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1.  Lack of association between Toxoplasma gondii infection and hypertensive disorders in pregnancy: a case–control study in a Northern Mexican population 
Parasites & Vectors  2014;7:167.
The outcome of pregnancy is often threatened by hypertension disorders, i.e. eclampsia. Rate of infection with the protozoan parasite Toxoplasma gondii can be as high as 80% in pregnant women, and infection acquired during pregnancy can lead to fetal death. Very little is known about a potential association between infections, i.e. those with Toxoplasma gondii, and hypertensive disorders during pregnancy.
Through a case–control study design, we investigated the presence of anti-Toxoplasma IgG and anti-Toxoplasma IgM antibodies in 146 pregnant women suffering from hypertensive disorders (cases) and 146 age-matched normotensive pregnant women (controls) attending a public hospital in Durango City, Mexico. Obstetric and blood pressure characteristics from cases and controls were also obtained.
Seroprevalence of anti-Toxoplasma IgG antibodies and IgG titers did not differ significantly in controls (8/146; 5.5%) and cases (9/146; 6.2%). Anti-Toxoplasma IgM antibodies were found in 2 (1.2%) controls and none of the cases. Seroprevalence of T. gondii in controls (5.5%) was similar to seroprevalences found in patients with mild preeclampsia (4/27: 14.8%), severe preeclampsia (5/95: 5.3%), eclampsia (0/16: 0%) and HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count) (0/8: 0%) (P = 0.23).
Our results suggest that latent infection with T. gondii is not associated with hypertensive disorders in pregnant women in Northern Mexico. Further studies with larger sample sizes are needed to elucidate the association of infection with T. gondii with hypertensive disorders in pregnancy.
PMCID: PMC3976501
Toxoplasma gondii; Seroprevalence; Preeclampsia; HELLP syndrome; Eclampsia; Infection; Epidemiology
2.  Advantage of using colonic washouts for Blastocystis detection in colorectal cancer patients 
Parasites & Vectors  2014;7:162.
There have been previous studies associating microorganisms to cancer and with our recent findings of Blastocytsis antigen having a higher in vitro proliferation of cancer cells strengthens the suspicion. Collecting faecal samples alone to associate this parasite with cancer may not be accurate due to the phenomenon of irregular shedding and the possible treatment administrated to the cancer patients. Hence, this become the basis to search for an alternate method of sample collection. Colonic washout is an almost complete washed up material from colon and rectum which includes various microorganisms such as Blastocystis and other lodged material within the villi. The detection of parasite in colonic washouts will give a better reflection on the association between Blastocystis and CRC.
Blastocytsis detection was made by in vitro culture method using Jones’ medium, formal ether concentration technique and conventional polymerase chain reaction (PCR) on faecal samples and colonic washouts of 204 CRC patients from colonoscopy procedure. Faecal samples and colonic washouts from 221 normal individuals served as control.
We observed an increased detection of Blastocystis using colonic washouts (n = 53, 12.47%) than faecal samples (n = 26, 6.12%). Eleven faecal samples showed positive results for Blastocystis which were also found in colonic washouts using the PCR technique. This study for the first time showed a significant Blastocystis infection among CRC patients (n = 43, 21.08%) compared to the asymptomatic normal individuals (n = 22, 9.95%). Blastocystis subtype 3 infection was found to be significantly more prevalent (n = 26, 12.75%) compared to other subtypes namely subtype 1: n = 9 (4.41%), subtype 2: n = 1 (0.49%), subtype 5: n = 1 (0.49%) and mixed subtype: n = 6 (2.94%) among the CRC patients.
The study showed that colonic washouts provide a better alternative for Blastocystis detection in CRC patients compared to faecal samples as this prevents treatment regime and the phenomenon of irregular shedding from influencing the detection results obtained from faecal samples.
PMCID: PMC3977685
Colorectal cancer; Blastocystis; Subtypes
3.  Increase in number of helminth species from Dutch red foxes over a 35-year period 
Parasites & Vectors  2014;7:166.
The red fox (Vulpes vulpes) is host to a community of zoonotic and other helminth species. Tracking their community structure and dynamics over decades is one way to monitor the long term risk of parasitic infectious diseases relevant to public and veterinary health.
We identified 17 helminth species from 136 foxes by mucosal scraping, centrifugal sedimentation/flotation and the washing and sieving technique. We applied rarefaction analysis to our samples and compared the resulting curve to the helminth community reported in literature 35 years ago.
Fox helminth species significantly increased in number in the last 35 years (p-value <0.025). Toxascaris leonina, Mesocestoides litteratus, Trichuris vulpis and Angiostrongylus vasorum are four new veterinary-relevant species. The zoonotic fox tapeworm (E. multilocularis) was found outside the previously described endemic regions in the Netherlands.
Helminth fauna in Dutch red foxes increased in biodiversity over the last three decades.
PMCID: PMC3978201  PMID: 24708710
Helminth fauna; Red fox; Biodiversity; Molecular analysis; Echinococcus; Toxocara; Taenia; Alaria
4.  Candidatus Neoehrlichia mikurensis and its co-circulation with Anaplasma phagocytophilum in Ixodes ricinus ticks across ecologically different habitats of Central Europe 
Parasites & Vectors  2014;7:160.
Candidatus Neoehrlichia mikurensis is a newly emerging tick-borne bacterium from the family Anaplasmataceae. Its presence in Ixodes ricinus ticks was reported from various European countries, however, it’s ecology and co-circulation with another member of the same family, Anaplasma phagocytophilum has not been rigorously studied yet.
Candidatus N. mikurensis was detected in all sampling sites. In total, 4.5% of ticks were positive including larvae. The highest positivity was detected in Austria with a prevalence of 23.5%. The probability of Candidatus N. mikurensis occurrence increased with the proportion of ticks infected with Anaplasma phagocytophilum.
A positive association between the occurrences of Candidatus N. mikurensis and A. phagocytophilum indicates that both bacteria share similar ecology for their natural foci in Central Europe.
PMCID: PMC3984398  PMID: 24693971
Candidatus Neoehrlichia mikurensis; Anaplasma phagocytophilum; Ixodes ricinus; Human granulocytic anaplasmosis; Neoehrlichiosis
5.  Ecology of Rhodnius robustus Larrousse, 1927 (Hemiptera, Reduviidae, Triatominae) in Attalea palm trees of the Tapajós River Region (Pará State, Brazilian Amazon) 
Parasites & Vectors  2014;7:154.
The rising number of acute cases of Chagas disease in the State of Pará, reported in the past two decades, has been associated, in part, with the ingestion of juice of local palm tree fruits, mainly açaí berry and bacaba. Near the study area, in Santarém, Pará State, an outbreak of Chagas disease has been notified and investigations suggest the consumption of bacaba juice as the main source of infection with T. cruzi. The purpose of this study is to assess the aspects associated to the ecology of Rhodnius robustus in palm trees of three communities of the Tapajós region, in the State of Pará, Brazil.
Palm trees were cut down and dissected to search for triatomines. DNA from triatomines was extracted to investigate natural infection by Trypanosoma cruzi and T. rangeli. For statistical analyzes, data from infestation of palm trees, as well as the rates of natural infection by T. cruzi and T. rangeli were compared by Chi-square test. Triatomine density values were analyzed by the nonparametric Kruskal Wallis test and then comparisons between each pair of variables were made by the Mann–Whitney test assuming a confidence interval of 95%.
We dissected 136 palm trees, 60 at the end of the rainy period and 76 at the end of the dry period. Seventy-three of them (53.7%) were infested with triatomines and three species were found, namely: Rhodnius robustus, Rhodnius pictipes and Panstrongylus lignarius. We collected 743 triatomines, and R. robustus was predominant (n = 739). The identification of natural infection of the insects by trypanosomatids revealed that 125 triatomines were infected by T. cruzi, 69 by T. rangeli and 14 presented both parasites, indicating the presence of mixed infection in the same vector.
The results suggest that São Tomé is the community with greater density of triatomines and infestation of palm trees; also, it demonstrates the existence of an intense sylvatic cycle in the region, which demands intensive surveillance to prevent human transmission.
PMCID: PMC3974420  PMID: 24690302
Rhodnius robustus; Palm trees; Tapajós Region; Trypanosoma cruzi; Trypanosoma rangeli; Amazon Region; Brazil
6.  Seroprevalence and genetic characterization of Toxoplasma gondii in three species of pet birds in China 
Parasites & Vectors  2014;7:152.
Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is one of the most common zoonosis worldwide, affecting a wide range of warm-blooded mammals and birds worldwide. However, no information on T. gondii infection in pet birds in China is available. Therefore, this study was performed to determine the prevalence of T. gondii infection in pet birds in Gansu province, China.
A total of 687 blood samples were collected from pet birds (Carduelis spinus, Alauda gulgula, Cocothraustes migratorlus) in three representative administrative regions in Gansu province, northwest China between August 2011 and September 2012 T. gondii antibodies were determined using the modified agglutination test (MAT). Genomic DNA was extracted from the brain tissues of seropositive pet birds and T. gondii B1 gene was amplified using a semi-nested PCR.DNA samples giving positive B1 amplification were then genetically characterized using multi-locus PCR-RFLP.
The overall T. gondii seroprevalence was 11.21% (77/687). C. spinus had the highest T. gondii seroprevalence (11.65%), followed by A. arvensis (11.39%) and C. migratorlus (5.26%), these differences were not statistically significant (P > 0.05). Of 77 DNA samples, 8 were positive for the T. gondii B1 gene, four showed complete genotyping results. Only one genotype (the Type II variant: ToxoDB genotype #3) was identified.
The results of the present survey indicated the presence of T. gondii infection in pet birds in Gansu province, China. These data provide base-line information for the execution of control strategies against T. gondii infection in pet birds. To our knowledge, this is the first report documenting the occurrence of T. gondii prevalence and genotype in pet birds in China.
PMCID: PMC3974739  PMID: 24690251
Toxoplasma gondii; Toxoplasmosis; Pet birds; Seroprevalence; Genetic characterization
7.  Implications of low-density microfilariae carriers in Anopheles transmission areas: molecular forms of Anopheles gambiae and Anopheles funestus populations in perspective 
Parasites & Vectors  2014;7:157.
Previous studies have shown a general reduction in annual transmission potential (ATP) of Anopheles species after mass drug administration (MDA) in lymphatic filariasis endemic communities. Whereas results obtained from a monitoring programme after three years of MDA revealed a decrease in ATP of Anopheles funestus this was not the same for An. gambiae s.s. in Ghana. In this study, the ability of these vectors in transmitting Wuchereria bancrofti in nine lymphatic filariasis endemic communities in Gomoa District of Ghana after four rounds of MDA with ivermectin and albendazole was investigated.
After mass screening of inhabitants in these communities, twelve consenting volunteers with different intensities of microfilariae (mf) slept under partly opened mosquito nets as sources of mf blood meal. Hourly collection of mosquitoes and finger-pricked blood were taken from 21.00 to 06.00 hours the following day. For each hour, half of the mosquitoes collected were immediately killed and dissected for mf. The remaining half were maintained up to 13 days for parasite maturation. Parasitaemia and infection rates in the mosquitoes were determined by microscopy. The mosquitoes were identified by microscopy and molecular techniques.
A total of 1,083 participants were screened and the overall parasite prevalence was 1.6% with mf intensities ranging from 0 to 59 per 100 μl and geometric mean intensity of 1.1 mf per ml of blood. Of the 564 mosquitoes collected, 350 (62.1%) were Anopheles spp., from which 310 (88.6%) were An. funestus and 32 (9.1%) An. gambiae. Six anopheline mosquitoes (1.7%) were found infected with L1, but no larva was observed in any of the mosquitoes maintained up to 13 days. Molecular studies showed all An. gambiae s.l. to be An. gambiae s.s., of which 21 (70%) were of the M molecular form.
At low-level parasitaemia after 4 rounds of MDA, there was no recovery of infective stage larvae of W. bancrofti in An. funestus s.l. as well as M and S forms of An. gambiae.
PMCID: PMC3974918  PMID: 24690378
Mass drug administration; Low-density microfilariae carriers; Wuchereria bancrofti; Anopheles gambiae; Anopheles funestus
8.  A multicentric evaluation of the recombinant Leishmania infantum antigen-based immunochromatographic assay for the serodiagnosis of canine visceral leishmaniasis 
Parasites & Vectors  2014;7:136.
Visceral leishmaniasis (VL) is a serious public health challenge in Brazil and dogs are considered to be the main urban reservoir of the causative agent. The culling of animals to control VL in some countries makes the accurate diagnosis of canine VL (CVL) essential. Recombinant antigens rLci1A and rLci2B were selected from a cDNA library of Leishmania infantum amastigotes due to their strong potential as candidates in diagnostic testing for CVL. The present multicentric study aimed to evaluate the sensitivity of a prototype test using these antigens (DPP rLci1A/rLci2B) against 154 sera obtained from symptomatic dogs within three endemic areas of VL in Brazil. The specificity was evaluated using 40 serum samples from negative dogs and dogs infected with other pathogens. Sensitivity and specificity rates of DPP rLci1A/rLci2B prototype were compared to rates from other diagnostic tests currently in use by the Brazilian Ministry of Health, including DPP®LVC, EIE®LVC.
DPP rLci1A/rLci2B prototype offered similar performance to that offered by DPP®LVC rapid test, as follows: sensitivity of 87% (CI 81–91) and 88% (CI 82–93) and specificity of 100% (CI 91–100) and 97% (CI 87–100), respectively for DPP rLci1A/rLci2B and DPP®LVC. When results of these two tests were considered concomitantly, sensitivity increased to 93.5% (CI 89–96).
The recombinant antigens rLci1A and rLci2B represent promising candidates for use in a multi-antigen rapid test for CVL. The inclusion of novel antigens to the DPP rLci1A/rLci2B prototype model could offer additionally enhanced sensitivity to detect animals infected by L. infantum.
PMCID: PMC3972511  PMID: 24684857
Canine visceral leishmaniasis; Serological diagnosis; Recombinant antigen; Rapid test; Multicentric study
9.  Seasonal dynamics of Culicoides (Diptera: Ceratopogonidae) biting midges, potential vectors of African horse sickness and bluetongue viruses in the Niayes area of Senegal 
Parasites & Vectors  2014;7:147.
The African horse sickness epizootic in Senegal in 2007 caused considerable mortality in the equine population and hence major economic losses. The vectors involved in the transmission of this arbovirus have never been studied specifically in Senegal. This first study of the spatial and temporal dynamics of the Culicoides (Diptera: Ceratopogonidae) species, potential vectors of African horse sickness in Senegal, was conducted at five sites (Mbao, Parc Hann, Niague, Pout and Thies) in the Niayes area, which was affected by the outbreak.
Two Onderstepoort light traps were used at each site for three nights of consecutive collection per month over one year to measure the apparent abundance of the Culicoides midges.
In total, 224,665 specimens belonging to at least 24 different species (distributed among 11 groups of species) of the Culicoides genus were captured in 354 individual collections. Culicoides oxystoma, Culicoides kingi, Culicoides imicola, Culicoides enderleini and Culicoides nivosus were the most abundant and most frequent species at the collection sites. Peaks of abundance coincide with the rainy season in September and October.
In addition to C. imicola, considered a major vector for the African horse sickness virus, C. oxystoma may also be involved in the transmission of this virus in Senegal given its abundance in the vicinity of horses and its suspected competence for other arboviruses including bluetongue virus. This study depicted a site-dependent spatial variability in the dynamics of the populations of the five major species in relation to the eco-climatic conditions at each site.
PMCID: PMC3973751  PMID: 24690198
Vector-borne disease; Insect vectors; Spatial and temporal dynamics; Light traps; Culicoides oxystoma; Culicoides imicola; Africa; Arbovirus; Orbivirus; Equids
10.  Autochthonous canine leishmaniasis in Romania: neglected or (re)emerging? 
Parasites & Vectors  2014;7:135.
Canine leishmaniasis is a vector-borne zoonotic disease caused by the protozoan parasite Leishmania infantum. In Romania between 1955 and 2013, no cases of human autochthonous visceral leishmaniasis were reported. Data regarding canine leishmaniasis is similarly scarce. Since the first report of clinical autochthonous canine leishmaniasis in 1935, there were only three sporadic reports of positive dogs all without any clinical signs. Our study reports the first clinical case of autochthonous canine leishmaniasis in the last 80 years, stressing the importance of a targeted surveillance of Leishmania infection, as infected dogs act as the primary reservoir for zoonotic visceral leishmaniasis.
PMCID: PMC3974411  PMID: 24684827
Leishmania infantum; Dog; Romania; Canine leishmaniasis
11.  Expression of microRNA-454 in TGF-β1-stimulated hepatic stellate cells and in mouse livers infected with Schistosoma japonicum 
Parasites & Vectors  2014;7:148.
In the process of hepatic fibrosis, hepatic stellate cells (HSCs) can be activated by many inflammatory cytokines. The transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators. Recently, some studies have also shown that microRNAs (miRNAs) play essential roles in the progress of liver fibrosis by being involved in the differentiation, fat metabolism and ECM production of HSCs.
The expression of miR-454 in LX-2 cells treated with TGF-β1 and in the fibrotic livers with Schistosoma japonicum infection was detected by qRT-PCR. The role of miR-454 on LX-2 cells was then analyzed by Western blot, flow cytometry and luciferase assay.
The results showed that the expression of miR-454 was down-regulated in the TGF-β1-treated LX-2 cells and miR-454 could inhibit the activation of HSCs by directly targeting Smad4. However, we found that miR-454 had no effect on cell cycle and cell proliferation in TGF-β1-treated LX-2. Besides these, miR-454 was found to be regulated in the process of Schistosoma japonicum infection.
All the results suggested that miR-454 could provide a novel therapeutic approach for treating liver fibrosis, especially the liver fibrosis induced by Schistosoma japonicum.
PMCID: PMC3974749  PMID: 24685242
miR-454; TGF-β1; Hepatic stellate cell; Hepatic fibrosis; Schistosoma japonicum
12.  Dengue transmission model by means of viremic adult immuno-competent mouse 
Parasites & Vectors  2014;7:143.
Dengue virus infection manifests in three distinct forms in humans: dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Infection with the virus is a fatal disease; no vaccine is available and prevention depends on interruption of the chain of transmission. The study of dengue viral transmission by mosquitoes is hindered due to the lack of an affordable animal model. In general, immuno-competent mice are used as a simple and inexpensive animal model, but mice are not susceptible to dengue virus infection and therefore viremia will not occur following the inoculation of the virus in such mice. Here, we report a method for creating artificial viremia in immuno-competent mice, and further demonstrate the use of viremic mice to simultaneously infect a large number of Aedes aegypti.
We infected K562 cells with DENV-2 in the presence of an antibody against DENV-4. We then incubated the cells for 2 d before injecting the infected cells into C3H mice. After 5 h incubation, we allowed 100–150 female Aedes aegypti to feed on blood from the mice directly. We collected blood samples from the mice and from randomly selected Ae. aegypti at 2, 6, 12, and 24 h post-blood meal and screened the samples for DENV-2 genome as well as for virus concentration.
Our procedure provided high virus concentrations in the mice for at least 7 h after viral inoculation. We found that 13 out of 14 randomly picked mosquitoes were infected with DENV-2. High concentrations of virus were detected in the mosquitoes until at least 12 h post-infection.
Using the viremic immuno-competent mouse, we show that mass infection of Ae. aegypti is achievable. Compared to other infection techniques using direct inoculation, membrane-feeding, or immuno-deficient/humanized mice, we are confident that this method will provide a simpler and more efficient infection technique.
PMCID: PMC3976050  PMID: 24685121
Aedes aegypti; Dengue virus type 2; Mass-infection; Viremia; Immuno-competent mouse
13.  Host-biting rate and susceptibility of some suspected vectors to Leishmania braziliensis 
Parasites & Vectors  2014;7:139.
American tegumentary leishmaniasis is a serious Brazilian public health problem. This diseases is attributed to seven species of Leishmania, however, the majority of cases are associated with Leishmania braziliensis. Some phlebotomine species have been implicated in the transmission of this parasite, nonetheless only Psychodopygus wellcomei has had its vectorial competence demonstrated. Thus this study sought to assess some parameters related to the vectorial capacity of anthropophilic species of sand fly occurring in São Paulo state: Pintomyia fischeri, Migonemyia migonei Nyssomyia intermedia, Nyssomyia whitmani, Expapillata firmatoi and Psychodopygus ayrozai, under laboratory conditions. These parameters were the duration of the gonotrophic cycle, proportion of females which feed on hamster, the rate of infection by L. braziliensis and the duration of the extrinsic incubation period.
The sandflies were collected in three regions of the São Paulo state: Greater São Paulo and the Mogi Guaçu and Iporanga municipalities. To assess the proportion of engorged females the insects were fed on hamsters to estimate the duration of the gonotrophic cycle. To estimate the susceptibility to infection of each species, their females were fed on hamsters infected with Leishmania braziliensis and dissected to ascertain the localization of the flagellates and estimate the extrinsic incubation period.
Low hamster attractiveness to Ps. ayrozai was observed. A high proportion of engorged females was observed when the hamster had its whole body exposed. The gonotrophic cycle ranged between three and eight days. Mg. migonei, Pi. fischeri, Ny. neivai, Ny. intermedia, Ny. whitmani and Ex.firmatoi presented susceptibility to infection by L. braziliensis. The highest infection rate (34.4%) was observed for Ny. whitmani and the lowest for Ny. intermedia (6.6%). Mg. migonei presented late-stage infection forms on the fifth day after feeding, but in the other species these forms were observed as from the fourth day.
Our results, together with other parameters of their behavior under natural conditions, suggest the potential role of Ex. firmatoi as vector of this parasite and reinforce that of Mg. migonei, Pi. fischeri, Ny. neivai, Ny. intermedia and Ny. whitmani in the areas in which they occur.
PMCID: PMC3976554  PMID: 24684943
Phlebotominae; Host biting rate; Vectorial capacity; Leishmania (Viannia) braziliensis; Vector
14.  Field comparison of circulating antibody assays versus circulating antigen assays for the detection of schistosomiasis japonica in endemic areas of China 
Parasites & Vectors  2014;7:138.
Schistosomiasis remains a serious public health problem in affected countries, and routine, highly sensitive and cost-effective diagnostic methods are lacking. We evaluated two immunodiagnostic techniques for the detection of Schistosoma japonicum infections: circulating antibody and circulating antigen assays.
A total of 1864 individuals (between 6 and 72 years old) residing in five administrative villages in Hubei province were screened by serum examination with an indirect hemagglutination assay (IHA). The positive individuals (titer ≥20 in IHA) were reconfirmed by stool examination with the Kato-Katz method (three slides from a single stool specimen). Samples of good serum quality and a volume above 0.5 ml were selected for further testing with two immunodiagnostic antibody (DDIA and ELISA) and two antigen (ELISA) assays.
The average antibody positive rate in the five villages was 12.7%, while the average parasitological prevalence was 1.50%; 25 of the 28 egg-positive samples were also circulating antigen-positive. Significant differences were observed between the prevalence according to the Kato-Katz method and all three immunodiagnostic antibody assays (P-value <0.0001). Similar differences were observed between the Kato-Katz method and the two immunodiagnostic antigen assays (P-value <0.0001) and between the antigen and antibody assays (P-value <0.0001).
Both circulating antibody and circulating antigen assays had acceptable performance characteristics. Immunodiagnostic techniques to detect circulating antigens have potential to be deployed for schistosomiasis japonica screening in the endemic areas.
PMCID: PMC3978087  PMID: 24684924
Schistosoma japonicum; Circulating antibody; Circulating antigen; China
15.  Laboratory and experimental hut evaluation of a long-lasting insecticide treated blanket for protection against mosquitoes 
Parasites & Vectors  2014;7:129.
Long-lasting insecticide treated blankets (LLIBs) may provide additional protection against malaria where use of long lasting insecticidal nets (LLIN) is low or impractical such as in disaster or emergency situations.
Initial efficacy testing of a new candidate LLIB was carried out at LSHTM and KCMUCo, before and after washing, in cone and ball bioassays and arm-in-cage tests against pyrethroid susceptible Anopheles gambiae. A small scale field trial was conducted using veranda-trap experimental huts in northern Tanzania against wild An. arabiensis and Culex quinquefasciatus mosquitoes. Treatments included unwashed and 5 times washed permethrin treated LLIB and blankets hand-treated with permethrin (ITB), untreated blankets, and a holed unwashed Olyset net.
Cone test mortality was 75% for LLIB when unwashed, but decreased to 32% after 5 washes and <10% after 10 washes. In arm-in-cage tests protection against biting was 100% for LLIBs regardless of the number of washes while reduction in landings was 79% when unwashed, 75% after 5 washes, but declined to 41% after 10 and 33% after 20 washes. In ball bioassays using pyrethroid resistant An. arabiensis, mortality was low in all treatments (<35%) and there was no significant difference in mortality between Olyset net, LLIB or ITB (p > 0.05). Percentage mortality of An. arabiensis in huts with LLIB unwashed (26%) was not statistically different to Olyset net (31%, p = 0.5). The 5 times washed LLIB reduced blood-feeding by 49% which was equivalent to Olyset net (p > 0.086). There was no significant difference in percentage blood-feeding between LLIB and ITB unwashed or 5 times washed (p = 0.147 and p = 0.346 respectively). The 5 times washed LLIB reduced blood-feeding of Culex quinquefasciatus by 40%, although the Olyset provided the greatest protection with 85% inhibition. ELISA analysis of a sub-sample of blood fed mosquitoes showed that not all had fed on humans in the huts, therefore blood-feeding inhibition may have been underestimated.
This trial demonstrated the potential of LLIBs to provide substantial personal protection even against pyrethroid resistant mosquitoes. LLIBs may prove particularly useful where LLINs are unsuitable or net usage is low.
PMCID: PMC3973002  PMID: 24679345
Insecticide treated blankets; Pyrethroids; Anopheles arabiensis; Vector control; Experimental hut; Tanzania
16.  Birds as potential reservoirs of tick-borne pathogens: first evidence of bacteraemia with Rickettsia helvetica 
Parasites & Vectors  2014;7:128.
Birds have long been known as carriers of ticks, but data from the literature are lacking on their role as a reservoir in the epidemiology of certain tick-borne disease-causing agents. Therefore, the aim of this study was to evaluate the presence of three emerging, zoonotic tick-borne pathogens in blood samples and ticks of birds and to assess the impact of feeding location preference and migration distance of bird species on their tick infestation.
Blood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing.
During the spring and autumn bird migrations, 128 blood samples and 140 ticks (Ixodes ricinus, Haemaphysalis concinna and a Hyalomma specimen) were collected from birds belonging to 16 species. The prevalence of tick infestation and the presence of tick species were related to the feeding and migration habits of avian hosts. Birds were shown to be bacteraemic with Rickettsia helvetica and Anaplasma phagocytophilum, but not with Candidatus Neoehrlichia mikurensis. The prevalence of rickettsiae was high (51.4%) in ticks, suggesting that some of them may have acquired their infection from their avian host.
Based on the present results birds are potential reservoirs of both I. ricinus transmitted zoonotic pathogens, R. helvetica and A. phagocytophilum, but their epidemiological role appears to be less important concerning the latter, at least in Central Europe.
PMCID: PMC3976504  PMID: 24679245
Ground feeding birds; Migratory birds; Ticks; Rickettsia helvetica; Anaplasma phagocytophilum; Candidatus Neoehrlichia mikurensis
17.  Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity 
Parasites & Vectors  2014;7:130.
The arthropod-borne Mayaro virus (MAYV) causes ‘Mayaro fever’, a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud.
Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay.
The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity.
B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus.
PMCID: PMC3973022  PMID: 24678592
Bauhinia longifolia; Flavonoids; Quercetin derivatives; Antiviral; MAYV
18.  Evaluation of the efficacy of DDT indoor residual spraying and long-lasting insecticidal nets against insecticide resistant populations of Anopheles arabiensis Patton (Diptera: Culicidae) from Ethiopia using experimental huts 
Parasites & Vectors  2014;7:131.
Indoor Residual Spraying (IRS) and Long-Lasting Insecticidal nets (LLINs) are major malaria vector control tools in Ethiopia. However, recent reports from different parts of the country showed that populations of Anopheles arabiensis, the principal malaria vector, have developed resistance to most families of insecticides recommended for public health use which may compromise the efficacy of both of these key vector control interventions. Thus, this study evaluated the efficacy of DDT IRS and LLINs against resistant populations of An. arabiensis using experimental huts in Asendabo area, southwestern Ethiopia.
The susceptibility status of populations of An. arabiensis was assessed using WHO test kits to DDT, deltamethrin, malathion, lambda-cyhalothrin, fenitrothion and bendiocarb. The efficacy of LLIN (PermaNet® 2.0), was evaluated using the WHO cone bioassay. Moreover, the effect of the observed resistance against malaria vector control interventions (DDT IRS and LLINs) were assessed using experimental huts.
The findings of this study revealed that populations of An. arabiensis were resistant to DDT, deltamethrin, lambda-cyhalothrin and malathion with mortality rates of 1.3%, 18.8%, 36.3% and 72.5%, respectively but susceptible to fenitrothion and bendiocarb with mortality rates of 98.81% and 97.5%, respectively. The bio-efficacy test of LLIN (PermaNet® 2.0) against An. arabiensis revealed that the mosquito population showed moderate knockdown (64%) and mortality (78%). Moreover, mosquito mortalities in DDT sprayed huts and in huts with LLINs were not significantly different (p > 0.05) from their respective controls.
The evaluation of the efficacy of DDT IRS and LLINs using experimental huts showed that both vector control tools had only low to moderate efficacy against An. arabiensis populations from Ethiopia. Despite DDT being replaced by carbamates for IRS, the low efficacy of LLINs against the resistant population of An. arabiensis is still a problem. Thus, there is a need for alternative vector control tools and implementation of appropriate insecticide resistance management strategies as part of integrated vector management by the national malaria control program.
PMCID: PMC3973027  PMID: 24678605
Ethiopia; An. arabiensis; Insecticide resistance; Experimental huts; Long-lasting insecticide treated nets
19.  Comparison of serological and molecular panels for diagnosis of vector-borne diseases in dogs 
Parasites & Vectors  2014;7:127.
Canine vector-borne diseases (CVBD) are caused by a diverse array of pathogens with varying biological behaviors that result in a wide spectrum of clinical presentations and laboratory abnormalities. For many reasons, the diagnosis of canine vector-borne infectious diseases can be challenging for clinicians. The aim of the present study was to compare CVBD serological and molecular testing as the two most common methodologies used for screening healthy dogs or diagnosing sick dogs in which a vector-borne disease is suspected.
We used serological (Anaplasma species, Babesia canis, Bartonella henselae, Bartonella vinsonii subspecies berkhoffii, Borrelia burgdorferi, Ehrlichia canis, and SFG Rickettsia) and molecular assays to assess for exposure to, or infection with, 10 genera of organisms that cause CVBDs (Anaplasma, Babesia, Bartonella, Borrelia, Ehrlichia, Francisella, hemotropic Mycoplasma, Neorickettsia, Rickettsia, and Dirofilaria). Paired serum and EDTA blood samples from 30 clinically healthy dogs (Group I) and from 69 sick dogs suspected of having one or more canine vector-borne diseases (Groups II-IV), were tested in parallel to establish exposure to or infection with the specific CVBDs targeted in this study.
Among all dogs tested (Groups I-IV), the molecular prevalences for individual CVBD pathogens ranged between 23.3 and 39.1%. Similarly, pathogen-specific seroprevalences ranged from 43.3% to 59.4% among healthy and sick dogs (Groups I-IV). Among these representative sample groupings, a panel combining serological and molecular assays run in parallel resulted in a 4-58% increase in the recognition of exposure to or infection with CVBD.
We conclude that serological and PCR assays should be used in parallel to maximize CVBD diagnosis.
PMCID: PMC3972965  PMID: 24670154
Canine vector-borne diseases; Serology; Molecular testing; Diagnostic panel
20.  The oxidative status and inflammatory level of the peripheral blood of rabbits infested with Psoroptes cuniculi 
Parasites & Vectors  2014;7:124.
Psoroptes cuniculi can parasitise the ear canal of the rabbit, and cause the afflicted animals to cease feeding and become severely debilitated, sometimes resulting in death. In this study, we examined the oxidative status and inflammatory level of the peripheral blood of rabbits infested with Psoroptes cuniculi and investigated the pathogenesis of this disease.
A total of 24 rabbits were divided into a healthy rabbit group and two infested rabbit groups. After weighing the rabbits, approximately 5 ml of blood was obtained from each animal. Then, the blood serum was extracted and used to assess the levels of antioxidant enzymes and inflammatory factors.
Compared to the healthy rabbits, the activities of catalase and glutathione-S-transferase and the level of malonyldialdehyde were increased, but the activity of superoxide dismutase was reduced in the infested rabbits. At the same time, a variety of inflammatory cells were activated, and the levels of inflammatory factors such as prostaglandin E2, interleukin-6, interleukin-8 and transforming growth factor-β1 were increased in peripheral blood.
Animal acariasis was associated with immunosuppressive disorders and inflammatory reaction. These results advance our understanding of the pathogenesis of Psoroptes cuniculi infestation in rabbits and can help guide the effectual treatment of this disease in clinics.
PMCID: PMC3972963  PMID: 24667000
Rabbits; Psoroptes cuniculi; Oxidative status; Superoxide dismutase; Inflammatory factor
21.  Parasitic antigens alter macrophage polarization during Schistosoma japonicum infection in mice 
Parasites & Vectors  2014;7:122.
Schistosome eggs are trapped in host liver and elicit severe hepatic granulomatous inflammation, which can lead to periportal fibrosis, portal hypertension, hemorrhage, or even death in the host. It was reported that the macrophage plays an important role in host immune responses to schistosome infection. Nitric oxide (NO) produced by classically activated macrophages (M1 macrophages) is cytotoxic to schistosomula and can prevent hepatic schistosomal fibrosis, while arginase-1 (Arg-1) expressed by alternatively activated macrophages (M2 macrophages) promotes hepatic schistosomal fibrosis. However, the dynamics of macrophage polarization, as well as the possible factors that regulate macrophage polarization, during schistosome infection remain unclear.
We first analyzed M1 and M2-phenotypic markers of peritoneal macrophages from mice infected with Schistosoma japonicum (S. japonicum) at indicated time points using flow cytometry (FCM) analysis and real-time PCR. Then we treated peritoneal macrophages from normal mice with schistosome worm antigen (SWA) or schistosome soluble egg antigen (SEA) and determined M1 and M2-phenotypic markers, in order to identify macrophage polarization in responding to schistosomal antigens.
In this study, we showed that macrophages were preferentially differentiated into the M1 subtype during the acute stage of S. japonicum infection. However, the level of M1 macrophages decreased and M2 macrophages significantly increased during the chronic stage of infection. Furthermore, we showed that SWA favors the generation of M1 macrophages, whereas SEA preferentially promotes M2-polarized phenotype.
These findings not only reveal the parasite antigen-driven dynamic changes in macrophage polarization, but also suggest that manipulation of macrophage polarization may be of therapeutic benefit in controlling excessive hepatic granulomas and fibrosis in the host with schistosomiasis.
PMCID: PMC3975460  PMID: 24666892
Schistosoma japonicum; Liver fibrosis; Macrophage polarization; Schistosomal antigen
22.  Bacterial and protozoal agents of feline vector-borne diseases in domestic and stray cats from southern Portugal 
Parasites & Vectors  2014;7:115.
Feline vector-borne diseases (FVBD) have emerged in recent years, showing a wider geographic distribution and increased global prevalence. In addition to their veterinary importance, domestic cats play a central role in the transmission cycles of some FVBD agents by acting as reservoirs and sentinels, a circumstance that requires a One Health approach. The aim of the present work was to molecularly detect feline vector-borne bacteria and protozoa with veterinary and zoonotic importance, and to assess associated risk factors in cats from southern Portugal.
Six hundred and forty-nine cats (320 domestic and 329 stray), from veterinary medical centres and animal shelters in southern Portugal, were studied. Anaplasma spp./Ehrlichia spp., Babesia spp., Bartonella spp., Borrelia burgdorferi sensu lato, Hepatozoon spp. and Leishmania spp. infections were evaluated by polymerase chain reaction (PCR) in blood samples.
One hundred and ninety-four (29.9%) cats were PCR-positive to at least one of the tested genera or complex of FVBD agents. Sixty-four (9.9%) cats were positive to Leishmania spp., 56 (8.6%) to Hepatozoon spp., 43 (6.6%) to Babesia spp., 35 (5.4%) to Anaplasma spp./Ehrlichia spp., 19 (2.9%) to Bartonella spp. and 14 (2.2%) to B. burgdorferi s.l. Thirty-three (5.1%) cats were positive to two (n = 29) or three (n = 4) genera/complex. Babesia vogeli, Bartonella clarridgeiae, Bartonella henselae, Ehrlichia canis, Hepatozoon felis and Leishmania infantum were identified by DNA sequencing.
The occurrence of FVBD agents in southern Portugal, some of them with zoonotic character, emphasizes the need to alert the veterinary community, owners and public health authorities for the risk of infection. Control measures should be implemented to prevent the infection of cats, other vertebrate hosts and people.
PMCID: PMC3972989  PMID: 24655431
Cats; Feline vector-borne diseases; Bacteria; Protozoa; Portugal
23.  Serological and molecular survey of Leishmania infection in dogs from Luanda, Angola 
Parasites & Vectors  2014;7:114.
Canine leishmaniosis (CanL) due to Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania infection.
One hundred-and-three dogs presented to a veterinary medical centre in Luanda were serologically and molecularly assessed for Leishmania with the direct agglutination test (DAT) and polymerase chain reaction (PCR). Two dogs were seropositive, with DAT titres of 800 and ≥6400; the latter was also found to be PCR-positive and confirmed to be infected with L. infantum by DNA sequence analysis. No other dog was found to be PCR-positive. The first dog had been imported from Portugal, but the latter had never left Angola (neither had its parents), strongly suggesting an autochthonous infection.
Although other cases of CanL have previously been described in the country, this is the first reported study of canine Leishmania infection at the population level, as well as the first report on the molecular characterization of L. infantum in dogs from Angola.
PMCID: PMC3972991  PMID: 24655415
Dogs; Luanda; Angola; Direct agglutination test; Polymerase chain reaction; Leishmania infantum; Canine leishmaniosis
24.  Serological and molecular prevalence of selected canine vector borne pathogens in blood donor candidates, clinically healthy volunteers, and stray dogs in North Carolina 
Parasites & Vectors  2014;7:116.
Canine vector borne diseases (CVBDs) comprise illnesses caused by a spectrum of pathogens that are transmitted by arthropod vectors. Some dogs have persistent infections without apparent clinical, hematological or biochemical abnormalities, whereas other dogs develop acute illnesses, persistent subclinical infections, or chronic debilitating diseases. The primary objective of this study was to screen healthy dogs for serological and molecular evidence of regionally important CVBDs.
Clinically healthy dogs (n = 118), comprising three different groups: Gp I blood donor candidates (n = 47), Gp II healthy dog volunteers (n = 50), and Gp III stray dogs (n = 21) were included in the study. Serum and ethylenediamine tetraacetic acid (EDTA) anti-coagulated blood specimens collected from each dog were tested for CVBD pathogens.
Of the 118 dogs tested, 97 (82%) dogs had been exposed to or were infected with one or more CVBD pathogens. By IFA testing, 9% of Gp I, 42% of Gp II and 19% of Gp III dogs were seroreactive to one or more CVBD pathogens. Using the SNAP 4DX® assay, Gp I dogs were seronegative for Anaplasma spp., Ehrlichia spp., and B. burgdorferi (Lyme disease) antibodies and D. immitis antigen. In Gp II, 8 dogs were Ehrlichia spp. seroreactive, 2 were infected with D. immitis and 1 was B. burgdorferi (Lyme disease) seroreactive. In Gp III, 6 dogs were infected with D. immitis and 4 were Ehrlichia spp. seroreactive. Using the BAPGM diagnostic platform, Bartonella DNA was PCR amplified and sequenced from 19% of Gp I, 20% of Gp II and 10% of Gp III dogs. Using PCR and DNA sequencing, 6% of Gps I and II and 19% of Gp III dogs were infected with other CVBD pathogens.
The development and validation of specific diagnostic testing modalities has facilitated more accurate detection of CVBDs. Once identified, exposure to vectors should be limited and flea and tick prevention enforced.
PMCID: PMC3972993  PMID: 24655461
Healthy dogs; CVBDs; Co-infection; Blood donors
25.  Management of canine leishmaniosis in endemic SW European regions: a questionnaire-based multinational survey 
Parasites & Vectors  2014;7:110.
Canine leishmaniosis (CanL) caused by Leishmania infantum is a widespread endemic disease in SW Europe. This study was designed to determine how veterinarians clinically manage CanL in this region by analysing information collected in a questionnaire completed by local veterinarians working in clinics in France, Portugal, Greece, Spain, Italy and Slovenia.
Over the period 2004–2011, a questionnaire on CanL was sent to 12,546 small animal clinics located in the six countries surveyed. The questionnaire with 10 items comprising open and closed questions sought to obtain comparable data regarding the main clinical manifestations of CanL, the diagnostic methods used, the treatment regimens selected, recommended preventive measures and awareness of the important public health implications of CanL.
The data collected reflect similarities in the clinical manifestations reported although there was some variation in the concurrent diseases described, and wide variation in the clinical management of CanL among the countries examined in terms of dosing regimens, therapeutic agents and the criteria used to diagnose CanL. Most veterinarians properly informed dog owners about the preventive measures available and about the zoonotic implications of CanL.
This survey describes the current situation in SW endemic countries in Europe regarding the clinical management of CanL. The data collected reveal a need to unify criteria from evidence-based medicine to determine and similarly apply the best diagnostic and treatment methods available for this disease in the different countries.
PMCID: PMC3974741  PMID: 24656172
Canine leishmaniosis; Survey; Clinical signs; Diagnosis; Therapy; Prevention; Europe

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