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.     

Polarization of macrophages induced by Toxoplasma gondii and its impact on abnormal pregnancy in rats

Toxoplasma gondii infection is the leading cause of fetal intrauterine growth retardation among the five kinds of pathogens termed as TORCH, including Toxoplasma, Rubella virus, Cytomegalo virus, herpes virus and others during pregnancy. Pathogens infect the fetus through the placenta. T. gondii infection may result in congenital toxoplasmosis, miscarriage, stillbirth, and preemie, and increase pregnancy complications. Adaptive immune response induced by T. gondii infection stimulates T cells and macrophages to produce high levels of cytokines. Physiologically, the microenvironment of pregnancy was Th2-dominant. Here we set up a pregnant Sprague-Dawley rat model, and reported the polarization of macrophages induced by genotype Chinese 1 strain (Wh6) of Toxoplasma, and its adverse impact on pregnancy. The results showed that Wh6 infection pre- or in-gestation both led to abnormal pregnancy outcomes. Peritoneal macrophages in pre-gestation infection were polarized toward classically activated macrophages (M1), while in-gestation infection drove macrophages to polarize toward M2 activation. The Th2-dominant immune response in pregnant rat somewhat inhibits the excessive bias of the macrophages toward M1, and partially, toward M2. Infection of pre- and in-gestation may alter the physiological immune microenvironment in pregnant rats, giving rise to abnormal pregnancy outcomes.     

Epitope analysis,expression and protection of SAG5A vaccine against Toxoplasma gondii

Bioinformatics approaches were used to identify B-cell epitopes and T-cell epitopes on SAG5A protein. Compared to SAG1, SAG5A with good B-cell epitopes and T-cell epitopes had a potentiality to become a more successful vaccine against Toxoplasma gondii. Thereafter, SAG5A DNA vaccine was constructed successfully and was injected into mice with peptide to evaluate the immunoprotection. Compared to the control groups, the vaccine (DNA/peptide) could induce more effective cellular and humoral immune responses in immunized mice. Furthermore, a significant reduction of brain cyst was detected in the mice vaccinated with peptide (732 ± 160), pSAG5A (815 ± 197), or pSAG5A/peptide (436 ± 174) compared by the mice injected by PBS (1260 ± 241) or pEGFP-C1 (1350 ± 268). The number of cysts in brains was 35% reduced in the mice immunized with DNA/peptide than in the control mice treated by PBS. The results indicated that the DNA vaccine encoding SAG5A significantly induced immune responses and enhanced protection against cysts of PRU strain, especially with the help of peptide.     

Seroprevalence and risk factors of Toxoplasma gondii infection in domestic sika deer (Cervus nippon) in northeastern China

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, which can infect warm-blooded animals and humans. A serological survey was undertaken to examine the seroprevalence and risk factors associated with T. gondii infection in sika deer in northeastern China. 114 (13.46%, 95% CI 11.16–15.76) out of 847 serum samples were positive to T. gondii by modi?ed agglutination test (MAT) at a 1:25 cut-off, with titers of 1:25 in 44, 1:50 in 32, 1:100 in 17, 1:500 in 11, 1:1500 or higher in 10. These samples were collected between November 2012 and October 2013 from Inner Mongolia, Jilin and Heilongjiang provinces in China. However, statistically signi?cant differences were not observed between T. gondii seroprevalence and genders or regions of sika deer in the logistic regression analysis (P > 0.05) and left out of the ?nal model. Seroprevalence of T. gondii infection in male sika deer was 14.07% (95% CI 11.14–17.01), slightly higher than that in the female (12.38%) (95% CI 8.69–16.06) and seroprevalence of T. gondii infection in Harbin, Changchun city, Jilin city and Chifeng city were 12.02% (95% CI 7.60–16.44), 15.51% (95% CI 11.52–19.50), 12.27% (95% CI 7.23–17.31) and 12.50% (95% CI 7.38–17.63), respectively. Seasons of sampling were considered as main risk factors associated with T. gondii infection, autumn (15.32%) were more than two times (OR = 1.98, 95% CI = 1.18–3.33, P = 0.01) at risk of acquiring T. gondii infection compared to winter (8.37%). Our results indicated a widespread exposure to T. gondii among sika deer in China. To our knowledge, this is the first report of T. gondii seroprevalence in sika deer in China.     

Matrix metalloproteinase-2 and -9 lead to fibronectin degradation in astroglia infected with Toxoplasma gondii

Toxoplasma gondii is a zoonotic parasite and its infection in human can induce toxoplasmic encephalitis in immune disorders. In this study, astroglia were infected with the TS-4 strain of T. gondii tachyzoite in vitro to investigate the changes of matrix metalloproteinase (MMP)-2, MMP-9 and their substrate fibronectin. MMP-2 and MMP-9 were significantly increased at 1 h, 6 h and 12 h post-infection (PI) in the cell homogenates, and increased at 6 h, 12 h, 24 h and 48 h PI in the cell-cultured supernatants. Fibronectin degradation also occurred at the same time points. In addition, immunocytochemistry showed that MMP-2 and MMP-9 localized in the cytoplasm, and confocal scanning laser microscopy revealed co-labeled patterns of MMP-2 and MMP-9 with fibronectin. MMP-2 and MMP-9 interacted with fibronectin, respectively. These results suggest that MMP-2 and MMP-9 induction from astroglia may contribute to extracellular matrix (ECM) degradation occurring in toxoplasmosis. Thus, we hypothesize that MMP-2 and MMP-9 cleave fibronectin and may contribute to the astroglia reaction and leukocyte migration to the sites of T. gondii replication during toxoplasmic encephalitis.     

Perfil serológico en gestantes extranjeras frente a VIH,VHB, VHC,virus de la rubéola, Toxoplasma gondii,Treponema pallidum, y Trypanosoma cruzi

Introduction

The increase in immigration is changing the prevalence of mother to child infectious diseases. Our aim is to determine the serological profile of foreign pregnant women against these infections.

Methods

A retrospective cross sectional study was performed in a tertiary hospital from Madrid between August 2007 and October 2008. The seroprevalence against HIV, HBV, HCV, rubeola, T. gondii, T. pallidum and T. cruzi was determined in every pregnant immigrant, as well as in a representative group of Spanish pregnant women.

Results

A total of 2526 immigrant and 157 Spanish pregnant women were studied. None of the Spanish and 0.5% of the foreigners showed antibodies against HIV; 18.9% of them were Sub-Saharan women. Antigen HBs was detected in 2% of the immigrant women and in 1.1% of the Spanish women. Asian women had the highest rate of type B Hepatitis (10.9%). There was 0.9% of type C Hepatitis among the immigrants and 1% among the Spanish. Within the cases with RPR ≥ 1/8, 1.6% were immigrants, most of whom were Latin American. Thirty-one per cent of the immigrants showed antibodies against T. gondii (37.5% from Central America, 2.5% from the Far East). More than 95% of the Spanish women had antibodies against Rubella, this being lower in the rest of the areas (75.5% in Sub-Saharan Africa). T. cruzi infection was detected in 12.1% of the Bolivian women studied.

Conclusion

The prevalence of mother-to-child transmitted infections depends on the origin of pregnant women. Knowledge of these differences may lead to improved control these diseases.     

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.     

Efficacy of azithromycin in a murine toxoplasmosis model,employing a Toxoplasma gondii strain from Turkey

A murine toxoplasmosis model with Balb/C mice was used to investigate the therapeutic and prophylactic efficacy of azithromycin in a native strain of Toxoplasma gondii. Initially, seven groups--four studies and three controls--were established and 10(3) tachyzoites of this native strain of T. gondii were injected intraperitoneally to the mice in groups 1, 2, 3, 4 and 7. Azithromycin was given to groups 1-4 at different times of infection orally between 100 and 300 mg/kg/day for 10 days. Azithromycin was found to be effective at 200 mg/kg/day and above in the prophylaxis, at 250 mg/kg/day and above in the treatment of toxoplasmosis. These results suggest that azithromycin is effective in the prophylaxis and early infection of a highly virulent strain of T. gondii, and it doubled the survival time in the late infection. Azithromycin could be an alternative treatment regimen for human toxoplasmosis, if supported by further clinical investigations.     

Antidermatophytic and antileishmanial activities of essential oils from Lippia gracilis Schauer genotypes

Lippia gracilis, popularly known in Brazil as ‘alecrim-de-tabuleiro’, is used for many purposes, especially antimicrobial and antiseptic activities. The leaves of three L. gracilis genotypes, including LGRA-106, LGRA-109 and LGRA-110 were collected from the Active Germplasm Bank located in the “Campus Rural da UFS” research farm at the São Cristóvão country, Sergipe State, Brazil. The essential oils were obtained from leaves of L. gracilis plants by hydrodistillation. Chemical analysis of the essential oils was performed by gas chromatography–mass spectrometry (GC–MS). The susceptibility of Trichophyton rubrum strains, MYA3108 and TruMDR2, to the two L. gracilis genotypes (LGRA-106 and LGRA-109) essential oils was determined by the serial microdilution method. Leishmanicidal activity of essential oil from LGRA-106 and LGRA-110 was assayed by tetrazolium-dye (MTT) colorimetric method. The oxygenated monoterpene thymol was the main component of the essential oil from genotype LGRA-106, while Carvacrol was more abundant in LGRA-109 and LGRA-110. The concentrations of LGRA-106 and LGRA-109 essential oils that completely eliminate the fungi were determined and these concentrations were similar to those observed for fluconazole, a common antifungal drug. Among the genotype tested, LGRA-106 essential oil exhibited the best fungicidal activity at 46.87 μg mL⁻¹. Regarding to leishmanicidal activity, the IC50, for LGRA-106 and LGRA-110, was 86.32 and 77.26 μg mL⁻¹, respectively. The results showed that L. gracilis essential oil, rich in thymol and thymol itself presented best antidermatophytic activity, while the best leishmanicidal activity was obtained with essential oil from genotype rich in Carvacrol and Carvacrol itself.     

Identification and quantification of the Acanthamoeba species and genotypes from reservoirs in Taiwan by molecular techniques

The occurrence of Acanthamoeba was investigated from 21 main reservoirs of Taiwan with 12 (57.1%) testing positive. Analysis of the 18S rRNA gene PCR product was performed in order to identify the Acanthamoeba isolates. Acanthamoeba spp. concentrations were determined according to TaqMan real-time qPCR. Acanthamoeba genotypes of all isolates were identified T4. The species were categorized to Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba hatchetti. The concentration of Acanthamoeba spp. in detected positive reservoir water samples was in the range of 3.0–1.8 × 10³ cells/L. These results highlight the importance of Acanthamoeba in reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.     

Direct genotyping of animal and human isolates of Toxoplasma gondii from Colombia (South America)

Genetic analysis of the SAG2 locus was performed to determine the prevalence of the main genotypes of Toxoplasma gondii (SAG2 types I, II, and III) associated with humans, cats, birds and guinea pig toxoplasmosis in Colombia. This typing was directly performed on clinical samples and autopsy material from human or animals. A total of 50 from 146 samples were positive by specific B1 Toxoplasma PCR assay and then were analyzed by restriction fragment length polymorphism in PCR-amplified SAG2 products. Characterization of the SAG2 gene was successful in 33 (66%) of the samples. Genotyping indicated that 31 (93.9%) were SAG2 type I, 1 was SAG2 type III and 1 was atypical. In birds and cats all samples were SAG2 type I. Results support a predominance of the Toxoplasma SAG2 type I circulating in human and animals in South America.     

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.     

Temporal trends and epidemiological aspects of ciguatera in French Polynesia: a 10-year analysis

OBJECTIVES: The purpose of this study was to report the temporal trends of the incidence of ciguatera poisoning from 1992 to 2001 in French Polynesia. METHODS: This retrospective study analysed 7842 cases of ciguatera disease recorded over a period of 10 years. RESULTS: The annual incidence varied from 26.3 to 41.9 per 10,000 person-years. An analysis of cases grouped by archipelago revealed differences in incidences (P < 0.0001) with the most remote archipelagos having the highest incidences. A detailed analysis on a sub-sample of recorded cases for which clinical information was available (n = 1824) confirmed the neurological and gastrointestinal nature of this seafood poisoning. CONCLUSION: The incidence of ciguatera poisoning appeared relatively stable during the 10 years of the study period. However, the gradient of remoteness observed suggests an adaptation of management of ciguatera disease to each archipelago.     

Trypanosoma cruzi congenital transmission in wild bats

Trypanosoma cruzi congenital transmission in wild bats (Molossus molossus), associated with infected Rhodnius prolixus in a natural habitat from a rural locality in western Venezuela, is reported. T. cruzi blood circulating trypomastigotes in a pregnant bat were detected by parasitological methods. Polymerase chain reaction (PCR) assays carried out in samples from the heart and the fetus of the same infected female, revealed the presence of T. cruzi-specific DNA in both of the tissues, demonstrating transmission of the infection from the mother to the offspring. Eighty percent of the captured bats and 100% of the examined fetuses from pregnant specimens were shown to be infected by T. cruzi, indicating that M. molossus is a very susceptible species for this parasite, and that T. cruzi congenital transmission is a common phenomenon in nature. To our knowledge, this seems to be the first report on congenital T. cruzi transmission in wild bats in Venezuela. The circulation of T. cruzi lineage I in the study area was demonstrated by typing the isolates from bats and triatomine bugs captured in the same habitat. The potential epidemiological implication of these findings in areas where Chagas disease is endemic is discussed.     

New Cryptosporidium parvum subtypes of IIa subfamily in dairy calves from Brazil

Bovine cryptosporidiosis is mainly caused by four distinct species: Cryptosporidium parvum, C. bovis, C. ryanae and C. andersoni. The first, C. parvum, is a major concern in livestock causing economic losses, in addition to public health impact because of its zoonotic characteristics. The present study aimed to determine the occurrence of different species and subtypes of Cryptosporidium using molecular techniques. A total of 143 fecal samples were collected from calves from three dairy farms located in the state of Rio de Janeiro, Brazil. Saturated sugar centrifugal flotation method was used for the microscopic evaluation of the samples. Among these samples, 19.6% (28) were positive by microscopy, and 82.1% (23) of these 28 samples had their diagnosis confirmed by PCR using 18S as gene target. After sequencing, three species of Cryptosporidium were found to infect calves in different age groups. In pre-weaning phase (<2 months), 10% (3/30) of the calves were infected with C. parvum, whereas 14.2% (16/113) of post-weaning calves (≥2 months) were observed to be infected with C. andersoni and 1.8% (2/113) by C. ryanae with the latter diagnosed for the first time in the state of Rio de Janeiro. Those samples identified as C. parvum were further characterized at the GP60 locus, and PCR products were cloned. Eight different subtypes (IIaA20G2R1, IIaA20G2R2, IIaA19G2R1, IIaA19G2R2, IIaA18G1R1, IIaA18G2R2, IIaA16G3R2 and IIaA14G2R2) of C. parvum were identified, all belonging to the IIa family subtype, which is considered of high zoonotic potential. The subtypes mentioned above have not yet been detected in Brazilian cattle, and four of these subtypes (IIaA20G2R2, IIaA19G2R2, IIaA18G2R2 and IIaA14G2R2) had not been diagnosed elsewhere in calves until this study.     

Trypanosoma rangeli isolates of bats from Central Brazil: Genotyping and phylogenetic analysis enable description of a new lineage using spliced-leader gene sequences

Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T. rangeli, T. dionisii, T. cruzimarinkellei and T. cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus planirostris were identified as T. rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T. rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T. rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T. rangeli and T. cruzi.     

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).     

vacA genotypes of Helicobacter pylori in the oral cavity and stomach of patients with chronic gastritis and gastric ulcer

Introduction

Helicobacter pylori adheres to various components of the human saliva. Therefore, the objective of this research was to simultaneously detect H. pylori in saliva and in gastric biopsy, and to determine the agreement between the vacA genotypes in both saliva and gastric biopsy.

Materials and methods

A total of 162 patients with chronic gastritis and 34 with gastric ulcer were studied, and saliva and biopsy samples were collected from each patient. H. pylori DNA was detected by conventional PCR and nested PCR was used for vacA genotyping.

Results

In 24% of the patients (47/196) H. pylori DNA was found in saliva and in biopsy; 52.5% (103/196) were saliva^negative/biopsy^positive and 6.6% (13/196) were saliva^positive/biopsy^negative. In either or both H. pylori vacAs1m1 or s1m2 genotypes were detected in saliva in 41.5% of the patients with chronic gastritis. Forty-seven percent had >1 genotype, and the s1m1/s1m2 combination was found in 36% of them. H. pylori vacAs1m1 and s1m2 were also found in the saliva and biopsy of patients with gastric ulcer. The genotypes found in saliva and biopsy of the same patient had 51.1% agreement. In 27.6% of the 47 patients saliva^positive/biopsy^positive two genotypes were found in saliva, and one or both in the stomach.

Conclusions

The s1m1/s1m2 genotypes, alone or together, are found simultaneously in saliva and gastric biopsy of the same patient. These results suggest that H. pylori reaches the oral cavity by various ways, and that saliva can be the transmitting and re-infecting vector.