Gold nanoparticle-carboxymethyl cellulose nanocolloids for detection of human immunodeficiency virus type-1 (HIV-1) using laser light scattering immunoassay.

It is estimated that over 100 million people have been infected with human immunodeficiency virus (HIV-1) resulting in approximately 30 million deaths globally. Herein, we designed and developed novel nano-immunoconjugates using gold nanoparticles (AuNPs) and carboxymethylcellulose (CMC) biopolymer, which performed simultaneously as an eco-friendly in situ reducing agent and surface stabilizing ligand for the aqueous colloidal process. These AuNPs-CMC nanocolloids were biofunctionalized with the gp41 glycoprotein receptor (AuNPs-CMC-gp41) or HIV monoclonal antibodies (AuNPs-CMC_PolyArg-abHIV) for detection using the laser light scattering immunoassay (LIA). These AuNPs-CMC bioengineered nanoconjugates were extensively characterized by morphological and physicochemical methods, which demonstrated the formation of spherical nanocrystalline colloidal AuNPs with the average size from 12 to 20 nm and surface plasmon resonance peak at 520 nm. Thus, stable nanocolloids were formed with core-shell nanostructures composed of AuNPs and biomacromolecules of CMC-gp41, which were cytocompatible based on in vitro cell viability results. The AuNPs-CMC-gp41 nanoconjugates were tested against HIV monoclonal antibodies conjugates (AuNPs-CMC_PolyArg-abHIV) using the light scattering immunoassay (LIA) where they behaved as active nanoprobes for the detection at nM level of HIV-1 antigenic proteins. This strategy offers a novel nanoplatform for creating bioprobes using green nanotechnology for the detection of HIV-1 and other virus-related diseases.

Vaccinia virus detection in dairy products made with milk from experimentally infected cows.

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.

Use of atomic force microscopy as a diagnostic tool to identify orthopoxvirus.

Atomic force microscopy (AFM) is a versatile technique that permits the imaging of surfaces and generates topographical images from a variety of materials. Due to the fact that AFM requires minimum sample manipulation, it is a valuable tool for studying biological materials such as cells, DNA, bacteria and viruses. The aim of the present study was to standardize the AFM technique as a diagnostic tool for detection of naturally occurring orthopoxviruses. The samples analyzed were collected during natural outbreaks of Vaccinia virus (VACV) in dairy cattle in Brazil. These viruses are zoonotic infections; and therefore safe manipulation of all samples is required. The AFM technique would provide a more secure way to diagnose infection. By using the "in air" AFM technique after purification and inactivation process, relatively crude preparations of viruses were visualized rapidly. Details for efficient sample preparation and AFM imaging are described. The AFM technique provides a rapid and biosecure tool for the diagnosis of emerging orthopoxviruses and has potential as a tool for screening bioterrorism samples.

In vitro and in vivo assessment of nanotoxicity of CdS quantum dot/aminopolysaccharide bionanoconjugates.

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 5days) and three colloidal concentrations (10nM, 50nM, and 100nM). 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 30days, 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.

Serological Survey of Porcine circovirus-2 in Captive Wild Boars (Sus scrofa) from Registered Farms of South and South-east Regions of Brazil.

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.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

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.

Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens.

The role of individual viral proteins in the immune response to bluetongue virus (BTV) is not clearly understood. To investigate the contributions of the outer capsid proteins, VP2 and VP5, and possible interactions between them, these proteins were expressed from recombinant vaccinia viruses either as individual proteins or together in double recombinants, or with the core protein VP7 in a triple recombinant. Comparison of the immunogenicity of the vaccinia expressed proteins with BTV expressed proteins was carried out by inoculation of rabbits and sheep. Each of the recombinants was capable of stimulating an anti-BTV antibody response, although there was a wide range in the level of response between animals and species. Vaccinia-expressed VP2 was poorly immunogenic, particularly in rabbits. VP5, on the whole, stimulated higher ELISA titers in rabbits and sheep and in some animals in both species was able to stimulate virus neutralizing antibodies. When the protective efficacy of VP2 and VP5 was tested in sheep, vaccinia-expressed VP2, VP5 and VP2 + VP5 were protective, with the most consistent protection being in groups immunized with both proteins.

Infection by Neospora caninum associated with bovine herpesvirus 1 and bovine viral diarrhea virus in cattle from Minas Gerais State, Brazil.

Neospora caninum is a canine parasite which is considered a significant cause of bovine abortion. Two cattle herd groups were serologically studied with the objective of studying the prevalence of infection by N. caninum associated with BHV1 and BVDV infections. In group I, 15 dairy herds (476 samples) naturally infected by the three infectious agents were analyzed,. In group II, three dairy herds (100 samples) of cows vaccinated for two viruses were analyzed, in order to determine the infection prevalence by N. caninum. In the first group, an infection prevalence of 12.61, 34 and 28.3% was determined for N. caninum BHV1 and BVDV, respectively. In the second group, a seropositive prevalence of 46, 85 and 76%, respectively, was determined for N. caninum, BVH1 and BVDV. In the first group, the virus and N. caninum had shown in the first group 4.41% positive samples in association with BVH1, 3.15% with BVDV, and 8.41% with BVH1 and BVDV.

The ovine cytotoxic T lymphocyte responses to bluetongue virus.

This study uses recombinant vaccinia viruses expressing truncated or entire bluetongue virus (BTV) proteins to map the location of epitopes recognized by cytotoxic T lymphocytes (CTL) from Australian merino sheep. The non-structural protein, NS1, was recognised by CTL from all sheep, while VP2, VP3, VP5 and VP7 were recognised by CTL from only some sheep. The remaining proteins (except for VP1, which was not tested) did not contain CTL epitopes. When truncated genes were used to map the location of CTL epitopes, it was found that sheep often have CTL that recognise more than one epitope in NS1 or VP2. Overall there was considerable diversity in the CTL recognition patterns in the sheep tested.

Lack of antibody protection against Porcine circovirus 2 and Porcine parvovirus in naturally infected dams and their offspring.

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.

Detection of Vaccinia virus in blood and faeces of experimentally infected cows.

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.

Looking back: a genetic retrospective study of Brazilian Orf virus isolates.

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.

Brazilian IgY-Bothrops antivenom: Studies on the development of a process in chicken egg yolk.

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.

The effects of sow viremia and maternal antibodies in porcine circovirus 2 on viral infection and weight of piglets / Efeito da viremia da porca e de anticorpos maternos para circovírus suíno 2 na infecção e no peso da leitegada

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 log10 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...

O objetivo deste estudo foi caracterizar o efeito do infecção pelo circovírus suíno 2 (PCV2) em porcas gestantes na viremia e no peso da leitegada. Vinte porcas e 100 leitões recém-nascidos foram acompanhados. Amostras de colostro e soro das porcas foram obtidas no dia do parto. Amostras de leite foram coletadas no dia pós-parto 20. Os leitões foram pesados e tiveram amostras de soro coletadas nos dias um, 20, 42, 63 e 84 pós-parto. Soro, colostro e leite foram testados para carga viral do PCV2. Soro foi avaliado para presença de anticorpos neutralizantes. O DNA do PCV2 foi encontrado em 14 de 20 amostras de colostro e em 11 de 20 amostras de leite. No dia pós-parto 1, 29% dos leitões foram virêmicos. A carga viral do PCV2 variou 3,02-6,75 log10 cópias / mL, considerando todos os dias amostrados. Não houve correlação entre viremia das porcas e os níveis de anticorpos no soro ou na carga de PCV2 no colostro e na viremia dos leitões com um dia de vida. A carga de PCV2 no colostro e no leite foi associada à viremia em leitões do desmame até 84 dias pós-parto. A carga viral do PCV2 em leitões não foi associada com o peso ao longo deste estudo...

Utilização de minerais iônicos ou complexos orgânicos de minerais no pré-parto de vacas Holandesas / Use of ionic minerals or organic mineral complexes in the dry period of Holsteins cows

Os efeitos do uso de minerais complexados durante o pré-parto sobre a ocorrência de retenção de placenta foram avaliados em 135 vacas Holandesas de dois ou mais partos: grupo mineral iônico (69 animais) e grupo mineral complexado (66 animais). Em 55 desses animais foram também avaliadas as concentrações séricas da imunoglobulina G (IgG), Zn, Cu e a qualidade do colostro. O experimento foi realizado em delineamento inteiramente ao acaso, em arranjo em parcelas subdivididas. As concentrações séricas de IgG e dos microminerais foram avaliadas por análise de variância, sendo utilizados, respectivamente, os testes de Duncan e Fisher. A taxa de erro α admitida foi de 7%. Não foram observadas diferenças entre os grupos para ocorrência de retenção de placenta, qualidade do colostro, concentrações séricas de Zn e IgG (P>0,07), sendo observada diferença para a concentração de Cu (P<0,07). As concentrações de IgG foram diferentes nas semanas pré-parto avaliadas (P<0,07).

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).

Anticorpos contra vírus do grupo da língua azul em caprinos e ovinos do sertão de Pernambuco e inferências sobre sua epidemiologia em regiões semiáridas / Antibodies against bluetongue-virus group in goats and sheep from Pernambuco state and inferences on bluetongue epidemiology under tropical conditions

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 percent for goats (n = 410) and 4.3 percent for sheep (n = 400). The prevalences of seropositive animals were low in the mesoregion of Sertão Pernambucano (4.8 percent for goats and 4.1 percent for sheep) and São Francisco Pernambucano (1.0 percent for goats and 4.5 percent 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.

In vitro evaluation of Clostridium septicum alpha toxoid / Avaliação in vitro de toxoide alfa de Clostridium septicum

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 percent, indicating that cell culture is a viable alternative in the evaluation of vaccine potency, screening of vaccinal seeds, and Clostridium septicum alpha toxin titration.

Padronizou-se um teste para detecção de anticorpos neutralizantes in vitro, em cultura de células. O modelo in vitro mostrou-se mais sensível que os testes com animais, permitindo a detecção de títulos de toxina e antitoxina alfa mais baixos. Os títulos observados na soroneutralização in vivo e in vitro, apresentaram correlação de 99,12 por cento, demonstrando ser a cultura de células uma alternativa viável na avaliação da potência de vacinas, triagem de sementes vacinais e titulação de toxina alfa de Clostridium septicum.

Prevalence of antibodies to selected viruses in bovine embryo donors and recipients from Brazil, and its implications in international embryo trade.

The prevalence of serum antibody to enzootic bovine leukosis (EBL), blue-tongue (BT), bovine herpesvirus 1 (BHV 1) and bovine virus diarrhoea (BVD) viruses in bovine embryo donors (D) and recipients (R) from Minas Gerais State, Brazil was investigated. Of 451 sera (130 D plus 321 R) tested for antibodies against EBL virus 104 (23.1%) were positive. Of 410 sera (130 D plus 280 R) tested for antibody to BT, BHV 1 and BVD viruses the respective number of positive sera were 313 (76.3%), 209 (51.0%) and 153 (37.3%). Donors had significantly (Chi-square test, p < 0.05) higher prevalence rates of antibody than recipients to EBL and BVD viruses. The donors were 60 Bos indicus and 70 Bos taurus purebred cows. Antibody to EBL virus was significantly less common among Bos indicus.

Meningoencefalite por Herpesvirus bovino 5 em Minas Gerais: relato de caso clínico / Meningoencephalitis by Bovine herpesvirus 5 in Minas Gerais state: clinical case report

Relata-se um caso de meningoencefalite causada por Herpesvirus bovino 5 (BoHV-5) heritabilityem uma vaca com cinco anos de idade. O animal manifestou quadro clínico inicial de síndrome medular baixa, caracterizada por incoordenação dos membros pélvicos, sinais estes ainda não descritos para a enfermidade. Dentro de pouco tempo a doença evoluiu para síndrome cerebral, e o óbito ocorreu seis dias após o inicio dos sintomas. Na histopatologia, evidenciou-se meningoencefalite difusa, não supurada, e a confirmação do diagnóstico foi feita por reação em cadeia de polimerase e sequenciamento do segmento parcial da glicoproteína G do vírus. O trabalho confirma a presença do BoHV-5 em Minas Gerais, descreve características clínicas novas para a enfermidade e ressalta sua importância no diagnóstico diferencial das neuropatias bovinas.

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.

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

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.