi and 0.008 for those identified as An. dangi. The mean genetic distance between the two nominal species was 0.006, compared with 0.077 for any group versus the outgroup taxa Anopheles dirus and Anopheles minimus, and the specimens of the two forms clustered in a single strongly supported clade. Consequently, An. dangi is merely a morphological variant of An. crawfordi and is deemed to be a synonym of that nominal species.     

Asociación en un mismo plásmido de blaVIM-1 y qnrS2 en Klebsiella pneumoniae y Klebsiella oxytoca aisladas en Sevilla

Background

Combined resistance to quinolones and β-lactams is common in Enterobacteriaceae. The appearance in enterobacteria coding for metallo-β-lactamases and determinants of plasmid-mediated quinolone resistance are an emerging problem in our country.

Methods

The susceptibility was determined by E-test. The resistance genes were detected by PCR and the corresponding plasmids were characterised.

Results

This study describes 2 strains (1 Klebsiella oxytoca, 1 Klebsiella pneumoniae) carrying the genes qnrS2 and blaVIM-1 in a transferable plasmid of 70-Kb isolated in surveillance cultures at the University Hospital Virgen Macarena in Seville.

Conclusion

This is the first combination of qnrS2 and bla_VIM-1 on the same non-typeable plasmid isolated in our centre.     

Stormwater drains and catch basins as sources for production of Aedes aegypti and Culex quinquefasciatus

We present data showing that structures serving as drains and catch basins for stormwater are important sources for production of the mosquito arbovirus vectors Aedes aegypti and Culex quinquefasciatus in Mérida City, México. We examined 1761 stormwater drains – located in 45 different neighborhoods spread across the city – over dry and wet seasons from March 2012 to March 2013. Of the examined stormwater drains, 262 (14.9%) held water at the time they were examined and 123 yielded mosquito immatures. In total, we collected 64,560 immatures representing nine species. The most commonly encountered species were Cx. quinquefasciatus (n = 39,269) and Ae. aegypti (n = 23,313). Ae. aegypti and Cx. quinquefasciatus were collected during all 11 months when we found water-filled stormwater drains, and both were found in stormwater drains located throughout Mérida City. We also present data for associations between structural characteristics of stormwater drains or water-related characteristics and the abundance of mosquito immatures. In conclusion, stormwater drains produce massive numbers of Ae. aegypti and Cx. quinquefasciatus across Mérida City, both in the wet and dry seasons, and represent non-residential development sites that should be strongly considered for inclusion in the local mosquito surveillance and control program.     

The life cycle of Ascocotyle (Phagicola) longa (Digenea: Heterophyidae), a causative agent of fish-borne trematodosis

The complete life cycle of the trematode Ascocotyle (Phagicola) longa (Digenea: Heterophyidae) is elucidated by natural observation validated by experimental infections. The natural first intermediate host of A. (P.) longa, an agent of human heterophyiasis in Brazil, is the cochliopid snail Heleobia australis (new first intermediate host). Metacercariae were found encysted in the body musculature, heart, stomach, liver, kidney, spleen, gonads and mesentery of mullets Mugil liza. Hamsters Mesocricetus auratus were experimentally infected with metacercariae of A. (P.) longa obtained from the mullets, and the adults recovered were used to infect the snails H. australis. Rediae and cercariae of A. (P.) longa are described for the first time. The ultrastructure of the tegument of A. (P.) longa shows a change in spination pattern from the cercaria with single-pointed spines to the metacercaria and adult with multipointed, brush-shaped spines. The life cycle of A. (P.) longa is related to estuaries and coastal lagoons where the recruitment of mugilid juveniles occurs. The high prevalence (100%) of A. (P.) longa encysted in the mullets examined within the urban area of Rio de Janeiro indicates the potentially great public health impact of the consumption of raw mullets.     

Two cases of zoonotic cryptosporidiosis in Spain by the unusual species Cryptosporidium ubiquitum and Cryptosporidium felis

Introduction

Two cases of infection by zoonotic transmission of unusual species of Cryptosporidium were detected in 2010–2011 in Spain (León and Zaragoza).

Materials and methods

Cryptosporidium spp. was detected by microscopic examination of modified Ziehl–Neelsen stained fecal smears. PCR-RFLP of the SSUrDNA gene and sequencing of the amplified fragment confirmed the species.

Results

C. ubiquitum and C. felis were identified in samples from an immunocompetent child and from a HIV-positive adult, respectively.

Conclusions

This is the first report of human infection by C. ubiquitum (cervine) and autochthonous C. felis, identified in Spain.     

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.     

Occurrence of Neospora caninum and Toxoplasma gondii DNA in brain tissue from hoary foxes (Pseudalopex vetulus) in Brazil

The hoary fox (Pseudalopex vetulus) is a wild canid native to Brazil and is commonly found in the semiarid northeastern area living in contact with cattle. The main purpose of this study was to investigate the presence of Neospora caninum and Toxoplasma gondii DNA in hoary foxes, in the state of Paraíba, Brazil. Brain tissue samples were collected from 49 hoary foxes. From the samples, DNA extraction and the polymerase chain reaction (PCR) were performed using specific primers for N. caninum and T. gondii. The prevalences found were 14.3% (7/49) for T. gondii and 12.2% (6/49) for N. caninum. The molecular identities of the amplified products were confirmed by means of the sequencing reaction. This study demonstrated the presence of N. caninum and T. gondii DNA in free-ranging hoary foxes in Brazil for the first time, thus confirming that this species is an intermediate host.     

Monitoring of in vitro susceptibilities and molecular markers of resistance of Plasmodium falciparum isolates from Thai-Myanmar border to chloroquine, quinine, mefloquine and artesunate

Malaria is one of the major causes of morbidity and mortality worldwide. The major factor which has aggravated the situation is the emergence of multidrug resistant Plasmodium falciparum malaria. To successfully deal with the problem, thorough understanding of the molecular bases for reduced parasite sensitivity to existing antimalarial drugs is of considerable importance. The objective of this work was to broaden the insight into the molecular mechanisms of resistance of P. falciparum to quinoline-containing antimalarials and artemisinin derivatives. Polymorphisms of the candidate genes pfmdr1 and pfcrt were investigated in relation to the susceptibility (in vitro sensitivity) of P. falciparum isolates to chloroquine (CQ), mefloquine (MQ), quinine (QN) and the artemisinin derivative - artesunate (AS). A total of 26 P. falciparum isolates were successful cultured. In vitro sensitivity results indicate the increase in susceptibility of P. falciparum strains in Thailand to CQ, while the susceptibility to MQ and QN was markedly declined. The pattern of cross-resistance was observed between MQ vs QN vs AS. Only one point mutation in the pfmdr1 gene, i.e., N86Y was observed with low prevalence of 7.7% (2/26). In contrast, the mutations at positions 76T, 220S, 271E, 326S, 356T and 371I in the pfcrt gene were identified in almost all isolates (25 isolates, 96.2%). The association between polymorphisms of the pfmdr1 and susceptibility of the parasite to MQ and QN was observed (increased susceptibilities to MQ and QN in isolates with mutations). Moreover, the correlation between pfmdr1 gene amplification and susceptibility of the parasite to MQ, QN and AS was observed (decreased susceptibilities to MQ, QN and AS in isolates with increased pfmdr1 copy number).