Research Items (140)
- Nov 2018
Many human diseases, including metabolic, immune, and central nervous system disorders, as well as several types of cancers, are the consequence of an important alteration in lipid-related metabolic biomolecules. Although recognized that one of the most important metabolic hallmarks of cancer cells is deregulation of lipid metabolism, the multiple complex signaling pathways are poorly understood yet. Thus, in this research, novel nanoconjugates made of ZnS quantum dots (QDs) were directly synthesized in aqueous media using phosphoethanolamine (PEA) as the capping ligand, which is an important biomolecule naturally present in cells for de novo biosynthesis of fatty acids and phospholipids involved in the cell structure (e.g., membrane), differentiation, and cancer growth. These QD-PEA bio-nanoconjugates were characterized by spectroscopical and morphological techniques. The results demonstrated that fluorescent ZnS nanocrystalline QDs were produced with uniform spherical morphology and estimated sizes of 3.3 ± 0.6 nm. These nanoconjugates indicated core-shell colloidal nanostructures (ZnS QD-PEA) with the hydrodynamic diameter (HD) of 26.0 ± 3.5 nm and ζ-potential centered at -30.0 ± 4.5 mV. The cell viability response using mitochondrial activity assay in vitroconfirmed no cytotoxicity at several concentrations of PEA (biomolecule) and the ZnS-PEA nanoconjugates. Moreover, these nanoconjugates effectively behaved as fluorescent nanomarkers for tracking the endocytic pathways of cancer cells using confocal laser scanning microscopy bioimaging. Hence, these results proved that biofunctionalized ZnS-PEA nanoprobes offer prospective tools for cellular bioimaging with encouraging forecast for future applications as active fluorescent biomarker conjugates in metabolic-related cancer research.
- Oct 2018
Mosquito‐borne arboviruses are a major public health concern worldwide and are responsible for emerging and re‐emerging diseases. Taken together, the arboviruses have a strong impact on public health and are the most common causes of equine encephalitis. In‐depth diagnostic investigation of equine viral encephalitis is of utmost importance for the epidemiological surveillance and control of this disease. Regarding neurological disorders in equids, in April‐May 2018, at least 12 cases of equid mortality with acute neurological signs were reported in six farms from Espirito Santo state, Brazil. To investigate the etiological agent of this neurological disease outbreak, central nervous system (CNS) fragments from two horses and two donkeys were submitted for virologic diagnosis. Rabies, equine herpesvirus‐1, and arbovirus‐associated encephalomyelitis were investigated using differential diagnosis techniques. West Nile virus (WNV) was detected by nested RT‐PCR in CNS fragments from each of the four animals in the study and confirmed by nucleotide sequencing. This is the first case of neurological disease in equids confirmed to be associated with WNV infection in Brazil. This finding unveils a new and urgent field of research and the need to understand the epidemiological and clinical characteristics of the disease and the risk to public health. This article is protected by copyright. All rights reserved.
Bela Vista Biological Sanctuary (RBV) is a protected area of Itaipu Binacional, a hydroelectric power company located on the border of Brazil and Paraguay. A captive population of Brazilian dwarf brocket deer (Mazama nana, Cervidae, Artiodactyla) is maintained for conservation purposes. Despite the reproductive success of the animals, outbreaks of a fatal hemorrhagic disease have been registered over the years, compromising conservation eﬀorts. In order to identify the etiological agents of these hemorrhagic diseases, 32 captive Brazilian dwarf brockets were sampled to investigate bluetongue virus (BTV), epizootic hemorrhagic disease (EHD), and adenovirus hemorrhagic disease (AHD), in 2015. Only one deer (1/32; 3.12%) was seropositive for BTV. After this survey, ﬁve animals died in the early autumn of 2015 and 2016, again presenting clinical signs of hemorrhagic disease. Using RT-qPCR, RT-PCR and DNA sequencing, ﬁve BTV serotypes (3, 14, 18, 19, and 22) were identiﬁed in blood and tissues collected during necropsies. These BTV serotypes had not been previously described or isolated in Brazil, either in wild or domestic ruminants. Additionally, diﬀerential diagnosis was performed for EHD and AHD, but all samples were negative for both diseases. The multiple distinct BTV serotypes identiﬁed in these outbreaks resulted in a high lethality (100%) of Brazilian dwarf brockets and indicated that various BTV serotypes are circulating in the area.
- Jul 2018
Several studies have shown the occurrence of poxvirus infections associated with exanthematic lesions in cattle from many Brazilian states. Coinfection between viruses belonging to 2 genera, Orthopoxvirus (OPXV) and Parapoxvirus (PPV), was already identified from the lesions of affected cows and humans. The DNA and infectious viral particles of Vaccinia virus, an OPXV, have been detected in milk of naturally and experimentally infected cows. However, to date no reports have described the detection of Pseudocowpox virus, a PPV, in milk. Thus, we investigated the presence of PPV and OPXV in milk samples obtained from dairy cows from a Brazilian region with exanthematic disease outbreaks. From 2011 to 2014, 6 dairy farms with exanthematic disease outbreaks involving dairy cows, calves, and humans were visited. Twelve crusts of cows' teat lesions and 60 milk samples were collected. The crusts and milk samples were analyzed by PCR to detect OPXV or PPV DNA. According to the analyzed crusts, we detected PPV infection in 4 of the 6 visited farms, from which we investigated the PPV contamination in milk. From the 40 milk samples tested, PPV DNA was detected in 12 samples. Of these milk samples, 8 were positive for both PPV and OPXV. This is the first report of PPV DNA detection in milk samples from affected cows, indicating that the virus may be present in milk and potentially contaminating dairy products associated or not with OPXV. In addition to the lesions caused by direct contact, the presence of 2 or more poxvirus species in milk showed that the effect of zoonotic exanthematic diseases on public health and animal husbandry is relevant and cannot be overlooked.
- May 2018
Polymer-drug conjugation is an attractive approach for target delivering insoluble and highly toxic drugs to tumor sites to overcome the side-effects caused by cancer chemotherapy. In this study we designed and synthesized novel polymer-drug-peptide conjugates for improved specificity on targeting cancer cells. Chemically modified polysaccharide, carboxymethylcellulose (CMC), was conjugated with doxorubicin (DOX) anticancer drug by amide bonds and dually biofunctionalized with integrin-target receptor tripeptide (RGD) and L-arginine (R) as cell-penetrating amino acid for synergistic targeting and enhancing internalization by cancer cells. These bioconjugates were tested as prodrugs against bone, breast and brain cancer cell lines (SAOS, MCF7 and U87) and a normal cell line (HEK 293T, reference). The physicochemical characterization showed the formation of amide bonds between carboxylates (-RCOO-) from CMC biopolymer and amino groups (-NH2) from DOX and peptides (RGD or R). Moreover, these polymer-drug-peptide bioconjugates formed nanoparticulate colloidal structures and behaved as “smart” drug delivery systems (DDS) promoting remarkable reduction of the cytotoxicity towards normal cells (HEK 293T) while retaining high killing activity against cancer cells. Based on cell viability bioassays, DNA-staining and confocal laser microscopy, this effect was assigned to the association of physicochemical aspects with the difference of the endocytic pathways and the drug release rates in live cells caused by the biofunctionalization of the macromolecule-drug systems with RGD and L-arginine. In addition, chick chorioallantoic membrane (CAM) assay was performed as an in vivo xenograft model test, which endorsed the in vitro results of anticancer activities of these polymer-drug systems. Thus, prodrug nanocarriers based on CMC-DOX-peptide bioconjugates were developed for simultaneously integrin-targeting and high killing efficacy against cancer cells, while preserving healthy cells with promising perspectives in cancer chemotherapy.
- Mar 2018
Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51–CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.
Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions in the teats of dairy cows and the hands of milkers and is an important public health issue. Severe VACV-induced lesions in the teats and udder of cows and buffaloes could lead to mastitis and other secondary infections, thereby reducing productivity and resulting in economic losses to the dairy industry. In Brazil, BV re-emerged in the late 1990s and is now endemic in most of the Brazilian territory. In the last 15 years, much effort has been made to know more about this disease and its epidemiology, etiologic agents, and interactions with the host and the environment. In this review, we describe the known dynamics of VACV infection in cattle and the viral shedding routes, as well as the relevance of BV for animal and public health.
Since 1999 Vaccinia virus (VACV) outbreaks involving bovines and humans have been reported in Brazil; this zoonosis is known as Bovine Vaccinia (BV) and is mainly an occupational disease of milkers. It was only in 2008 (and then again in 2011 and 2014) however, that VACV was found causing natural infections in Brazilian equids. These reports involved only equids, no infected humans or bovines were identified, and the sources of infections remain unknown up to date. The peculiarities of Equine Vaccinia outbreaks (e.g., absence of human infection), the frequently shared environments, and fomites by equids and bovines in Brazilian farms and the remaining gaps in BV epidemiology incited a question over OPV serological status of equids in Brazil. For this report, sera from 621 equids-representing different species, ages, sexes and locations of origin within Minas Gerais State, southeast Brazil-were examined for the presence of anti-Orthopoxvirus (OPV) antibodies. Only 74 of these were sampled during an Equine Vaccinia outbreak, meaning some of these specific animals presented typical lesions of OPV infections. The majority of sera, however, were sampled from animals without typical signs of OPV infection and during the absence of reported Bovine or Equine Vaccinia outbreaks. Results suggest the circulation of VACV among equids of southeast Brazil even prior to the time of the first VACV outbreak in 2008. There is a correlation of OPVs outbreaks among bovines and equids although many gaps remain to our understanding of its nature. The data obtained may even be carefully associated to recent discussion over OPVs history. Moreover, data is available to improve the knowledge and instigate new researches regarding OPVs circulation in Brazil and worldwide.
Bluetongue (BT) is a vector-borne viral disease caused by the Bluetongue virus (BTV), an Orbivirus from the Reoviridae family, affecting domestic and wild ruminants. BTV circulation in Brazil was first reported in 1978, and several serological surveys indicate that the virus is widespread, although with varied prevalence. In 2014, BT outbreaks affected sheep flocks in Rio Grande do Sul state, causing significant mortality (18.4%; 91/495) in BTV-infected sheep. In total, seven farms were monitored, and one or two sheep from each farm that died due to clinical signs of BT were necropsied. Apathy, pyrexia, anorexia, tachycardia, respiratory, and digestive disorders were noted. Additionally, an abortion was recorded in one of the monitored farms. The main gross lesions observed were pulmonary edema, anterior-ventral pulmonary consolidation, muscular necrosis in the esophagus and in the ventral serratus muscle, and hemorrhagic lesions in the heart. The blood and tissue samples were tested for BTV RNA detection by RT-qPCR targeting the segment 10. Positive samples were used for viral isolation. The isolated BTVs were typed by conventional RT-PCR targeting the segment 2 of the 26 BTV serotypes, followed by sequencing analysis. BTV-1, BTV-4 and BTV-17 were identified in the analyzed samples. Double or triple BTV co-infections with these serotypes were detected. We report the occurrence of BT outbreaks related to BTV-1, BTV-4 and BTV-17 infections and co-infections causing clinical signs in sheep flocks in Southern Brazil, with significant mortality and lethality rates.
- Aug 2017
Seneca Valley virus (SVV) is the etiological agent of vesicular disease in pigs, clinically indistinguishable of classical viral vesicular infections, including foot-and-mouth disease. The first outbreaks of SVV infection in Brazil were reported in 2014. However, it was not known whether the virus was circulating in Brazilian pig herds before this year. This study is a retrospective serological investigation of porcine health status to SVV in Brazil. Serum samples (n = 594) were grouped in before (2007–2013, n = 347) and after (2014–2016, n = 247) SVV outbreaks in Brazil. Twenty-three pig herds were analyzed, of which 19 and 4 were sampled before and after the beginning of SVV outbreaks, respectively. Two herds sampled after 2014 presented animals with SVV-associated clinical manifestations, while the other two housed asymptomatic pigs. Anti-SVV antibodies were evaluated by virus neutralization test. The results demonstrated that pig herds of different Brazilian geographical regions and distinct pig categories were negative to anti-SVV antibodies in sera obtained before 2014. Antibodies to SVV were detected only in serum samples obtained after 2014, particularly in herds with the presence of pigs with SVV-clinical signs. These results present robust serological evidence that the SVV was not present in the major Brazilian pig producing regions prior to 2014.
- Jul 2017
Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 10² pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d.
- Jun 2017
Vaccinia virus (VACV) is the agent of bovine vaccinia (BV), an emerging zoonosis that causes exanthematic lesions on the teats of dairy cows and on the hands of milkers. The virus has been detected in the milk of naturally infected cows. The objective of this study was to investigate and quantify VACV DNA as well as the presence of infectious virus particles in samples of cheese curd, cheese whey and pasteurized milk produced using milk from cows experimentally inoculated with VACV-GP2, a Brazilian isolate of VACV (VACV-BR). VACV DNA was detected in samples of cheese and pasteurized milk at different time points, even after the resolution of the typical lesions caused by VACV, which occurred after 22 days post-infection (dpi), on average. Moreover, it was possible to detect infectious viral particles in cheese samples on alternate days until 27 dpi. The presence of both VACV DNA and infectious viral particles in cheese samples throughout the clinical course of BV and even after the disappearance of the typical clinical signs of disease draws attention to the risk associated with consumption of the cheese. Furthermore, VACV-contaminated milk and cheese may represent an occupational risk to cheesemakers who often manipulate milk and cheese curd without wearing gloves.
O plasma sanguíneo em pó (PSP), produto natural de indústria frigorífica, tem mostrado efeitos benéficos sobre o crescimento e desempenho de leitões desmamados precocemente. Atualmente, embora o circovírus suíno 2 (PCV2) tenha grande importância para a suinocultura, não há informações sobre o impacto do uso de PSP e a resposta imune ao PCV2 em infecções naturais. Este trabalho avaliou diferentes níveis de inclusão de PSP em dietas de leitões e as cargas virais de PCV2 correspondentes. Quatro níveis de inclusão de PSP foram testados em dois períodos consecutivos: 0, 2, 4 ou 6% durante o período 1 (14 aos 28 dias de idade) e 1, 2 ou 3% de PSP durante o período 2 (29 a 42 dias de idade). No período 3 (42 aos 56 dias de idade), todos os leitões foram alimentados com dieta isenta de PSP. Amostras de soro foram coletadas semanalmente e testadas para anticorpos anti-PCV2 e carga de DNA de PCV2. As concentrações de 6% e 3% de PSP fornecidas nas rações durante o período 1 e 2, respectivamente, influenciaram na carga viral de PCV2 de suínos naturalmente infectados.
- Apr 2017
Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that mainly affects lactating cows and dairy farm milkers. The epidemiological role(s) of other cattle categories such as dry cows, bulls, and heifers in BV remains unclear. This study was performed to investigate VACV in affected dairy cattle herds and perifocal farms during an outbreak in Brazil. Crusts from lesions of cows' teats were collected from all farms with BV outbreaks. Milk, feces, blood, and serum were collected from symptomatic and asymptomatic lactating cows. Blood and serum were also sampled from other cattle categories (calves, heifers, dry cows, and bulls). The samples were tested for VACV by PCR, and to confirm VACV viability, VACV-positive samples were inoculated in BSC-40 cells and stained using immunoperoxidase. Neutralizing antibodies were investigated using plaque reduction neutralization test. Viral DNA was detected in milk, blood, and feces samples of symptomatic and asymptomatic dairy cows and in blood samples from other cattle categories on farms with and without confirmed BV outbreak. In affected farms, viable virus was identified in feces and milk samples from lactating cows and in blood samples from asymptomatic dry cows. Viable VACV was also identified in feces from lactating cows and one bull's blood sample from perifocal farms. Neutralizing antibodies were detected in 81.6% of the herds affected by BV and in 53.8% of the herds on perifocal farms. The presented data indicate a potential source of viral dissemination, which contributes to the persistence and spread of VACV in the environment.
- Mar 2017
Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions on the teats of dairy cows and the milkers’ hands. Since 1999, due to the occurrence of many BV outbreaks in dairy farms across all Brazilian regions, there is a need to improve the control and prevention measures of the disease. Vaccination is one of the major tools to prevent viral diseases, and it could be an alternative for BV prevention. The main objective of this study was the development of vaccine formulations against BV using the inactivated VACV strain GP2 as antigen combined with different adjuvants. Potency tests were performed in mice, which were vaccinated with two doses at a 21-day interval, and then challenged with the vaccine homologous virus. VACV strain GP2 inactivated by beta-propiolactone (BPL) in association with adjuvants was effective in inducing a humoral immune response against VACV, as measured by neutralizing antibody (NA) titers, and was variable depending on the adjuvant used in each vaccine formulation. The vaccine formulation containing aluminum hydroxide (AH) associated with saponin as adjuvant induced the production of high NA titers in all vaccinated mice, giving 100% protection in Balb/c murine model after challenge with homologous virus.
The complete genome sequence of Bluetongue virus (BTV) serotype 17 strain 17/BRA/2014/73, isolated from a sheep in Brazil in 2014, is reported here. All segments clustered with western topotype strains and indicated reassortment events with other BTV from the Americas. The strain 17/BRA/2014/73 represents a novel reference strain for BTV-17 from South America.
- Oct 2016
The nanotoxicity of Cd-containing quantum dots (QDs) for biomedical applications is very controversial and not completely understood. In this study, we evaluated the cytotoxicity of surface-biofunctionalized CdS QDs with chitosan directly synthesized via aqueous route at room temperature. These core-shell CdS-chitosan nanoconjugates showed different degrees of cytotoxic responses using MTT cell proliferation assay toward three human cell cultures, human osteosarcoma cell line (SAOS), non-Hodgkin's B cell lymphoma (Toledo), and human embryonic kidney cell line (HEK293T), under three exposure times (1, 3, and 5 days) and three colloidal concentrations (10 nM, 50 nM, and 100 nM). The results clearly demonstrated that the CdS QDs, regardless to the fact that they were coated with a biocompatible aminopolysaccharide shell, induced a severe dose- and time-dependent inhibition of cell viability. In addition, the HEK293T and SAOS cell lines showed much more sensitive response compared to Toledo, which indicated that the cytotoxicity was also cell-type dependent. The exceptional resistance of Toledo cells to toxic effects of CdS nanoconjugates even at severe test conditions was assigned to specific role of B-lineage cells of the immune defense system. Remarkably, no conclusive evidence of toxicity of CdS nanoconjugates was observed in vivo using intravenous injections of CdS nanoconjugates in BALB/c mouse animal models for 30 days, but localized fluorescence was detected in ex-vivo liver tissue samples. Therefore, these results prove that there is no guarantee of “risk-free” use of CdS nanoconjugates for in vivo applications, even when functionalized with biopolymer ligands, as they can pose an excessive threat due to unpredicted and uncorrelated responses under in vitro and in vivo biological assays with highly toxic cadmium ions.
In January 2013, an outbreak of Bluetongue (BT) affecting a Lacaune sheep flock occurred in Vassouras, Rio de Janeiro state, Brazil. From March to August 2013, blood samples collection and clinical examination were performed monthly, in order to monitor the epidemiological profile of Bluetongue virus (BTV) circulation and clinical disease in the flock. Agar gel immunodiffusion (AGID) and reverse transcription polymerase chain reaction (RT‑PCR) targeting BTV segment 10 were used as diagnostic assays. Additionally, insect trapping was conducted in the farm from May to July 2013. The flock serological prevalence to BTV was 80% since the first month of monitoring, with a variation in the serological rate depending on the sheep age categories. The number of susceptible lambs increased with time, probably due to the decrease of passive immunity. Viral RNA was detected in blood samples, demonstrating viral circulation, prolonged viraemia, and potential source for virus transmission in the region, even in a dry and cool season. The presence of Culicoides pusillus and Culicoides insignis was confirmed in the farm. The emergence of this outbreak in a Brazilian endemic area for BTV emphasises an urgent need of animal surveillance and BTV epidemiological studies. © 2016, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ’G. Caporale’. All rights reserved.
Alexandra AP Mansur,1 Herman S Mansur,1 Sandhra M de Carvalho,1–3 Zélia IP Lobato,2 Maria IMC Guedes,2 Maria F Leite3 1Center of Nanoscience, Nanotechnology, and Innovation-CeNano2I, Department of Metallurgical and Materials Engineering, 2Department of Preventive Veterinary Medicine, Veterinary School, 3Department of Physiology and Biophysics, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Abstract: Herein, for the first time, we demonstrated that novel biofunctionalized semiconductor nanomaterials made of Cd-containing fluorescent quantum dot nanoconjugates with the surface capped by an aminopolysaccharide are not biologically safe for clinical applications. Conversely, the ZnS-based nanoconjugates proved to be noncytotoxic, considering all the parameters investigated. The results of in vitro cytotoxicity were remarkably dependent on the chemical composition of quantum dot (CdS or ZnS), the nature of the cell (human cancerous and embryonic types), and the concentration and time period of exposure to these nanomaterials, caused by the effects of Cd2+ on the complex nanotoxicity pathways involved in cellular uptake. Unexpectedly, no decisive evidence of nanotoxicity of CdS and ZnS conjugates was observed in vivo using intravenous injections in BALB/c mice for 30 days, with minor localized fluorescence detected in liver tissue specimens. Therefore, these results proved that CdS nanoconjugates could pose an excessive threat for clinical applications due to unpredicted and uncorrelated in vitro and in vivo responses caused by highly toxic cadmium ions at biointerfaces. On the contrary, ZnS nanoconjugates proved that the “safe by design” concept used in this research (ie, biocompatible core–shell nanostructures) could benefit a plethora of applications in nanomedicine and oncology. Keywords: fluorescent nanoparticles, semiconductor quantum dots, nanotoxicity, bionanoconjugates, nanoprobes
- Jun 2016
Bioengineered hybrids are emerging as a new class of nanomaterials consisting of a biopolymer and inorganic semiconductors used in biomedical and environmental applications. The aim of the present work was to determine the cytocompatibility of novel water-soluble Bi2S3 quantum dots (QDs) functionalized with chitosan and O-carboxymethyl chitosan (CMC) as capping ligands using an eco-friendly aqueous process at room temperature. These hybrid nanocomposites were tested for cytocompatibility using a 3-(4,5-dimethylthiazol-2yl) 2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay with cultured human osteosarcoma cells (SAOS), human embryonic kidney cells (HEK293T cells) and a LIVE/DEAD® viability-cytotoxicity assay. The results of the in vitro assays demonstrated that the CMC and chitosan-based nanohybrids were not cytotoxic and exhibited suitable cell viability responses. However, despite the "safe by design" approach used in this research, we have proved that the impact of the size, surface charge and biofunctionalization of the nanohybrids on cytotoxicity was cell type-dependent due to complex mechanisms. Thus, these novel bionanocomposites offer promising prospects for potential biomedical and pharmaceutical applications as fluorescent nanoprobes.
South America (SA) has the ideal climatic conditions for occurrence of Bluetongue virus (BTV) and other orbiviruses. Based on serological evidence of BTV circulation, the virus is widespread across SA. However, little knowledge has been acquired about BTV origin and distribution, and circulation of specific serotypes is almost non‑existent. The first barrier to a better understanding of Bluetongue (BT) in SA concerns its recognition in the field, as most infections of seropositive ruminants are unapparent in endemic areas. There are few reports on BTV isolation in SA, many of them from viraemic asymptomatic animals. Among the known competent BTV vectors, Culicoides insignis is the most abundant species in SA. However, information about biological characteristics and competence of various other Culicoides species described in SA is missing. The few reports on BT outbreaks lead to an underestimation of the disease impact on the continent economy. To overcome these major gaps, it is necessary to: improve diagnostic structure and disease recognition in the field; identify BTV serotypes and their distribution in different SA areas; and to study the biology and epidemiology of Culicoides. Furthermore, it is important to have a surveillance system for BT, as well as contingency plans for possible outbreaks in SA.
RESUMO Objetivou-se avaliar o percentual de casos suspeitos de circovirose à histologia que realmente estavam relacionados a esta doença, baseado na imunoistoquímica, e avaliar a intensidade, freqüência e distribuição de antígenos virais em lesões histológicas de diferentes órgãos. Foram utilizados 133 casos da rotina com lesões histológicas sugestivas de circovirose suína em diferentes órgãos. A técnica de imunoistoquímica para PCV2 foi padronizada utilizando anticorpo policlonal produzido em suíno com diluições variando de 1:100 a 1:2500. Todas as amostras dos 133 casos foram submetidas a esta coloração imunoistoquímica e foi realizada avaliação da freqüência de casos positivos e negativos para PCV2. Também foi avaliada a freqüência de marcação de cada órgão submetido à imunoistoquímica e a intensidade de marcação dos órgãos nesta técnica. Oitenta e dois casos (61,7%) apresentaram marcação positiva para PCV2. Linfonodos foram os órgãos mais freqüentemente acometidos pela infecção, 66 casos (56,9%), seguidos por intestinos (55,5%), rins (46,6%), pulmões (37,5%), baço (34,4%) e fígado (26,6%). Apesar de ter sido pequeno o número de casos com amostras de intestino, chama a atenção a elevada freqüência de acometimento deste órgão. Demonstrou-se claramente a necessidade da confirmação da suspeita histológica pela técnica de imunoistoquímica. PALAVRAS-CHAVE: Suíno, circovírus tipo 2, circovirose, imunoistoquímica. ABSTRACT RETROSPECTIVE STUDY OF PORCINE CIRCOVIRUS 2 ASSOCIATED DISEASES DIAGNOSED BY IMMUNOHISTOCHEMICAL TECHNIQUE. The aim of this study was to evaluate the accuracy of routine hematoxylin and eosin histology stain to diagnosis circovirosis, compared to immunohistochemistry, and to evaluate the intensity and frequency of PCV2 infection in different organs. One hundred thirty-three routine histologic cases with circovirosis-suggestive lesions in different organs were used. A porcine anti-PCV2 polyclonal antibody was used for optimizing an indirect immunohistochemistry (IHC) technique with dilutions ranging from 1:100 to 1:2500. This IHC stain was performed in all studied cases for detection of positive and negative samples. Intensity of infection was also determined in each organ. Eight-two cases were positive for PCV2 by IHC. Infection was most often detected in lymph nodes, 66 cases (56.9%), followed by intestines (55.5%), kidneys (46.6%), lungs (37.5%), spleen (34.4%) and liver (26.6%). Despite of the reduced number of intestine samples (n = 9), the high frequency of infection in this organ was surprising. The results clearly demonstrated the need for IHC in order to confirm routine histologic cases with suspicion of circovirosis.
Background Lawsonia intracellularis is the etiologic agent of proliferative enteropathy, which causes diarrhea in several animal species, including swine. Serology can be used both to determine the prevalence of antibodies against a specific pathogen in a herd and to obtain the serological profile, which is used to determine the dynamics of infection in the herd. The objective of this study was to determine the serological profile and seroprevalence of anti-L. intracellularis antibodies in swine herds from intensive production regions of Minas Gerais, Brazil, and to identify the risk factors related to the herd-level seropositivity. Results A total of 2999 serum samples were collected for this cross–sectional study in the four major regions of intensive swine production in Minas Gerais, Brazil. To obtain better estimates and increase the external validity of the seroprevalence, the sample data were weighted based on the pig population of each herd, the stratum in which the herd was classified and the swine population of the region where each herd was located. A questionnaire was used to identify potential risk factors related to this herd-level seropositivity. The overall weighted prevalence in Minas Gerais was 34.7 % (95 % confidence interval: 32.12 - 37.20 %), and there was no significant difference among the sampled regions, with the seroprevalence rates ranging between 32.06 and 37.66 %. Finishing pigs were the most prevalent among the sampled categories. Among the evaluated risk factors, “cleaning before disinfecting” had a negative impact in the seroprevalence (p < 0.05) and was considered a protective factor. Conclusions The anti-L. intracellularis antibodies were detected in all of the investigated herds in Minas Gerais, which indicated a wide distribution of the agent in the state. The predominant serological profile was consistent with the dynamics of infection previously observed in pig herds in other countries with similar antimicrobial usage, in which the nursery pigs usually show the lowest seroprevalence and the finishing pigs exhibit the highest. Herds that adopt the practice of “cleaning before disinfection” can decrease their L. intracellularis antibody seropositivity.
Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects lactating cows and milkers. VACV DNA and infectious particles have been detected in milk of naturally infected cows. However, the period and pattern of VACV shedding in milk is unknown, as is whether the presence of VACV in milk is due to a localized or a systemic infection. To address those questions, eight lactating cows were inoculated with VACV in previously scarified teats. The experiment was divided in two phases. In Phase 1, milk samples were collected daily for 33 days, and in Phase 2, four animals from the first phase were immunosuppressed. In both phases, milk was collected with a sterile catheter on even days and by hand milking on odd days. All animals showed typical BV lesions in the inoculated teats. All milk samples were subjected to nested polymerase chain reaction (PCR) and real-time quantitative PCR to detect VACV DNA. PCR-positive samples were subjected to virus isolation. VACV DNA was intermittently detected in milk in both phases and infectious viral particles could be detected only in phase 2, on the 69th, 73rd, 74th, 77th, 79th, and 81st days postinfection. Despite the possibility of propagation of VACV through milk, it is known that milk continues to be drawn and marketed normally during outbreaks of the disease. The detection of both VACV DNA and infectious particles in milk samples draws attention to the potential public health risk associated with the consumption of milk from BV outbreaks. Detection of VACV in the milk from noninfected teats demonstrated that VACV shedding in milk might be related to a systemic infection. Moreover, it was shown that VACV DNA and viral infectious particles could be detected in milk even after healing of the lesions, demonstrating that VACV may cause a persistent infection in cattle.
- Jul 2015
Cancer remains one of the world's most devastating diseases with millions of fatalities and new cases every year. In this work, we attempted to develop a facile "enzyme-free" fluoroimmunoassay based on the novel nanoconjugates composed of CdS quantum dots (QDs) as the fluorescent inorganic core and an antibody-modified polysaccharide as the organic shell, modeling their possible application for the in vitro diagnosis of non-Hodgkin lymphoma (NHL) cancer. Chitosan was conjugated with an anti-CD20 polyclonal antibody (pAbCD20) by the formation of covalent amide bonds. In the sequence, these chitosan-antibody conjugates were utilized as direct ligands for the surface biofunctionalization of CdS QDs (CdS/chitosan-pAbCD20) using a single-step colloidal process in aqueous medium at room temperature. The most relevant physico-chemical properties of these nanoconjugates were assessed by morphological and spectroscopic techniques. The results indicated that CdS nanocrystals were produced with an average diameter of 2.5nm and with cubic zinc blende crystalline nanostructure. The CdS-immunoconjugates (CdS/chitosan-pAbCD20) presented colloidal hydrodynamic diameter (HD) of 15.0±1.2nm. In addition, the results evidenced that the "enzyme-free" QD-linked immunosorbent assay (QLISA) was effective for the in vitro detection against the antigen CD20 (aCD20) based on fluorescent behavior of the CdS nanoconjugates. Moreover, the CdS-immunoconjugates were successfully used for fluorescence bioimaging of NHL cancer cells. Finally, the cell viability results using different cell cultures based on LDH, MTT and Resazurin bio-assays have demonstrated no cytotoxicity of the new CdS-chitosan bioconjugates relative to the standard controls. Thus, CdS conjugates may offer a promising platform for the future development of in vitro and in vivo applications for the detection and diagnosis of NHL cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.
Porcine Respiratory Disease Complex (PRDC) is a group of diseases that cause high losses in the swine industry. Several infectious agents are related to PRDC including porcine circovirus 2 (PCV-2), pseudorabies virus (SuHV-1),Haemophilus parasuis (HP), Mycoplasma hypneumoniae (MH) and Pasteurela multocida (PM). The aim of this study was to develop real-time PCRs (qPCR) for the detection of these infectious agents. Oligonucleotides were designed for each specific infectious agent and labeled with different fluorophores to amplify specific parts of the genome. This was done in two groups of reactions—a duplex qPCR for SuHV-1 and PCV-2 and a multiplex qPCR to detect the three bacteria simultaneously. The reactions were tested in 142 pooled samples of swine lymph nodes and lungs with clinical signs of PRDC. There were 135 samples that tested positive for PCV-2, 61 for HP, 29 for PM, 30 for MH and zero for SuHV-1. We recorded 76 cases of co-infection. The qPCRs developed in this study are useful tools in the diagnosis of PRDC.
Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit without exhibiting any clinical signs.
This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified. Copyright © 2015. Published by Elsevier Ltd.
- Mar 2015
In this study, we designed and developed novel biocompatible ZnS quantum dot (QD) nano-immunoconjugates to detect cancer cells strictly via an environmentally-friendly chemistry process. ZnS QDs were capped by chitosan-based ligands that were chemically conjugated at room temperature with an antibody-specific cancer biomarker for non-Hodgkin lymphoma using a one-pot aqueous colloidal route. The nano-immunoconjugates were extensively characterised by several spectroscopic and morphological approaches and biological assays. The results demonstrated that ultra-small colloidal ZnS nanocrystals with an average diameter of 3.7 nm were produced and stabilised by the chitosan-antibody conjugates. In addition, they exhibited intense fluorescence activity and were effective for specific targeting, labelling, and bioimaging of cancerous lymphocyte B-cells. Moreover, the results of in vitro cell viability assays using fibroblast cell line indicated that the ZnS-nanoconjugates were cytocompatible. These may be used in numerous applications in oncology diagnosis and nanomedicine.
Background Swine influenza virus (SIV) is the cause of an acute respiratory disease that affects swine worldwide. In Brazil, SIV has been identified in pigs since 1978. After the emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds.Objective The objective of this study was to evaluate the serological profile for influenza virus in farrow-to-finish pig farms in Minas Gerais state, Brazil.Methods Thirty farms with no SIV vaccination history were selected from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/Southwest and the Belo Horizonte metropolitan area). At each farm, blood samples were randomly collected from 20 animals in each production cycle category: breeding animals (sows and gilts), farrowing crate (2–3 weeks), nursery (4–7 weeks), grower pigs (8–14 weeks), and finishing pigs (15–16 weeks), with 100 samples per farm and a total of 3,000 animals in this study. The samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A/swine/Iowa/8548-2/98) reference strain.ResultsThe percentages of seropositive animals for H1N1pdm09 and H3N2 were 26.23% and 1.57%, respectively, and the percentages of seropositive herds for both viruses were 96.6% and 13.2%, respectively.Conclusions The serological profiles differed for both viruses and among the studied areas, suggesting a high variety of virus circulation around the state, as well as the presence of seronegative animals susceptible to influenza infection and, consequently, new respiratory disease outbreaks.This article is protected by copyright. All rights reserved.
This study aimed to survey captive wild boars for antibodies against Porcine circovirus-2 (PCV-2) in registered farms. Serum samples (n = 1305) were collected from 90-day-old wild boars from 118 farms of the Brazilian South-east region, including the states of Minas Gerais and São Paulo, and South region, including the states of Paraná, Rio Grande do Sul and Santa Catarina. All herds (100%) presented reactive animals, in varying numbers and from low-to-high antibody titres, with the occurrence ranging from 82 to 89%. Considering farms, the average prevalence was of 84.9% (P < 0.05) and ranged from 54.1 to 94.95%. Regarding the geographic regions studied, the prevalence was of 100%, with PCV2 antibodies detected in wild boars of all regions. This study provides the first evidence of PCV2 antibodies in captive wild boars in Brazil.
- Jul 2014
Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome (PMWS). The PCV2 capsid (Cap) protein is a leading antigen candidate for vaccine and serological diagnostic testing, due to its immunogenic properties. In this study, the codon-optimized PCV2 Cap gene was cloned into a pPICZαA vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The screening of recombinant yeasts was followed by detection of the recombinant Cap (rCap) protein by Western blot, using sera from pigs naturally infected with PCV2. The rCap secreted protein was used without prior purification as a coating antigen in the ELISA test, with high discrimination between PCV2-positive and negative sera. These results reveal a high confidence in the specific immunoreactivity of the secreted antigen and show the antigenicity of the recombinant protein. The feasibility of the P. pastoris expression system for the production of PCV2 Cap as secreted protein and its apparent bioactivity, suggests there are good prospects for the use of this antigen in the investigation of PCV2 infections and testing for vaccine purposes.
##Assembly-Data-START## Assembly Method :: Staden v. v.5 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
- Jun 2014
Despite undeniable advances in medicine in recent decades, cancer is still one of the main challenges faced by scientists and professionals in the health sciences as it remains one of the world's most devastating diseases with millions of fatalities and new cases every year. Thus, in this work, we endeavored to synthesize and characterize novel multifunctional immunoconjugates composed of quantum dots (QDs) as the fluorescent inorganic core and antibody-modified polysaccharide as the organic shell, focusing on their potential applications for in vitro diagnosis of non-Hodgkin lymphoma (NHL) cancer tumors. Chitosan was covalently conjugated with anti-CD20 polyclonal antibody (pAbCD20) via the formation of amide bonds between amines and carboxyl groups. In the sequence, these biopolymer-antibody immunoconjugates were utilized as direct capping ligands for the biofunctionalization of CdS QDs (CdS/chitosan-pAbCD20) using a single-step process in aqueous medium at room temperature. The nanostructures were characterized by UV-visible spectroscopy, Photoluminescence Spectroscopy (PL), FTIR, and transmission electron microscopy (TEM) with selected area electron diffraction. The TEM images associated with the UV-vis optical absorption results indicated the formation of ultra-small nanocrystals with average diameters in the range of 2.5-3.0 nm. Also, the PL results demonstrated that the immunoconjugates exhibited "green" fluorescent activity under ultraviolet excitation. Moreover, using in vitro Laser light scattering immunoassay (LIA), the QDs/immunoconjugates have shown binding affinity against antigen CD20 (aCD20) expressed by lymphocyte-B cancer cells. In summary, innovative fluorescent nano-immunoconjugate templates were developed with promising perspectives to be used in the future for the detection and imaging of cancer tumors.
The aim of this study was to characterize the porcine circovirus 2 ( PCV2) infections in farrowing sows and to evaluate an association with piglet viremia and weight. Twenty sows and 100 newborn piglets were studied. Colostrum and serum of the sows were obtained on the day of parturition. Milk samples were collected on day 20 postpartum. Blood samples were taken and the piglets were weighed on days 1, 20, 42, 63 and 84 postpartum. Colostrum, milk and serum were evaluated for PCV2 DNA load. Serum was evaluated for neutralizing antibodies. PCV2 DNA was found in 17/20 serum samples, 14/20 colostrum samples and 11/20 milk samples. On day 1 postpartum 29% of piglets were viremic. PCV2 viral load ranged from 3.02 to 6.75 log(10) copies/ mL considering all sampled days. There was no correlation between sow viremia, antibody levels or PCV2 load in colostrum and piglet viremia on day 1 postpartum. The PCV2 load in colostrum and milk was associated with viremia in piglets from weaning to 84 days postpartum. Piglets' PCV2 viremia and viral load could not be associated with weight throughout this study.
In vitro potency test for evaluation of Clostridium perfringens type D epsilon toxoid Avaliação in vitro da potência de toxoide épsilon de Clostridium perfringens tipo D ABSTRACT: The control of enterotoxemia caused by the epsilon toxin, produced by Clostridium perfringens type D, is based on vaccination with epsilon toxoid. The potency test for this immunogen is conducted using seroneutralization in mice. Here, an in vitro test for detection of neutralizing antibodies with Madin-Darby Canine Kidney (MDCK) cells was standardized in order to study alternative methodologies for the potency test. Titers observed in the in vivo and in vitro seroneutralization tests had a correlation of 99.73%.
This study investigated the adjuvant potential of W/O/W multiple emulsions and microemulsions, comparing them with traditional aluminum hydroxide and oil-in-water emulsion adjuvants against bluetongue vaccine (BTV). Local inflammatory reactions were assessed in rabbits by measuring the temperature of the animals and the skin thickness at the site of application. Antibodies titers were determined by serum-neutralization test. Histological analyses of lesions at the site of adjuvants application were done. Results showed that multiple emulsion and microemulsion maintained their stability even in the presence of complex components and presented adequate characteristics for subcutaneous administration. They were able to induce immune response against BTV, but it was smaller than the traditional adjuvants. Despite microemulsion adjuvant showed lower antibodies titre, it was easier to prepare more stable at 4°C and it was the only one that did not induce any local reaction.
- Jun 2013
Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an important zoonosis that affects dairy cattle. There are many aspects of the disease that remain unknown, and aiming to answer some of these questions, the clinical, hematological, and biochemical parameters of VACV experimentally infected cows were evaluated. In the first part of the study, lactating cows were infected with VACV-GP2 strain. In the second part, animals previously infected with VACV-GP2 were divided into two treatment groups: Group 1, immunosuppressed cows; and Group 2, re-infected cows. In this study, BV could be experimentally reproduced, with similar lesions as observed in natural infections. Moreover, a short incubation period and local lymphadenopathy were also observed. VACV could be detected by PCR and isolated from scabs taken from teat lesions of all inoculated and re-inoculated animals. Lymphocytosis and neutrophilia were observed in all animals from the first part of the experiment, and lymphopenia and relative neutrophilia were observed in the immunosuppressed animals. Detection of viral DNA in oral mucosa lesions suggests that viral reactivation might occur in immunosuppressed animals. Moreover, clinical disease with teat lesions may occur in previously VACV-infected cows under the experimental conditions of the present study.
Influenza A virus (IAV) is a respiratory pathogen of pigs and is associated with the porcine respiratory disease complex (PRDC), along with other respiratory infectious agents. The aim of this study was to diagnose and to perform a clinic-pathological characterization of influenza virus infection in Brazilian pigs. Lung samples from 86 pigs in 37 farrow-to- -finish and two farrow-to-feeder operations located in the States of Minas Gerais, São Paulo, Paraná, Rio Grande do Sul, Santa Catarina, and Mato Grosso were studied. Virus detection was performed by virus isolation and quantitative real time reverse-transcription PCR (qRT-PCR). Pathologic examination and immunohistochemistry (IHC) were performed in 60 lung formalin-fixed paraffin-embedded tissue fragments. Affected animals showed coughing, sneezing, nasal discharge, hyperthermia, inactivity, apathy, anorexia, weight loss and growth delay, which lasted for five to 10 days. Influenza virus was isolated from 31 (36.0%) lung samples and 36 (41.9%) were positive for qRT-PCR. Thirty-eight (63.3%) lung samples were positive by IHC and the most frequent microscopic lesion observed was inflammatory infiltrate in the alveoli, bronchiole, or bronchi wall or lumen (76.7%). These results indicate that influenza virus is circulating and causing disease in pigs in several Brazilian states.
Background: Influenza A viruses circulating in pigs in Brazil are still not characterized, and only limited data are available about swine influenza epidemiology in the country. Therefore, we characterized the hemagglutinin (HA) and neuraminidase (NA) genes of influenza viruses isolated from Brazilian pigs. We also evaluated one case of probable swine-to-human transmission. Methods: Twenty influenza viruses isolated from pigs during 2009-2010 in five Brazilian states (Minas Gerais, Sao Paulo, Parana, Rio Grande do Sul, and Mato Grosso) were used. One human isolate, from a technician who became ill after visiting a swineherd going through a respiratory disease outbreak, was also used in the study. Phylogenetic analysis for the HA and NA genes and hemagglutinin amino acid sequence alignment were performed. Results: All isolates clustered with pandemic H1N1 2009 (pH1N1) viruses and appeared to have a common ancestor. Genetic diversity was higher in the HA than in the NA gene, and the amino acid substitution S203T in one of HA's antigenic sites was found in most of the samples. The human isolate was more related to swine isolates from the same herd visited by the technician than to other human isolates, suggesting swine-to-human transmission. Conclusion: Our results show that pH1N1 was disseminated and the predominant subtype in Brazilian pigs in 2009-2010.
In 2011, vaccinia virus caused an outbreak of bovine vaccinia, affecting dairy cattle and dairy workers in Brazil. Genetic and phenotypic analyses identified this isolate as distinct from others recently identified, thereby reinforcing the hypothesis that different vaccinia virus strains co-circulate in Brazil.
Vaccinia virus outbreak in Serro County, Minas Gerais state, southeastern Brazil. Appendix shows location of outbreak, lesions on human patient, clinical signs of control and experimentally infected mice, and results of phenotypic plaque-forming testing of vaccinia viruses on epithelial kidney cells BSC-40.
- Oct 2012
The aim of this study was to evaluate Porcine parvovirus (PPV) and Porcine circovirus 2 (PCV2) infection in dams and their offspring and the role of antibody protection on these infections. Sera were collected from gilts and sows by venipuncture and from umbilical cord of newborn pre-suckle piglets for the detection of PCV2 and PPV antibodies by immunoperoxidase monolayer and haemmaglutination inhibition assays, respectively. Gilts and sows sera were submitted to viral detection by PCR, as well as heart, lung, tonsil and lymph nodes samples from stillborn and mummified fetuses. High antibody titers before artificial insemination (AI) (>5.120 and >2.560 UHA for PCV2 and PPV, respectively), were found associated with viremia and fetal exposure for both PCV2 and PPV, respectively, in gilts and sows, regardless of pregnancy stage. These infections resulted in litters with mummified, stillborn, as well as seropositive and viable newborns. These findings bring new evidence about the lack of antibody protection against PCV2 and PPV infections in dams, indicating that more studies are necessary about the role of humoral response against both pathogens.
Orf virus (ORFV), the prototype of the genus Parapoxvirus, is the aetiological agent of contagious ecthyma (CE), a pustular dermatitis that afflicts domestic and wild small ruminants. CE is one of the most widespread poxvirus diseases in the world, causing public health impacts. Outbreaks of ORFV have been observed in all geographical regions of Brazil, affecting ovine and caprine herds. The origins, epidemiology and identity of Brazilian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In the present study, we revisited CE outbreaks which occurred until 32 years ago, and we assessed, genetically, five viral isolates. We performed the sequencing and analysis of the three ORFV molecular markers: B2L gene, virus interferon resistance gene (VIR) and the vascular endothelial growth factor gene. Nucleotide and amino acid analysis of the analysed genes demonstrated that Brazilian ORFVs do not form a unique cluster, and presented more similarity to other worldwide ORFV samples than with each other. These data raise the questions of whether there are different worldwide ORFVs circulating in Brazil, or if all the Brazilian ORFV samples are of the same virus taken at distinct time points.
The maned wolf, Chrysocyon brachyurus, is an endangered Neotropical canid that survives at low population densities. Diseases are a potential threat for its conservation but to date have been poorly studied. We performed clinical evaluations and investigated the presence of infectious diseases through serology and coprologic tests on maned wolves from Galheiro Natural Private Reserve, Perdizes City, Minas Gerais State, southeastern Brazil. Fifteen wolves were captured between 2003 and 2008. We found high prevalences of antibody to canine distemper virus (CDV; 13/14), canine parvovirus (CPV; 4/14), canine adenovirus type 2 (13/14), canine coronavirus (5/11), canine parainfluenza virus (5/5), and Toxoplasma gondii (6/8), along with Ancylostomidae eggs in all feces samples. Antibodies against Leishmania sp. were found in one of 10 maned wolves, and all samples were negative for Neospora caninum. Evidence of high exposure to these viral agents was also observed in unvaccinated domestic dogs from neighboring farms. High prevalence of viral agents and parasites such as CDV, CPV, and Ancylostomidae indicates that this population faces considerable risk of outbreaks and chronic debilitating parasites. This is the first report of exposure to canine parainfluenza virus in Neotropical free-ranging wild canids. Our findings highlight that canine pathogens pose a serious hazard to the viability of maned wolves and other wild carnivore populations in the area and emphasize the need for monitoring and protecting wildlife health in remaining fragments of the Cerrado biome.
The effects of the use of complex minerals on the occurrence of retained placenta during pre-partum were valued on 135 Holstein cows from two or more deliveries. The animals were divided in two groups: ionic mineral (69 animals) and complexed mineral (66 animals). In 55 of these animals serum concentrations of imunoglobulin G (IgG), Zn and Cu and colostrum quality were also evaluated. The experiment was conducted in complete randomized split-plot design, serum IgG and trace minerals were evaluated by analysis of variance and used, respectively, Duncan's test and Fisher. The α error rate of 7% was accepted. There were no differences between groups for the occurrence of retained placenta, colostrum quality and serum concentrations of Zn and IgG (P>0.07), a difference was observed for Cu (P<0.07) concentrations. The IgG concentrations were different on the weeks pre partum evaluated (P <0.07).
- Sep 2012
Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that affects dairy cattle and milkers, causing economic losses and impacting animal and human health. Based on the clinical presentation, BV appears to be a localized disease, with lesions restricted to the skin of affected individuals. However, there are no studies on the pathogenesis of the disease in cows to determine if there is a systemic spread of the virus and if there are different ways of VACV shedding. The objective of this work was to study if there is a systemic spread of VACV in experimentally infected cows and to study the kinetics of VACV circulation in the blood and shedding in the feces of these animals. To this end, eight crossbred lactating cows were used. Three teats of each cow were inoculated with the GP2V strain of VACV. All animals were monitored daily, and blood and fecal samples were collected for 67days post-infection (dpi). After this period, four of these previously infected cows were immunosuppressed using dexamethasone. Viral DNA was continuously detected and quantified in the blood and feces of these animals in an intermittent way, even after the resolution of the lesions. At slaughter, tissues were collected, and viral DNA was detected and quantified in the mesenteric and retromammary lymph nodes, ileum, spleen and liver. The detection of VACV DNA in the feces for a longer period (67dpi) and in the lymphatic organs provides new evidence about VACV elimination and suggests that BV could be a systemic infection with a chronic course and viral shedding through the feces.
The aims of the present study were to determine the prevalence of bluetongue virus (BTV) antibodies in sheep from Distrito Federal. Sera from 606 sheep of 18 herds were submitted to the agar-gel immunodiffusion (AGID) for bluetongue virus antibodies. The prevalences of bluetongue infection found in Distrito Federal were 100% (CI 95%: 84.67 to 100.00) for flocks and 52.37% (389/606) (CI 95%: 35.76 to 68.98) for animals. Thus, data from the present study showed that infection by bluetongue virus is highly widespread in sheep flocks in the Distrito Federal, which intensifies the need for assessments on the impact of this disease in Brazil.
Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. Despite such impacts, there are no experimental studies about the pathogenesis of BV in cows to assess whether there is a systemic spread of the virus and whether there are different ways of VACV shedding. Trying to answer some of these questions, a study was proposed using experimental inoculation of VACV in cows. All experimentally infected cows developed lesions compatible with VACV infection in cattle. Two of the six animals presented VACV DNA in blood and faecal samples, starting at the 2nd and the 3rd day post-infection (d.p.i.), respectively, and lasting until the 36th d.p.i., in an intermittent way. This study provides new evidence that VACV can be detected in blood and faeces of infected cows, suggesting that BV could be a systemic disease, and also bringing new information about the epidemiology and pathogenesis of BV.
The aim of this study was to describe early infections with porcine circovirus type 2 (PCV2) in naturally infected piglets and the piglets' serologic profiles. A total of 20 sows (15 PCV2-vaccinated and 5 unvaccinated) and 100 newborn piglets were studied. Colostrum and serum of the sows and serum of the presuckling piglets were obtained on the day of parturition. Milk samples were collected on day 20 postpartum. Blood samples were taken and the piglets weighed on days 1, 20, 42, 63, and 84 postpartum. Colostrum and milk were evaluated for infectious PCV2 and for PCV2 total antibody (TA), neutralizing antibody (NA), and IgA. Serum samples were evaluated for PCV2 TA, NA, IgA, IgM, and DNA. The sows had high levels of TA and NA in serum and colostrum; however, 11 and 5, respectively, of the 20 colostrum and milk samples contained infectious PCV2. In the serum, PCV2 DNA and IgM were detected in 17 and 5, respectively, of the 20 sows. Nine piglets were born with PCV2 antibodies, which indicates in utero transmission of PCV2 after the period of immunocompetence (> 70 d of gestation). On day 1 postpartum, PCV2 DNA was detected in 29 of the 100 serum samples from the piglets. There was no difference between the weights of viremic and nonviremic piglets throughout the study. In conclusion, even on farms with sows that have high PCV2 antibody titers, vertical transmission of PCV2 may occur, resulting in piglet infection.
- Apr 2012
Please cite this paper as: Rajão et al. (2013). Serological evidence of swine influenza in Brazil. Influenza and Other Respiratory Viruses 7(2), 109–112. The aim of this work was to detect serum antibodies specific to influenza viruses in swine in Brazil. Serum samples of 355 pigs from 17 herds in Minas Gerais state were tested by hemagglutination inhibition (HI) for antibodies against H1N1 swine (SIV) and human influenza viruses, and H3N2 SIV. HI revealed that 158 animals (44·5%) and 11 herds (64·7%) were positive for H1N1 SIV, 36 animals (10·1%) and four herds (23·5%) were positive for H3N2 SIV, and 136 animals (38·3%) and 10 herds (58·8%) were positive for H1N1 human. This study indicates that swine influenza is disseminated throughout Minas Gerais state, Brazil.
Vaccinia virus (VACV) has been associated with zoonotic exanthemic outbreaks affecting bovids and human beings, with significant public health and economic impacts. Rapid and reliable diagnostic methods are needed to detect and epidemiologically monitor antibodies to VACV. The current study describes the development of an immunoperoxidase monolayer assay (IPMA) for detection of total VACV antibodies in bovine serum. The assay was validated by comparison with a plaque reduction neutralization test (PRNT). Kappa index of agreement, diagnostic sensitivity, specificity, and accuracy of the IPMA were -1.008, 100%, 96%, and 98%, respectively, when compared with PRNT on 148 field bovine sera. Repeatability tests on 32 field-positive serum samples revealed that intraclass coefficient correlation was 0.86. In experimentally infected cattle, VACV antibodies were detectable by IPMA 4 days postinfection, which was more than 2 weeks earlier than with the PRNT, indicating that IPMA could be a more sensitive test than the latter. In 4 naturally VACV-diseased cows monitored for 13 months, IPMA could detect VACV antibodies up to 13 months, a longer time than PRNT. The IPMA is simpler to produce and perform when compared with PRNT and is time saving and suitable for large-scale surveys of VACV infection in bovine.
We surveyed 49 free-living collared peccaries (Pecari tajacu) in Brazil for antibodies against bluetongue virus (BTV) and porcine circovirus 2 (PCV2). Antibodies against BTV were detected in 19/49 (39%) samples. All samples were negative for PCV2. The importance of antibodies to BTV in collared peccaries remains to be determined.
The prevalence of antibodies against bluetongue virus was investigated in 41 dairy goats and 40 sheep herds in the semi-arid region of Pernambuco state and the conditions for insect Culicoides maintenance, considering climate dynamics and vector competence, were evaluated. The percents of seropositive herds in agar gel immunodiffusion test for bluetongue virus group were 24 for goats and 27.5 for sheep. The estimated prevalences of seropositive animals were 3.9% for goats (n = 410) and 4.3% for sheep (n = 400). The prevalences of seropositive animals were low in the mesoregion of Sertão Pernambucano (4.8% for goats and 4.1% for sheep) and São Francisco Pernambucano (1.0% for goats and 4.5% for sheep). There were no significant differences between species and regions. Considering the social and economic importance of goats and sheep raising in the semi-arid region, it is essential to establish preventive measures to control imports of ruminants from these areas.
Vaccinia virus (VACV) has been associated with several exanthematic outbreaks in bovine, human, and equine species in Brazilian rural areas. Little is known about VACV reservoirs, although it is believed that rodents could be associated with VACV outbreaks. With the goal of filling one more gap in the VACV ecological puzzle, the present work aimed at mimicking a potential transmission route of VACV between cows and rodents, both known as natural VACV hosts. Balb/c mice were exposed to feces of experimentally VACV infected cows for 20 days, and samples from these mice were examined by using molecular and serological tests. VACV DNA was detected in feces and blood samples after several days of exposure; infectious VACV particles were also detected in the feces. The presence of anti-VACV neutralizing antibodies in murine sera further suggested horizontal transmission. If the transmission model described here can be applied to natural environments, exposure to bovine feces could be considered a risk factor for the spread of VACV; consequently, the traditional use of bovine manure as a fertilizer in agricultural activities may be promoting the infection of rodents.
Vaccinia virus (VACV) is the cause of bovine vaccinia (BV), an emerging zoonotic disease that affects dairy cows and milkers. Some chemical disinfectants have been used on farms affected by BV to disinfect cow teats and milkers' hands. To date, there is no information about the efficacy of disinfectants against VACV. Therefore, this study aimed to assess the virucidal activity of some active disinfectants commonly used in the field. Sodium hypochlorite, quaternary ammonium combined with chlorhexidine, and quaternary ammonium combined with glutaraldehyde were effective in inactivating the virus at all concentrations tested. Iodine and quaternary ammonium as the only active component were partially effective. The presence of bovine feces as organic matter and light decreased the effectiveness of sodium hypochlorite. These results show that an appropriated disinfection and asepsis of teats and hands may be helpful in the control and prevention of BV and other infections with VACV.
- May 2011
Serum antibodies and shedding of porcine circovirus type 2 (PCV2) into lacteal secretions were examined in naturally infected sows. Total (TA) and neutralising (NA) antibodies against PCV2 were evaluated in serum and colostrum from 20 vaccinated (Vac) and 21 unvaccinated (N-vac) sows. Anti-PCV2 IgA titres and PCV2 infectious titres were determined in colostrum and milk. All sows had high TA and NA levels in serum and colostrum. Infectious PCV2 was detected in 22/41 colostrum samples (7/20 Vac and 15/21N-Vac sows) and 5/20 milk samples (1/5 Vac and 4/15N-Vac sows). Anti-PCV2 IgA was found in high levels in colostrum and varying levels in milk. Infectious PCV2 may be present in milk and colostrum of naturally infected sows, even in the presence of NA.
A virological and serological cross-sectional study (E1) and a longitudinal study (E2) were performed on herds with (G2 and G3) and without (G1) post weaning multisystemic syndrome (PMWS) in Brazil. Blood, serum, nasal and rectal swabs samples were collected of sows, farrowing piglets, nursery, growing and finishing pigs. In E1, were sampled 40 animals in each category (G1a and G2). In E2, (G1b and G3), 35 farrowing piglets were identified and sampled along the production cycle. Porcine circovirus type 2 (PCV2) antibodies were assayed. A PCR was used to detected PCV2 genome in blood and swabs. In E1, sows had high rates of viremic and seropositives animals, with percentage of sows with high antibodies titers greater than G2. Passive antibodies decline occurred between nursery and growing area with increased viral shedding in swabs and subsequent seroconversion in G1. In G2, the passive antibodies decay occurred in nursery, with a reduction in viral shedding. In E2, the decline of maternal immunity occurred between the 1st and 2nd collection in G1b, and between 2nd and 3rd collections in G3. In both herds, the decay of passive immunity coincided with increased viremia and viral shedding; and seroconversion occurred between the 3rd and 4th collection in both herds with decline of viremia. Viremia and viral shedding was detected in all samples days, 42% of animals sampled in E2 were viremic and all tissue samples collected at slaughter were positive for PCV2. This study confirms the persistence of viremia even in the presence of high titers of antibodies and the serological profile in a herd with or without PMWS may be different, especially with regard to the passive immunity duration.
Padronização de teste de potência in vitro para vacinas que contenham toxoide alfa de Clostridium novyi tipo B. obtido pelas técnicas in vitro e in vivo foi de 98,38%, indicando ser possível a utili-zação do modelo estudado na substituição do modelo animal para teste de potência de toxoide alfa de C. novyi tipo B. Palavras-chave: Clostridium novyi tipo B, soroneutralização, vacinas, bioética. aBstract standardization of an in vitro PotencY test for vaccines containinG Clostridium novyi tYPe B alPha-toxoid. Clostridium novyi type B is the pathogen re-sponsible for necrotizing hepatitis caused by the action of alpha toxin. The control of this disease is based on immunization of animals with vaccines containing alpha toxoid of C. novyi type B in its composition, and the evaluation of this toxoid is made by seroneutralization in mice. Therefore, the present study was aimed to standardize a test of the power of alpha toxoid of C. novyi type B cell line VERO, as an alternative to animal bioassay The correlation coefficient obtained by the in vitro and in vivo techniques was 98.38%, indicating the possible use of the model to replace the animal model to test the power of alpha toxoid of C. novyi type B.
Bovine vaccinia is an emergent zoonosis caused by the Vaccinia virus (VACV). The disease is characterized by the appearance of exanthematic lesions that occur in humans and dairy cows. Previous studies have revealed the presence of infectious viral particles in milk samples during an outbreak of bovine vaccinia in Brazil, indicating the possibility of disease transmission through raw milk. To assess the viability of the virus in milk after thermal treatment and processing procedures, milk samples were experimentally contaminated with 10(3) plaque forming units (PFU)/mL (group I) and 10(5) PFU/mL (group II) VACV Guarani P2 virus, and the third group was not contaminated and served as a control. The samples were submitted to storage temperatures in a cold chamber, freezer for 48 hours, and to low temperature long-time treatment. Moreover, the viral viability was evaluated in cheese produced with contaminated milk using 10(4) PFU/mL VACV Guarani P2. Notably, the virus remained viable in milk after storage for 48 hours in both the cold chamber and the freezer, with a reduction in viral titer of 14.49% and 25.86%, respectively. Group II showed a viral reduction in titer of 61.88% and 75.98%, respectively. Thermal treatment 65°C for 30 minutes showed a reduction of viral titer of 94.83% and 99.99%, respectively, in group I and group II, but still showed remaining viable virus particles. In addition, it was possible to recover infectious viral particles from both the solid curds and the whey of the cheese produced with experimentally contaminated milk. The cheese shows a reduction in viral titer of 84.87% after storage at 4°C for 24 hours. The presence of viable viral particles in milk after both thermal treatment and cheese production indicates a potential public health risk.
Wild canids are under many pressures, including habitat loss, fragmentation and disease. The current lack of information on the status of wildlife health may hamper conservation efforts in Brazil. In this paper, we examined the prevalence of canine pathogens in 21 free-ranging wild canids, comprising 12 Cerdocyon thous (crab-eating fox), 7 Chrysocyon brachyurus (maned wolf), 2 Lycalopex vetulus (hoary fox), and 70 non-vaccinated domestic dogs from the Serra do Cipó National Park area, Southeast Brazil. For wild canids, seroprevalence of antibodies to canine parvovirus, canine adenovirus, canine coronavirus and Toxoplasma gondii was 100 (21/21), 33 (7/21), 5 (1/19) and 68 (13/19) percent, respectively. Antibodies against canine distemper virus, Neospora caninum or Babesia spp. were not found. We tested domestic dogs for antibodies to canine parvovirus, canine distemper virus and Babesia spp., and seroprevalences were 59 (41/70), 66 (46/70), and 42 (40/70) percent, respectively, with significantly higher prevalence in domestic dogs for CDV (P<0.001) and Babesia spp. (P=0.002), and in wild canids for CPV (P<0.001). We report for the first time evidence of exposure to canine coronavirus in wild hoary foxes, and Platynossomun sp. infection in wild maned wolves. Maned wolves are more exposed to helminths than crab-eating foxes, with a higher prevalence of Trichuridae and Ancylostomidae in the area. The most common ectoparasites were Amblyomma cajennense, A. tigrinum, and Pulex irritans. Such data is useful information on infectious diseases of Brazilian wild canids, revealing pathogens as a threat to wild canids in the area. Control measures are discussed. KeywordsBrazilian Cerrado-Canid conservation-Disease-Seroprevalence-Wildlife pathogens
The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.
- Jun 2010
Amino acid sequences of vaccinia virus (VACV) samples and comparison with homologous genes sequences from several orthopoxviruses, Brazil. A) Alignment of vaccinia growth factor gene sequences from 6 monkey serum samples showing 100% identity (horizontal box). VACV-TO_CA, sequence from Cebus apella; VACV-TO_AC, sequence from Allouata caraya; HPXV, horsepoxvirus; CPXV, cowpoxvirus; MPXV, monkeypoxvirus; VARV, variola virus; ECMV, ectromelia virus. B) Alignment of orthopoxvirus hemagglutinin gene amino acid sequences showing the deletion signature region (vertical box) in VACV-TO isolates and several VACV strains isolated during bovine vaccinia outbreaks. Arrow indicates polymorphism site in the hemagglutinin amino acid sequences between VACV-TO_CA and VACV-TO_AC. Alignments were made by using ClustalW (www.ncbi.nlm.nih.gov/pmc/articles/PMC308517) and MEGA version 3.1 software (www.megasoftware.net). HSPV, horsepoxvirus.
To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.
Aiming to investigate in vitro alternatives, a test for neutralizing antibody detection using cell culture was developed. This test was more sensitive than previous animal models, allowing for detection of substantially lower alpha toxin and anti-alpha toxin titers. Titers observed during in vivo and in vitro seroneutralization had a correlation of 99.12%, indicating that cell culture is a viable alternative in the evaluation of vaccine potency, screening of vaccinal seeds, and Clostridium septicum alpha toxin titration.
Enterotoxemia (also called pulpy kidney disease) is an enteric disease, that affect ruminants, produced by epsilon toxin from Clostridium perfringens type D, an anaerobic commonly isolated from soil and feces of healthy animals. The diagnostic is based on detection of this exotoxin in the intestinal content by soroneutralization in mice. Therefore, this study aimed to standardize a test for detection and titration of the toxin in vitro, and compare it with the phenomenon in vivo. A volume of epsilon toxin was titrated in mice and in some cell lines. After concluding the most sensitive cell line, were held in vitro titrations of dilutions from a toxin wich one had in vivo titer known. The results were grouped and a mathematical equation was developed. MDCK cell line showed that the phenomenon observed in vitro can be expressed by a mathematical equation wich shows a correlation of 98.33% with a minimum lethal dose determined in vivo. Therefore, the soroneutralization using MDCK allows a specific, sensitive, practical, fast, and doesn't requere use of animal titration of epsilon toxin.
Occupational exanthematic diseases represent an important cause of public health impact and economical losses. Among the viral exanthematic diseases, two caused by poxviruses are noteworthy: the bovine vaccinia (BV), caused by the Vaccinia virus (VACV); and the milker's nodule, in which the agent is the Pseudocowpox virus (PCPV). Both agents are zoonotic and have been associated with several cases of bovine infection. In Brazilian rural areas BV has been highly prevalent, particularly in milk herds. Farmers, milkers and their close contacts developed lesions on the hands, forearms, legs and face accompanied by several systemic symptoms. Although VACV and PCPV present with similar epidemiological and transmission patterns, no VACV and PCPV co-infection cases have to date been described. To describe the first case of zoonotic VACV and PCVP co-infection, based on serological and molecular methods. In this work we report a case of a Brazilian rural worker who presented with a large severely ulcerated-pustule skin lesion, associated with fever, headache, malaise, myalgia and axillary, inguinal and cervical limphadenopathy. The worker declared occupational contact with cattle that had notable injuries on their teats. Human and bovine clinical samples were collected and submitted to serological and molecular tests. PCR and phylogenetic analysis revealed the presence of VACV DNA and PCPV DNA in the patient's lesion. Serological tests indicated anti-VACV neutralizing antibodies and molecular assays showed the presence of VACV and PCPV DNA in the patient sera. VACV and PCPV also were detected in dairy cattle. Together, these results indicate a case of zoonotic VACV/PCPV co-infection. Epidemiological surveillance and appropriate medical treatment are essential for the control of both diseases, especially in the most severe cases, as described in the present study.
The vaccinia virus (VACV), which causes exanthemous lesions in dairy cattle and humans, has been associated with several bovine vaccinia outbreaks in Brazil. Currently, no data are available about the safety of milk produced in VACV-affected areas. In this study, 47 milk samples were collected during bovine vaccinia outbreaks and submitted to viral isolation, DNA detection, and nucleotide sequencing of the conserved tk gene. The appearance of characteristic white pocks on the chorioallantoic membranes of chicken eggs, in association with viral cytopathic effects in chicken embryo fibroblasts and phylogenetic data, strongly suggest milk contamination by VACV. This is the first report of VACV detection in and isolation from milk.
- Nov 2009
The aims of this study were to devise a process for raising antibodies against Brazilian Bothrops venom in chicken egg yolks, to determine the best delipidation method for the preparation of the aqueous extract and to define the best purification conditions for IgY bothropic antivenom produced in eggs from hens immunized with Brazilian standard bothropic antigen. A group of nine Single Comb White Leghorn laying hens were immunized with venom from five different species of pit vipers of the genus Bothrops. The immunization process was carried out in three cycles, each performed six weeks apart. For extraction, the egg yolk was diluted 1:10 in distilled water, adjusted to a pH of 5.0, subjected to a freeze-thaw cycle, centrifuged and filtered before being precipitated with 20%(w/v) ammonium sulfate salt. This methodology retrieved 2.57 mg of IgY/ml of yolk from eggs. This preparation yielded antibodies capable of neutralizing lethal toxic activity of the pool of Bothrops sp venoms from five species, with an effective dose (ED50) of 365 microL/2 LD50 and, 1.0 mL of IgY antivenom neutralized 0.154 mg of venom.
Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks - BV), affecting dairy cattle and milkers. Little is known about VACV's natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated. In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks. These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?
Sorologia para o vírus da língua azul em bovinos de corte, ovinos e veados campeiros no Pantanal sul-mato-grossense [Bluetongue virus serosurvey in beef cattle, sheep, and pampas deer from Brazilian Pantanal] A língua azul é uma doença viral, não contagiosa, transmitida por dípteros do gênero Culicoides, que afeta ruminantes domésticos e silvestres. A incidência da doença clínica é altamente variável. O vírus da língua azul (BTV) pode causar doença grave em certas raças de ovinos e em alguns cervídeos, mas raramente causa doença em bovinos, caprinos e na maioria dos ruminantes silvestres (Gibbs e Greiner, 1994). Em bovinos, os surtos são esporádicos, a morbidade variável (geralmente em torno de 5%) e a mortalidade muito baixa ou nula (Lobato, 1999). As principais consequências econômicas da infecção pelo BTV são perdas indiretas devido ao aborto, queda do desempenho reprodutivo e na produção de leite, e perda de condição corporal, além da restrição internacional de movimentação animal e seus germoplasmas. Grande parte do território brasileiro encontra-se inserido dentro da zona epidêmica para o BTV, marcado pelas condições climáticas adequadas ao desenvolvimento do vetor (Gibbs e Greiner, 1994). A primeira referência do BTV no Brasil e América do Sul data de 1978, quando foi relatada à Organização Mundial de Saúde Animal (OIE) evidência sorológica da ocorrência do vírus (Lobato, 1999; Lager, 2004). Em 1980, oito de um total de 60 bovinos exportados do Brasil para os
This investigation was carried out in beef cattle (n=219), sheep (n=55), and pampas deer (Ozotoceros bezoarticus) (n=49) from Nhecolândia, sub region of Brazilian Pantanal in Mato Grosso do Sul State, Brazil. It was aimed to assess the seropositivity of these species to bluetongue virus (BTV) by agar gel immunodiffusion test. Seropositivity rates were 42.0% for cattle and 10.9% for sheep. The pampas deer showed to be all seronegative. In cattle, seropositivity to BTV significantly increased with age (P<0.001). These data, the favorable environmental conditions to development of BTV vectors, and the bovine reproductive disorders reported by farmers may indicate that BTV infection occurrs in herds of Brazilian Pantanal, and probably induces to economical losses.
Orthopoxvirus (OPV) and Parapoxvirus (PPV) have been associated with worldwide exanthematic outbreaks. Some species of these genera are able to infect humans and domestic animals, causing serious economic losses and public health impact. Rapid, useful and highly specific methods are required to detect and epidemiologically monitor such poxviruses. In the present paper, we describe the development of a nested-multiplex PCR method for the simultaneous detection of OPV and PPV species directly from exanthematic lesions, with no previous viral isolation or DNA extraction. The OPV/PPV nested-multiplex PCR was developed based on the evaluation and combination of published primer sets, and was applied to the detection of the target pathogens. The method showed high sensitivity, and the specificity was confirmed by amplicon sequencing. Exanthematic lesion samples collected during bovine vaccinia or contagious ecthyma outbreaks were submitted to OPV/PPV nested-multiplex PCR and confirmed its applicability. These results suggest that the presented multiplex PCR provides a highly robust and sensitive method to detect OPV and PPV directly from clinical samples. The method can be used for viral identification and monitoring, especially in areas where OPV and PPV co-circulate.
Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.
Abstract The vaccinia virus (VACV), which causes exanthemous lesions in dairy cattle and humans, has been associated with several bovine vaccinia outbreaks in Brazil. Currently, no data are available about the safety of milk produced in VACV-affected areas. In this study, 47 milk samples were collected during bovine vaccinia outbreaks and submitted to viral isolation, DNA detection, and nucleotide sequencing of the conserved tk gene. The appearance of characteristic white pocks on the chorioallantoic membranes of chicken eggs, in association with viral cytopathic effects in chicken embryo fibroblasts and phylogenetic data, strongly suggest milk contamination by VACV. This is the first report of VACV detection in and isolation from milk.
This investigation was carried out in dairy cattle from four rural settlements of Corumba, MS, Brazil, in order to assess the seroprevalence of bovine herpesvirus 1 (BoHV-1), bluetongue virus (BTV), and Leptospira; and the prevalence was 50.9% (179/352), 51.3% (181/353), and 35.8% (125/349), respectively. Association between frequency of seropositive animals and evaluated rural settlements (P<0.01) was found for Leptospira, and indicated the influence of environmental factors in bovine infection. Age was significantly related to BoHV-1 (P<0.001), BTV (P<0.001), and Leptospira infection (P<0.01), showing higher number of infection among older animals. Once the bovine herds were not vaccinated, the presence of seropositive animals indicated that these infections occurred in rural settlements herds of Corumba assayed, and certainly induced economical loss.
Orf virus (ORFV), the prototype of the genus Parapoxvirus (PPV), is the etiological agent of contagious ecthyma, a severe exanthematic dermatitis that afflicts domestic and wild small ruminants. Although South American ORFV outbreaks have occurred and diagnosed there are no South American PPV major membrane glycoprotein B2L gene nucleotide sequences available. an outbreak of ovine contagious ecthyma in Midwest Brazil was investigated. The diagnosis was based on clinical examinations and molecular biology techniques. The molecular characterization of the virus was done using PCR amplification, cloning and DNA sequencing of the B2L gene. The phylogenetic analysis demonstrated a high degree of identity with ORFV strains, and the isolate was closest to the ORFV-India 82/04 isolate. Another Brazilian ORFV isolate, NE1, was sequenced for comparative analysis and also showed a high degree of identity with an Asian ORFV strain. Distinct ORFV strains are circulating in Brazil. This is the first report on the phylogenetic analysis of an ORFV in South America.
- Jun 2009
A comparative serologic study was performed in seven single site (SS) farrow-to-finish farms and four multi-site (MS) farrow-to-finish farms, with or without post-weaning multisystemic wasting syndrome (PMWS). In each farm, 30 blood samples were collected for each category of the production cycle: sows, farrowing crate, nursery, grower pigs, and finishing pigs. Sera were evaluated for the presence of antibodies to porcine circovirus type-2 (PCV2) via immunoperoxidase monolayer assay. Serologic profiles for PCV2 were different between SS and MS farms. Seroconversion following the decline in maternal antibodies occurred at a later stage on SS farms (grower pigs) than MS farms (nursery pigs). MS farms tended to have lower antibody titers than SS farms in the categories of sow, piglet, and nursery, while higher antibody titers were found in grower pigs. Characterization of serologic profiles for different farms may provide important information for the adoption of vaccination programs.
Neste estudo foi determinada a prevalência de anticorpos contra o vírus da língua azul em carneiros das mesorregiões do Sertão e da Borborema, semi-árido do Estado da Paraíba, bem como foram identificados os fatores de risco associados à infecção. A amostragem foi delineada para a determinação da prevalência de propriedades positivas (focos) e de animais soropositivos por mesorregião. Foi realizada uma seleção aleatória de unidades primárias, composta por 189 propriedades no Sertão e 100 propriedades na Borborema. Dentro das unidades primárias, foram amostrados todos os carneiros (unidades secundárias), resultando em 321 animais no Sertão e 185 na Borborema. Na ocasião da coleta, foi aplicado um questionário epidemiológico por propriedade. Para o diagnóstico sorológico, foi utilizada a prova de imunodifusão em gel de ágar (IDGA), com antígeno produzido na Escola de Veterinária da Universidade Federal de Minas Gerais). Uma propriedade foi considerada foco quando apresentou pelo menos um animal soropositivo. Na mesorregião do Sertão, as prevalências de focos e de animais soropositivos foram de 11,6% [7,8% - 17,1%] e 8,4% [5,7% - 12,3%], respectivamente. Na mesorregião da Borborema, a prevalência de focos foi de 0,0% [0,0% - 3,6%] e a prevalência de animais soropositivos foi de 0,0% [0,0% - 2,0%]. Os fatores de risco associados à língua azul foram a não realização de higiene das instalações (OR = 5,51) e a vermifugação dos animais duas a quatro vezes ao ano (OR = 4,44).
The prevalence of antibodies against Bluetongue virus in rams of the Sertão and Borborema mesoregions, semi-arid of the Paraíba state, Northeastern Brazil, was determined, and risk factors for the infection were identified. The sampling was delineated for the determination of the prevalence of positive herds and seropositive animals for each mesoregion. Herds (primary units) were randomly selected in the Sertão mesoregion (n = 189) and in the Borborema mesoregion (n = 100). Within the primary units, all rams (secondary units) were sampled, resulting in 321 animals of the Sertão mesoregion and in 185 animals of the Borborema mesoregion. During collection, an epidemiological questionnaire was applied to each herd. For serological diagnosis, the agar gel immunodiffusion test (AGID) with antigen produced in the School of Veterinary Medicine of the Federal University of Minas Gerais was carried out. Herds with at least one positive animal were considered positive. In Sertão mesoregion, the prevalence of positive herds and seropositive animals were 11.6% [7.8% - 17.1%] and 8.4% [5.7% - 12.3%], respectively. In Borborema mesoregion, the prevalence of positive herds was 0.0% [0.0% - 3.6%] and the prevalence of seropositive animals was 0.0% [0.0% - 2.0%]. The risk factors associated with Bluetongue were lack of cleaning and disinfection of facilities (OR = 5.51), and administration of anti-parasitic treatment twice to four times per year (OR = 4.44).
- Apr 2009
Objectives: The purpose of this work was to study the infection course and cross-protection in mice after intradermal injection of Vaccinia virus (VACV) strain Western Reserve and three Brazilian VACV strains: Aracatuba, Muriae and BeAn58058 isolated from cow, human and rodent, respectively. Methods: Balb/c mice were inoculated by footpad and back scarification and daily monitored regarding lesion development and weight loss. To check cross protection after intradermal VACV inoculation, mice were subsequently infected with different VACV strains and monitored to check lesion development. Serum neutralization assays were performed to check for the presence of antibodies against Orthopoxvirus. Results: After VACV intradermal inoculation the lesion development pattern was similar in mice infected with the different virus strains. By using the footpad scarification model, cross-protection among VACV strains was observed. Moreover, neutralizing antibodies against Orthopoxvirus were detected in sera from mice infected with all VACV strains. Conclusion: Although it was not possible to observe virulence differences among VACV strains isolated from cow, rodent and human using the murine model, this inoculation route showed to be an appropriated model to study lesions development since it mimics natural infections by VACV in nature.
Vaccinia virus was used as vaccine to eradicate smallpox. We report a zoonotic case of vaccinia virus infection in a 30-year-old patient who became infected after handling sick dairy cattle. The patient had inflamed lesions and systemic symptoms. Laboratory findings were indicative of down-modulated immune responses to the virus.
A clinical case of meningoencephalitis by Bovine herpesvirus 5 (BoHV-5) in a five-year-old cow was reported. The disease began with low spinal cord signs, characterized by incoordination, and these symptoms had never been related to this illness before. Signs of a brain syndrome were observed and the cow died in six days. At the histopathology, a spread non-supurative meningoencephalitis was diagnosed, and the virus identification was made by PCR and partial sequence of the glycoprotein G. This study confirm the BoHV-5 presence in the State of Minas Gerais, Brazil, describes new clinic characteristics, and show the importance of the disease in the differentiate diagnosis with others bovine central nervous system affections.
Vaccinia virus (VACV) has been associated with several bovine vaccinia outbreaks in Brazil, affecting cattle and humans. There are no available data about VACV environmental circulation or the role of wildlife in the emergence of an outbreak. Since VACV was isolated from rodents in Brazil, we investigated shedding and transmission of VACV strains in mice. The VACV excretion profile was assessed by PCR and chicken chorioallantoic membrane infection, revealing viral DNA and infectious virus in the faeces and urine of intranasally infected mice. Horizontal transmission was assessed by exposure of sentinel mice to wood shavings contaminated with excrement, to mimic a natural infection. Sentinel mice showed orthopoxvirus antibodies, and VACV DNA and infectious virus were detected in their faeces and intestines, even after six rounds of natural transmission. Together, these data suggest that murine excrement could play a relevant role in VACV spread and transmission, perhaps helping to explain how these viruses circulate between their natural hosts.
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