A metagenomic comparison of endemic viruses from broiler chickens with runting stunting syndrome and from normal birds

Runting-stunting syndrome (RSS) in broiler chickens is an enteric disease that causes significant economic losses to poultry producers worldwide due to elevated feed conversion ratios, decreased body weight during growth, and excessive culling. Of specific interest are the viral agents associated with RSS which have been difficult to fully characterise to date. Past research into the aetiology of RSS has implicated a wide variety of RNA and DNA viruses however, to date, no individual virus has been identified as the main agent of RSS and the current opinion is that it may be caused by a community of viruses, collectively known as the virome. This paper attempts to characterise the viral pathogens associated with 2 – 3 week old RSS-affected and unaffected broiler chickens using next-generation sequencing and comparative metagenomics. Analysis of the viromes identified a total of 20 DNA & RNA viral families, along with 2 unidentified categories, comprised of 31 distinct viral genera and 7 unclassified genera. The most abundant viral families identified in this study were the Astroviridae, Caliciviridae, Picornaviridae, Parvoviridae, Coronaviridae, Siphoviridae, and Myoviridae. This study has identified historically significant viruses associated with the disease such as chicken astrovirus, avian nephritis virus, chicken parvovirus, and chicken calicivirus along with relatively novel viruses such as chicken megrivirus and sicinivirus 1 and will help expand the knowledge related to enteric disease in broiler chickens, provide insights into the viral constituents of a healthy avian gut, and identify a variety of enteric viruses and viral communities appropriate for further study.

A Comparison of Two Brazilian Populations of Culex quinquefasciatus (Say, 1823) from Endemic and Non-endemic Areas to Infection with Wuchereria bancrofti (Cobbold, 1877)

Culex quinquefasciatus is known to be an efficient insect host of Wuchereria bancrofti. In Brazil Cx. quinquefasciatus is widely distributed throughout the country and is often abundant in and around human habitations. In contrast, Bancroftian filariasis is limited to three foci in Brazil. Experiments were undertaken to compare the vector capacities of Cx. quinquefasciatus originating from Maceió (Alagoas), one of the endemic areas of W. bancrofti infection in Brazil, and Belo Horizonte (Minas Gerais), a non endemic area. Laboratory-reared Cx. quinquefasciatus were dissected 20 days after blood feeding on microfilaraemic patients. Survival rates and the number of infective larvae that developed did not differ in female mosquitoes of different origins. Thus both populations of Culex were susceptible to infection with W. bancrofti

Mosquito traps designed to capture Aedes aegypti (Diptera: Culicidae) females: preliminary comparison of Adultrap, MosquiTRAP and backpack aspirator efficiency in a dengue-endemic area of Brazil

In this report, the efficiency of Adultrap under field conditions is compared to a CDC backpack aspirator and to MosquiTRAP. An urban dengue-endemic area of Rio de Janeiro was selected to evaluate the efficiency of mosquito traps in capturing Aedes aegypti females. Adultrap and aspirator captured similar numbers of Ae. aegypti females, with the former showing high specificity to gravid individuals (93.6%). A subsequent mark-release-recapture experiment was conducted to evaluate Adultrap and MosquiTRAP efficiency concomitantly. With a 6.34% recapture rate, MosquiTRAP captured a higher mean number of female Ae. aegypti per trap than Adultrap (Ç2 = 14.26; df = 1; p < 0,05). However, some MosquiTRAPs (28.12%) contained immature Ae. aegypti after 18 days of exposure in the field and could be pointed as an oviposition site for female mosquitoes. Both trapping methods, designed to collect gravid Ae. aegypti females, seem to be efficient, reliable and may aid routine Ae. aegypti surveillance.

A comparison of random sequence reads versus 16S rDNA sequences for estimating the biodiversity of a metagenomic library

The construction of metagenomic libraries has permitted the study of microorganisms resistant to isolation and the analysis of 16S rDNA sequences has been used for over two decades to examine bacterial biodiversity. Here, we show that the analysis of random sequence reads (RSRs) instead of 16S is a suitable shortcut to estimate the biodiversity of a bacterial community from metagenomic libraries. We generated 10,010 RSRs from a metagenomic library of microorganisms found in human faecal samples. Then searched them using the program BLASTN against a prokaryotic sequence database to assign a taxon to each RSR. The results were compared with those obtained by screening and analysing the clones containing 16S rDNA sequences in the whole library. We found that the biodiversity observed by RSR analysis is consistent with that obtained by 16S rDNA. We also show that RSRs are suitable to compare the biodiversity between different metagenomic libraries. RSRs can thus provide a good estimate of the biodiversity of a metagenomic library and, as an alternative to 16S, this approach is both faster and cheaper.

Comparison of the diagnosis of malaria by microscopy, immunochromatography and PCR in endemic areas of Venezuela

Whole blood samples (N = 295) were obtained from different locations in Amazonas and Sucre States, in Venezuela. Malaria was diagnosed by microscopy, OptiMAL™ and polymerase chain reaction (PCR), with Plasmodium vivax, P. falciparum, and P. malariae being detected when possible. We identified 93 infections, 66 of which were caused by P. vivax, 26 by P. falciparum, and 1 was a mixed infection. No infection caused by P. malariae was detected. The sensitivity and specificity of each diagnostic method were high: 95.7 and 97.9% for microscopy, 87.0 and 97.9% for OptiMAL, and 98.0 and 100% for PCR, respectively. Most samples (72.2%) showed more than 5000 parasites/µL blood. The sensitivity of the diagnosis by microscopy and OptiMAL decreased with lower parasitemia. All samples showing disagreement among the methods were reevaluated, but the first result was used for the calculations. Parasites were detected in the 6 false-negative samples by microscopy after the second examination. The mixed infection was only detected by PCR, while the other methods diagnosed it as P. falciparum (microscopy) or P. vivax (OptiMAL) infection. Most of the false results obtained with the OptiMAL strip were related to the P. falciparum-specific band, including 3 species misdiagnoses, which could be related to the test itself or to genetic variation of the Venezuelan strains. The use of the microscopic method for malaria detection is recommended for its low cost but is very difficult to implement in large scale, population-based studies; thus, we report here more efficient methods suitable for this purpose.

Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assays for comprehensive detection of human respiratory viruses

Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI = 0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation. Published by Elsevier B.V.

Metagenomic detection of viral pathogens in Spanish honeybees: co-infection by Aphid Lethal Paralysis, Israel Acute Paralysis and Lake Sinai Viruses

The situation in Europe concerning honeybees has in recent years become increasingly aggravated with steady decline in populations and/or catastrophic winter losses. This has largely been attributed to the occurrence of a variety of known and "unknown", emerging novel diseases. Previous studies have demonstrated that colonies often can harbour more than one pathogen, making identification of etiological agents with classical methods difficult. By employing an unbiased metagenomic approach, which allows the detection of both unexpected and previously unknown infectious agents, the detection of three viruses, Aphid Lethal Paralysis Virus (ALPV), Israel Acute Paralysis Virus (IAPV), and Lake Sinai Virus (LSV), in honeybees from Spain is reported in this article. The existence of a subgroup of ALPV with the ability to infect bees was only recently reported and this is the first identification of such a strain in Europe. Similarly, LSV appear to be a still unclassified group of viruses with unclear impact on colony health and these viruses have not previously been identified outside of the United States. Furthermore, our study also reveals that these bees carried a plant virus, Turnip Ringspot Virus (TuRSV), potentially serving as important vector organisms. Taken together, these results demonstrate the new possibilities opened up by high-throughput sequencing and metagenomic analysis to study emerging new diseases in domestic and wild animal populations, including honeybees.

Mathematical modelling of schistosomiasis japonica: comparison of control strategies in the People's Republic of China

We present the first mathematical model on the transmission dynamics of Schistosoma japonicum. The work extends Barbour's classic model of schistosome transmission. It allows for the mammalian host heterogeneity characteristic of the S. japonicum life cycle, and solves the problem of under-specification of Barbour's model by the use of Chinese data we are collecting on human-bovine transmission in the Poyang Lake area of Jiangxi Province in China. The model predicts that in the lake/marshland areas of the Yangtze River basin: (1) once-early mass chemotherapy of humans is little better than twice-yearly mass chemotherapy in reducing human prevalence. Depending on the heterogeneity of prevalence within the population, targeted treatment of high prevalence groups, with lower overall coverage, can be more effective than mass treatment with higher overall coverage. Treatment confers a short term benefit only, with prevalence rising to endemic levels once chemotherapy programs are stopped (2) depending on the relative contributions of bovines and humans, bovine treatment can benefit humans almost as much as human treatment. Like human treatment, bovine treatment confers a short-term benefit. A combination of human and bovine treatment will dramatically reduce human prevalence and maintains the reduction for a longer period of time than treatment of a single host, although human prevalence rises once treatment ceases; (3) assuming 75% coverage of bovines, a bovine vaccine which acts on worm fecundity must have about 75% efficacy to reduce the reproduction rate below one and ensure mid-term reduction and long-term elimination of the parasite. Such a vaccination program should be accompanied by an initial period of human treatment to instigate a short-term reduction in prevalence, following which the reduction is enhanced by vaccine effects; (4) if the bovine vaccine is only 45% efficacious (the level of current prototype vaccines) it will lower the endemic prevalence, but will not result in elimination. If it is accompanied by an initial period of human treatment and by a 45% improvement in human sanitation or a 30% reduction in contaminated water contact by humans, elimination is then possible. (C) 2002 Elsevier Science B.V. All rights reserved.

Comparison of small mammal prevalence of Leishmania (Leishmania) mexicana in five foci of cutaneous leishmaniasis in the State of Campeche, Mexico

In the Yucatan Peninsula of Mexico, 95% of the human cases of Cutaneous Leishmaniasis are caused by Leishmania (Leishmania) mexicana with an incidence rate of 5.08 per 100,000 inhabitants. Transmission is limited to the winter months (November to March). One study on wild rodents has incriminated Ototylomys phyllotis and Peromyscus yucatanicus as primary reservoirs of L. (L.) mexicana in the focus of La Libertad, Campeche. In the present study, the prevalence of both infection and disease caused by L. (L.) mexicana in small terrestrial mammals were documented during five transmission seasons (1994-2004) in five foci of Leishmaniasis in the state of Campeche. Foci separated by only 100 km, with similar relative abundances of small mammals, were found to differ significantly in their prevalence of both symptoms and infection. Transmission rates and reservoir species seemed to change in space as well as in time which limited the implementation of effective control measures of the disease even in a small endemic area such as the south of the Yucatan Peninsula.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

Comparison of conventional serology and PCR methods for the routine diagnosis of Trypanosoma cruzi infection

Introduction Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas disease, and it is estimated that approximately 5 million people in Brazil are infected with this parasite. This work aimed to compare the current diagnostic methods for Chagas disease, including conventional serological (IFAT and ELISA) and molecular techniques (PCR), to introduce PCR as an auxiliary technique. Methods A total of 106 chagasic patients were evaluated: 88 from endemic areas of Parana, 6 from São Paulo, 3 from Minas Gerais, 3 from Rio Grande do Sul, 1 from Bahia and 5 from the Santa Catarina T. cruzi outbreak. The samples were analyzed by conventional serological methods (IFAT, ELISA), hemoculture and PCR to confirm Chagas disease. Results When IFAT was used to determine antibody levels, the sensitivity was 81.7% for patients with the cardiac form of the disease and 100% for the other clinical forms. In contrast, ELISA showed 84% sensitivity and 100% specificity. The use of serological and molecular techniques and their implications for the diagnosis of Chagas disease in non-endemics area are discussed. Conclusions PCR constitutes an excellent support methodology for the laboratory diagnosis of Chagas disease due to its high sensitivity and specificity.

Cytokines and visceral leishmaniasis: a comparison of plasma cytokine profiles between the clinical forms of visceral leishmaniasis

It is not well established whether cytokine production differs in response to different clinical forms of visceral leishmaniasis (VL). In this work, we performed a cross-sectional study to investigate the plasma levels of cytokines [interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-10 and IL-12] involved in the pathogenesis of VL in 80 subjects from VL endemic areas, including subjects with active VL, subjects with asymptomatic infection, subjects with cured VL and uninfected controls. The patients were recruited by sampling from a referral hospital and by random selection from a population-based cohort study. The results showed significant differences in the plasma concentration of all cytokines between the groups (p < 0.05). Patients with the active disease had higher plasma levels of IL-10, IL-4, INF-γ and TNF-α relative to the other groups and they produced more IL-12 than asymptomatic and cured subjects. Only the IL-2 concentration was higher in the asymptomatic and cured subjects relative to the patients with active disease (p < 0.05). Our results suggest that these cytokines can be used as markers in epidemiological studies conducted in endemic areas to distinguish between different clinical forms of VL. However, their usefulness should be confirmed in investigations conducted in other endemic areas.

A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania) infantum

The aim of the present study was to detect natural infection by Leishmania (Leishmania) infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA), the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA) gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.

Comparison of Immunogenicity in Rhesus Macaques of Transmitted-Founder, HIV-1 Group M Consensus, and Trivalent Mosaic Envelope Vaccines Formulated as a DNA Prime, NYVAC, and Envelope Protein Boost.

An effective human immunodeficiency virus type 1 (HIV-1) vaccine must induce protective antibody responses, as well as CD4(+) and CD8(+) T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-recombinant NYVAC (rNYVAC) vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also, both consensus and mosaic immunogens induced significantly higher gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico-designed centralized immunogens for global HIV-1 vaccine development strategies. IMPORTANCE: There is an increasing appreciation for the importance of vaccine-induced anti-Env antibody responses for preventing HIV-1 acquisition. This nonhuman primate study demonstrates that in silico-designed global HIV-1 immunogens, designed for a human clinical trial, are capable of eliciting not only T lymphocyte responses but also potent anti-Env antibody responses.

Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis

More precise and rapid diagnostic methods for American cutaneous leishmaniasis (ACL) are necessary because of the growing number of cases observed in Brazil, including the northeastern region of the State of São Paulo. We applied PCR to 54 skin or mucosal biopsies from patients with a clinical and/or laboratory diagnosis of ACL using primers 13A and 13B, with positive results being obtained for 82% of the samples. When the PCR results were compared to those of histopathological leishmania detection, PCR showed superior results with 81.5% sensitivity and 95% CI of 68.0-95.1%. The Montenegro skin test (MST) was positive in 88.7% of patients. Since MST cannot be used as a diagnostic tool in endemic areas, the present results strongly suggest the use of PCR for the etiological confirmation of ACL, with emphasis on the mucosal form.