High reduction of staphylococcal biofilm by aqueous extract from marine sponge-isolated Enterobacter sp.

Staphylococcus aureus and Staphylococcus epidermidis are among the most important bacterial species responsible for biofilm formation on indwelling medical devices, including orthopaedic implants. The increasing resistance to antimicrobials, partly attributed to the ability to form biofilms, is a challenge for the development of new antimicrobial agents. In this study, the cell-free supernatant obtained from sponge-associated Enterobacter strain 84.3 culture inhibited biofilm formation (>65%) and dissociated mature biofilm (>85%) formed by S. aureus and S. epidermidis strains. The culture supernatant was subjected to solvent partitioning and the aqueous extract presented a concentration-dependent antibiofilm activity for each strain with a minimum biofilm eradication concentration (MBEC) ranging from 16 to 256 µg/mL. The effect of the aqueous extract on mature S. aureus biofilm was analyzed by confocal scanning laser microscopy, showing a significant reduction of the biofilm layer as well as diminished interactions among the cells. This extract is not toxic for mammalian cells (L929 cell line). Studies targeting substances with antibiofilm activity gained significant attention in recent years due to difficult-to-treat biofilm infections. Here, sponge-associated Enterobacter 84.3 proved to be a source of substances capable of eradicating staphylococcal biofilm, with potential medical use in the future.

Mycobacterium avium Subspecies paratuberculosis Infection in Zoo Animals: A Review of Susceptibility and Disease Process.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (ParaTB or Johne's disease), a contagious, chronic and typically fatal enteric disease of domestic and non-domestic ruminants. Clinically affected animals present wasting and emaciation. However, MAP can also infect non-ruminant animal species with less specific signs. Zoological gardens harbor various populations of diverse animal species, which are managed on limited space at higher than natural densities. Hence, they are predisposed to endemic trans-species pathogen distribution. Information about the incidence and prevalence of MAP infections in zoological gardens and the resulting potential threat to exotic and endangered species are rare. Due to unclear pathogenesis, chronicity of disease as well as the unknown cross-species accuracy of diagnostic tests, diagnosis and surveillance of MAP and ParaTB is challenging. Differentiation between uninfected shedders of ingested bacteria; subclinically infected individuals; and preclinically diseased animals, which may subsequently develop clinical signs after long incubation periods, is crucial for the interpretation of positive test results in animals and the resulting consequences in their management. This review summarizes published data from the current literature on occurrence of MAP infection and disease in susceptible and affected zoo animal species as well as the applied diagnostic methods and measures. Clinical signs indicative for ParaTB, pathological findings and reports on detection, transmission and epidemiology in zoo animals are included. Furthermore, case reports were re-evaluated for incorporation into accepted consistent terminologies and case definitions.

Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of Streptococcus suis.

Streptococcus (S.) suis is a major cause of economic losses in the pig industry worldwide and is an emerging zoonotic pathogen. One important virulence-associated factor is suilysin (SLY), a toxin that belongs to the family of cholesterol-dependent pore-forming cytolysins (CDC). However, the precise role of SLY in host-pathogen interactions is still unclear. Here, we investigated the susceptibility of different respiratory epithelial cells to SLY, including immortalized cell lines (HEp-2 and NPTr cells), which are frequently used in in vitro studies on S. suis virulence mechanisms, as well as primary porcine respiratory cells, which represent the first line of barrier during S. suis infections. SLY-induced cell damage was determined by measuring the release of lactate dehydrogenase after infection with a virulent S. suis serotype 2 strain, its isogenic SLY-deficient mutant strain, or treatment with the recombinant protein. HEp-2 cells were most susceptible, whereas primary epithelial cells were hardly affected by the toxin. This prompted us to study possible explanations for these differences. We first investigated the binding capacity of SLY using flow cytometry analysis. Since binding and pore-formation of CDC is dependent on the membrane composition, we also determined the cellular cholesterol content of the different cell types using TLC and HPLC. Finally, we examined the ability of those cells to reseal SLY-induced pores using flow cytometry analysis. Our results indicated that the amount of membrane-bound SLY, the cholesterol content of the cells, as well as their resealing capacity all affect the susceptibility of the different cells regarding the effects of SLY. These findings underline the differences of in vitro pathogenicity models and may further help to dissect the biological role of SLY during S. suis infections.

Identification of the Apa protein secreted by Mycobacterium avium subsp. paratuberculosis as a novel fecal biomarker for Johne's disease in cattle.

Paratuberculosis (PTB) or Johne's disease is a chronic intestinal infection of ruminants, caused by Mycobacterium avium subsp. paratuberculosis. The shedding of mycobacteria in the feces starts at the initial stages and increases with disease progression, suggesting that antigens secreted by mycobacteria could be excreted in the feces. Previously, we demonstrated that the alanine and proline-rich antigen (Apa), a secretory antigen of Map, could be detected in the intestine of cows with PTB using a monoclonal antibody. In this study, we verified whether this protein can be found in consistently detectable levels in the feces of cattle with PTB. Feces were obtained from cows with Johne's disease confirmed by laboratory tests, cows with suspected PTB based on seropositivity and from PTB-free control cows. Samples were immunoprecipitated using anti-Apa monoclonal antibody and analyzed by immunoblot. The Apa was detected as a 60/70 kDa doublet band in all samples obtained from animals with laboratory-confirmed disease and in a substantial proportion of seropositive asymptomatic animals, but not in the control samples. Additionally, the antigen was detected in the feces of animals with Johne's disease by ELISA. This study strongly suggests that Apa is a potential fecal biomarker of Johne's disease that could serve for immunodiagnosis.

A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family.

The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease. Using phage-displayed synthetic antibody libraries, we have conducted selections against 12 human IFNα subtypes to explore our ability to obtain fine-specificity antibodies that recognize and antagonize the biological signals induced by a single IFNα subtype. For the first time, we have isolated antibodies that specifically recognize individual IFNα subtypes (IFNα2a/b, IFNα6, IFNα8b and IFNα16) with high affinity that antagonize signaling. Our results show that highly specific antibodies capable of distinguishing between closely related cytokines can be isolated from synthetic libraries and can be used to characterize cytokine abundance and function.

Hydrocarbons preserved in a ~2.7 Ga outcrop sample from the Fortescue Group, Pilbara Craton, Western Australia.

The hydrocarbons preserved in an Archean rock were extracted, and their composition and distribution in consecutive slices from the outside to the inside of the rock were examined. The 2.7 Ga rock was collected from the Fortescue Group in the Pilbara region, Western Australia. The bitumen I (solvent-extracted rock) and bitumen II (solvent-extracted hydrochloric acid-treated rock) fractions have different hydrocarbon compositions. Bitumen I contains only trace amounts of aliphatic hydrocarbons and virtually no aromatic hydrocarbons. In contrast, bitumen II contains abundant aliphatic and aromatic hydrocarbons. The difference seems to reflect the weathering history and preservational environment of the investigated rock. Aliphatic hydrocarbons in bitumen I are considered to be mainly from later hydrocarbon inputs, after initial deposition and burial, and are therefore not indigenous. The lack of aromatic hydrocarbons in bitumen I suggests a severe weathering environment since uplift and exposure of the rock at the Earth's surface in the Cenozoic. On the other hand, the high abundance of aromatic hydrocarbons in bitumen II suggests that bitumen II hydrocarbons have been physically isolated from removal by their encapsulation within carbonate minerals. The richness of aromatic hydrocarbons and the relative scarcity of aliphatic hydrocarbons may reflect the original compositions of organic materials biosynthesised in ancient organisms in the Archean era, or the high thermal maturity of the rock. Cyanobacterial biomarkers were observed in the surficial slices of the rock, which may indicate that endolithic cyanobacteria inhabited the surface outcrop. The distribution of aliphatic and aromatic hydrocarbons implies a high thermal maturity, which is consistent with the lack of any specific biomarkers, such as hopanes and steranes, and the prehnite-pumpellyite facies metamorphic grade.

The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn's disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAPΔmptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAPΔmptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAPΔmptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAPΔmptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host.

Biotechnological potential of sponge-associated bacteria.

As sessile and filter-feeding metazoans, marine sponges represent an ecologically important and highly diverse component of marine benthic communities throughout the world. It has been suggested that marine sponges are hosts to many microorganisms which can constitute up to 40-60% of its biomass. Recently, sponges have attracted a high interest from scientific community because two important factors. First there is the fact that sponges have a wide range of associated bacteria; and, second, they are a rich source of bioactive substances. Since 1950, a number of bioactive substances with various pharmacological functions have been isolated from marine sponges. However, many of these substances were subsequently shown to be actually synthesized by sponge-associated bacteria. Bacteria associated with marine sponges constitute an interesting source of novel bioactive compounds with biotechnological potential such as antimicrobial substances, enzymes and surfactants. In addition, these bacteria may be biofilm forming and can act as bioindicators in bioremediation processes of environmental pollution caused by oil and heavy metals. This review focuses on the biotechnological applications of these microorganisms.

A multidisciplinary approach to diagnose naturally occurring bovine tuberculosis in Brazil / Uma abordagem multidisciplinar para o diagnóstico de tuberculose bovina em um rebanho naturalmente infectado no Brasil

A herd infected naturally with tuberculosis was investigated by different diagnostic methods. Ninety days after a screening test that identified 21 cows as skin test positive, a Comparative Intradermal Tuberculin Test (CITT) was performed in those 21 cows and in 29 other randomly selected skin test negative cows. Milk samples and nasal swabs were collected prior to the CITT for bacteriological culture and PCR, while blood samples were collected for IFN release and antibody responses to MPB70 and MPB83, at three time points post tuberculin injection. Animals positive by CITT were slaughtered and disease confirmation undertaken. Based on the Kappa test, IFN was comparable to the standard tests (culture, PCR and CITT) at all three sampling points. Results from both antibody ELISAs were similar but were not comparable to the standard tests. T-test analysis of the CITT, IFN and ELISAs demonstrated that their performances were not correlated. There is increasing recognition that individually, available diagnostic tests do not detect all infected cattle. Therefore, a comprehensive strategy for the diagnosis of bovine TB should include test results for the detection of both cellular and humoral immune responses where there may be animals at different stages of infection.

Um rebanho bovino naturalmente infectado por tuberculose foi analisado através de diferentes métodos diagnósticos. Um teste intradérmico simples (TIC) identificou 21 animais como positivos. Após 90 dias deste resultado, um teste intradérmico comparativo (TIC) foi aplicado nos 21 animais positivos ao TIS, além de outros 29 animais com resultados prévios negativos escolhidos aleatoriamente. De todos estes animais (50), foram coletadas amostras de leite e secreção nasal para isolamento e identificação de microrganismos por cultura e PCR; amostras de sangue de cada um dos animais foram coletadas para exames de ELISA: produção de Interferon-gama (IFN) e pesquisa de anticorpos frente aos antígenos MPB70 e MPB83. Tais amostras sanguíneas foram coletadas em três diferentes momentos: no dia da execução do TIC e nos dias dia 7 e dia 21 após a execução do TIC. Os animais que foram positivos a este teste foram abatidos; exames de identificação do agente, tais como cultivo e PCR foram realizados post-mortem para confirmação da doença. Baseado na análise Kappa, IFN apresentou resultados estatisticamente comparáveis aos resultados de isolamento e identificação bacteriana por cultura e PCR, além do TIC ao longo de todo o experimento. No entanto, TIC, ELISA e IFN não foram estatisticamente comparáveis. Tais resultados sugeriram que nenhum dos atuais métodos de diagnóstico para tuberculose possibilitou a identificação de todos os animais infectados. Por este motivo, uma estratégia mais abrangente deveria incluir métodos de diagnóstico que pudessem identificar a resposta imune celular e humoral, uma vez que animais de um mesmo rebanho poderiam se encontrar em diferentes estágios da infecção.

Pfatp6 molecular profile of Plasmodium falciparum isolates in the western Brazilian Amazon.

BACKGROUND: Anti-malarial drug resistance has emerged as one of the biggest challenges confronting the worldwide effort to control malaria. The appearance of chloroquine and multi-drug resistance had devastating effects on therapeutic efficacy of former first-line agents. Artemisinin has proven to be an excellent therapeutic alternative to fill the void in chemotherapeutic options left by resistance mechanisms. At the time of introduction, no resistance to artemisinins had been recorded, and artemisinins demonstrated excellent parasite reduction rates. In an attempt to protect artemisinin efficacy, the World Health Organization (WHO) made artemisinin-based combination therapy (ACT) its official first-line treatment recommendation for uncomplicated Plasmodium falciparum in 2006. In Brazil, artemether/lumefantrine became the Brazilian Malaria Control Programme's official treatment recommendation in 2007. The sarco/endoplasmic reticulum Ca2+ - ATPase ortholog of P. falciparum (pfatp6) has been suggested as one of the targets of artemisinins. Consequently, pfatp6 gene polymorphisms are being investigated as markers of artemisinin resistance elsewhere. The goal of this work was to describe the molecular profile of pfatp6 in P. falciparum isolates from different localities in the Amazonas State. METHODS: DNA polymorphisms of the pfatp6 gene in 80 P. falciparum isolates from 11 municipalities of the Amazonas State (Western Brazilian Amazon), before and after the introduction of ACT in the Brazilian anti-malarial guidelines, were analysed by automatic sequencing. Mutations in the pfatp6 gene were searched using Mutation Surveyor v3.25 software. RESULTS: The P. falciparum pfatp6 gene presented polymorphisms at codons 37, 630 and 898. The R37K mutation was found in 16% of the samples, A630S in 32% and I898I in 52%. No S769N mutation, however, was detected in the analysed samples. CONCLUSION: Despite the small number of samples, data presented here provide baseline information about polymorphisms of pfatp6 gene before and after exposure to ACT in a low transmission area, which will help to infer drug selection pressure in this area in the future.

Molecular assessment of UVC radiation-induced DNA damage repair in the stromatolitic halophilic archaeon, Halococcus hamelinensis.

The halophilic archaeon Halococcus hamelinensis was isolated from living stromatolites in Shark Bay, Western Australia, that are known to be exposed to extreme conditions of salinity, desiccation, and UV radiation. Modern stromatolites are considered analogues of very early life on Earth and thus inhabitants of modern stromatolites, and Hcc. hamelinensis in particular, are excellent candidates to examine responses to high UV radiation. This organism was exposed to high dosages (up to 500 J/m(2)) of standard germicidal UVC (254 nm) radiation and overall responses such as survival, thymine-thymine cyclobutane pyrimidine dimer formation, and DNA repair have been assessed. Results show that Hcc. hamelinensis is able to survive high UVC radiation dosages and that intact cells give an increased level of DNA protection over purified DNA. The organism was screened for the bacterial-like nucleotide excision repair (NER) genes uvrA, uvrB, uvrC, as well as for the photolyase phr2 gene. All four genes were discovered and changes in the expression levels of those genes during repair in either light or dark were investigated by means of quantitative Real-Time (qRT) PCR. The data obtained and presented in this study show that the uvrA, uvrB, and uvrC genes were up-regulated during both repair conditions. The photolyase phr2 was not induced during dark repair, yet showed a 20-fold increase during repair in light conditions. The data presented is the first molecular study of different repair mechanisms in the genus Halococcus following exposure to high UVC radiation levels.

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis.

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p<0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.

Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus: methicillin-resistant isolates are detected directly in blood cultures by multiplex PCR.

In this study, we standardized and evaluated a multiplex-PCR methodology using specific primers to identify Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus and their methicillin-resistance directly from blood cultures. Staphylococci clinical isolates (149) and control strains (16) previously identified by conventional methods were used to establish the multiplex PCR protocol. Subsequently, this methodology was evaluated using a fast and cheap DNA extraction protocol from 25 staphylococci positive blood cultures. A wash step of the pellet with 0.1% bovine serum albumin (BSA) solution was performed to reduce PCR inhibitors. Amplicons of 154bp (mecA gene), 271bp (S. haemolyticus mvaA gene) and 108 and 124bp (S. aureus and S. epidermidis species-specific fragments, respectively) were observed. Reliable results were obtained for 100% of the evaluated strains, suggesting that this new multiplex-PCR combined with an appropriate DNA-extraction method could be useful in the laboratory for fast and accurate identification of three staphylococci species and simultaneously their methicillin resistance directly in blood cultures.

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment.

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.

Analysis of intergenic spacer region length polymorphisms to investigate the halophilic archaeal diversity of stromatolites and microbial mats.

Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel intergenic spacer profiles. ARISA-PCR, performed directly on extracted DNA from different sample sites, provided significant insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in this environment.

Detection of Mycobacterium tuberculosis in sputum from Suruí Indian subjects, Brazilian Amazon.

This investigation aimed at the detection of Mycobacterium tuberculosis (MTB) in the sputum of Suruí Indian subjects from Amazonia, Brazil. Polymerase chain reaction analyses were positive for 12 samples, five of which were also culture-positive (N = 147). Four MTB genotypes were identified, one of which showed resistance to rifampicin and isoniazid. The study also highlighted one village complex as of particular importance, considering the relatively high number of tuberculosis cases reported and of MTB isolates obtained.

Detection of Mycobacterium tuberculosis in sputum from Suruí Indian subjects, Brazilian Amazon

This investigation aimed at the detection of Mycobacterium tuberculosis (MTB) in the sputum of Suruí Indian subjects from Amazonia, Brazil. Polymerase chain reaction analyses were positive for12 samples, five of which were also culture-positive (N = 147). Four MTB genotypes were identified, one of which showed resistance to rifampicin and isoniazid. The study also highlighted one village complex as of particular importance, considering the relatively high number of tuberculosis cases reported and of MTB isolates obtained.

Characterization of monocyte-derived dendritic cells maturated with IFN-alpha.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required, but the most potent reagents such as LPS or polyriboinosinic polyribocytidylic acid (Poly I:C) are not approved for clinical use. We tested the ability of type I interferon (IFN) to induce such maturation. We found that 24-h IFN-alpha co-culture of day 7 monocyte-derived DC generated with GM-CSF and IL-4 induces increased numbers of DC positive for CD54 and CD40 together with the co-stimulatory molecule CD80 but not the activation marker CD83. Also, IFN-alpha maturation leads to an increase in IP-10 and MCP-1 chemokine secretion, but only a minor increase in IL-12p40 secretion. In line with this, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials, but not as a single reagent.

Improvement of an in-house ELISA for bovine paratuberculosis serology in Brazil

Paratuberculose (doenca de Johne) se caracteriza por uma enterite crônica de ruminantes, causada por Mycobacterium avium subesp. paratuberculosis (Map). O padrão ouro diagnóstico da infeccão é a cultura e isolamento do microrganismo a partir de tecidos ou fezes. No entanto, seu crescimento demora de três a cinco meses e a excrecão de Map em níveis detectáveis em cultura é irregular e não ocorre durante os primeiros estágios da infeccão, o que diminui seus níveis de sensibilidade. No Brasil, existem poucos relatos de seu isolamento. Os testes imunológicos mais comuns para a identificacão da infeccão são: Fixacão de complemento, imunodifusão em gel de agarose e ensaio imunoenzimático do tipo ELISA. No Brasil, kits comerciais ELISA de diagnóstico são importados e seu uso não está ainda aprovado. O objetivo deste estudo é melhorar um ensaio ELISA-PPA in-house previamente desenvolvido (10) e determinar sua sensibilidade e especificidade. Na primeira etapa deste estudo, as alteracões do protocolo original contribuíram para diminuir as variacões encontradas entre pocos na mesma placa e entre placas em experimentos diferentes, o que demonstrou uma maior estabilidade e reprodutibilidade do teste. Na segunda etapa, um painel de 106 amostras de soros foi testado pelo protocolo modificado do ELISA-PPA para estimar seus índices de sensibilidade e especificidade. O ensaio apresentou sensibilidade de 76,9 per center e especificidade de 70,0 per center. Nosso estudo demonstrou que este ensaio pode ser recomendado como uma valiosa ferramenta para diagnóstico e controle da paratuberculose no Brasil e em outros países em desenvolvimento.

Diagnosis of Enterocytozoon bieneusi by the polymerase chain reaction in archival fixed duodenal tissue.

This study involved ninety five formalin-fixed paraffin-embedded duodenal biopsy specimens retrieved from hospital files that were microscopically observed for the presence of microsporidia. Eleven samples that revealed compatible organisms were analyzed by the polymerase chain reaction (PCR) with four different protocols for the detection of Enterocytozoon bieneusi. Amplicons of the right size were obtained by at least one method for nine samples, remaining two negative ones. We report a PCR methodology that allows the use of archival specimens obtained for traditional pathology.