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.

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.

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...

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.

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.

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.

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.

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.

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.

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.

Avaliação sorológica para Toxoplasma gondii pela imunofluorescência indireta e detecção do vírus da imunodeficiência felina pela nested PCR em felinos selvagens / Serological evaluation for Toxoplasma gondii by indirect immunofluorescence and detection of feline immunodeficiency virus by nested PCR in wild felines

Nineteen sera and blood samples from wild feline kept in captivity were tested for Toxoplasma gondii antibody and presence of feline immunodeficiency virus (FIV) DNA, respectively. Eighteen (94.7 percent) of the them were seropositive for toxoplasma. However, the only negative animal, a Leopardus pardalis, was the only FIV positve. These results suggest that the infection by FIV may have compromised its immune system and interfered with antibody production for toxoplasma.

Perfil sorológico para circovírus suíno tipo 2 em granjas comerciais de suínos no Brasil / Serological profiles to porcine circovirus type 2 antibodies in Brazil

A técnica de imunoperoxidase em monocamada de células (IPMC) para demonstração de anticorpos contra o circovírus suíno tipo 2 (CVS2) foi empregada para a investigação sorológica em oito granjas de suínos destinadas à produção comercial. Das 240 amostras de soros testadas, 229 (95,4%) foram reagentes com títulos que variaram de 20 até 10.240. Títulos de anticorpos foram detectados nas faixas etárias de duas a três semanas até animais acima de 24 semanas e encontrados em granjas com e sem a síndrome multissistêmica do definhamento (SMD). A média dos títulos de anticorpos revelou diferenças estatísticas (P<0,05) nas faixas etárias de 11 a 13 e 14 a 22 semanas nos animais oriundos de granjas com e sem a SMD. Os resultados refletem a importância de conhecer o perfil sorológico do rebanho e assegurar a implantação de um efetivo cronograma de vacinação contra o CVS2.

The immunoperoxidase in monolayer cells (IPMC) technique for the demonstration of antibodies against type 2 porcine circovirus (PCV2) was used for the serological investigation in eight industrial swine farms. Out of the 240 tested sera, 229 (95,4%) were reactive with titers which varied from 20 up to 10.240. Titers of antibodies were detected in pigs from two to three weeks of age up to above 24 weeks of age, and were observed in farms with and without clinical signs indicative of the post-weaning multisystemic wasting syndrome (PMWS). Analysing the mean titers, statistical differences (P<0.05) were evidentiated in two age intervals, of 11 to 13 and 14 to 22 weeks of age. The results indicate the importance of determining the serological profiles of commercial swine herds, in order to enable the implantation of effective hygiene and vaccination protocols for PCV2 prevention.

PCR multiplex para identificação de isolados de Clostridium chauvoei e Clostridium septicum / Multiplex PCR for identification of Clostridium chauvoei and Clostridium septicum

Padronizou-se uma técnica de reação em cadeia da polimerase múltipla (PCR multiplex) para detecção de Clostridium chauvoei e Clostridium septicum em culturas puras. Foram utilizados pares de iniciadores para segmentos específicos dos genes que codificam a flagelina de C. chauvoei e a toxina alfa de C. septicum. Para avaliaçã o da PCR multiplex, foram testados 16 isolados clínicos de C. chauvoei e 15 isolados de C. septicum provenientes de ruminantes, quatro sementes vacinais de cada um desses agentes. Amostras de referência de ambos os microrganismos foram usadas como controle. Para avaliar a especificidade, DNAs genômicos dos seguintes microrganismos foram usados: C. sordellii, C. novyi tipo A, C. novyi tipo B, C. perfringens tipo A, C. haemolyticum, C. botulinum tipo D, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli e Salmonella typhimurium. Todos os isolados e sementes vacinais de C. chauvoei e C. septicum foram detectados pela técnica. Não foram observadas reações cruzadas com as outras espécies de clostrídios, outras espécies bacterianas ou entre C. Chauvoei e C. septicum. As menores concentrações de DNA de C. chauvoei e C. septicum detectadas foram 45pg/µl e 30pg/µl, respectivamente. A PCR multiplex pode ser utilizada para a identificação específica de C. chauvoei e C. septicum em culturas puras.

Multiplex PCR was optimized to detect Clostridium chauvoei and Clostridium septicum in pure cultures. In each reaction, a pair of primers for a specific segment of the flagellin gene of C. chauvoei and a pair of primers for a specific segment of alpha toxin gene of C. septicum were employed. Reference strains of both microorganisms were used as control. The multiplex PCR was evaluated by testing 16 clinical isolates of C. chauvoei from ruminants, 15 clinical isolates of C. septicum from ruminants and, four vaccine strains of each one of these agents. Reference strains of both microorganisms were used as control. To evaluate the specificity, genomic DNA of the following microorganisms was used: C. sordellii, C. novyi type A, C. novyi type B, C. perfringens type A, C. haemolyticum, C. botulinum type D, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli, and Salmonella typhimurium. All the isolates and vaccine strains of C. chauvoei and C. septicum were positive by PCR assay and cross reactions were not observed with the other species of clostridia, the other bacterial species or amongst both investigated agents. The smallest concentrations of DNA detected from C. chauvoei and C. septicum were 45pg/µl and 30pg/µl, respectively. The multiplex PCR was useful for the specific identification of C. chauvoei and C. septicum in pure cultures.

Perfil clínico e imunológico de bovinos experimentalmente inoculados com veneno bruto e iodado de Bothrops alternatus / Clinical and immunological characteristics in cattle experimentally inoculated with crude and iodinated Bothrops alternatus venom

Dez novilhas mestiças, distribuídas em dois grupos experimentais (n=5) receberam na altura média da face cranial do membro anterior direito, entre as articulações umerorradioulnar e do carpo, por via intramuscular superficial, 0,15mg/kg de veneno de Bothrops alternatus bruto ou iodado. Todos os animais foram avaliados clinicamente antes - tempo zero - e às 6 e 10h, no 2º, 3º, 4º, 5º, 8º, 11º, 18º e 25º dias após a inoculação dos venenos. Dois animais do grupo que recebeu veneno bruto foram a óbito às 53h e 78h e os sobreviventes apresentaram apatia, letargia, anorexia, postura indicativa de dor, melena, petéquias e sufusões nas mucosas, aumento do tempo de preenchimento capilar, enfartamento ganglionar regional, aumento das freqüências respiratória e cardíaca, redução da freqüência de pulsação arterial periférica, elevação da temperatura retal e diminuição da movimentação ruminal. No local da inoculação do veneno bruto houve sangramento e ulceração dérmica, além de aumento significativo na circunferência e dobra da pele do membro inoculado, revelando formação de edema. Todos os animais também foram avaliados imunologicamente no 17º, 24º, 31º, 45º, 60º e 180º dia. Somente os que receberam veneno bruto produziram anticorpos, detectados até o 45º dia. Os que receberam veneno botrópico iodado apresentaram alterações gerais e locais de menor intensidade, porém sem produção de IgG nos tempos pesquisados, demonstrando que a iodação alterou a composição bioquímica do veneno, diminuindo sua toxicidade e imunogenicidade.

The effects of bothropic envenomation in 10 crossbred heifers, randomly divided into two groups, that received 0.15mg/kg of body weight of Bothrops alternatus crude or iodinated venom were studied. Behavior; attitude; appetite; defecation; urination; mucous membranes; capillary perfusion time; lymph nodes; respiratory, cardiac and pulse frequencies; rectal temperature and rumination frequency diameter and skin fold of the foreleg on the inoculation site were observed before(zero time) and at 6 and 10h and on the 2nd, 3rd, 4th, 5th, 8th, 11th, 18th and 25th day after venom inoculation. Two cattle of Bothrops crude venom group, died at 53 and 78h and the surviving animals showed apathy, lethargy, anorexia, pain indicative attitudes, melena, hemorrhagic spots and suffusions on mucous membranes, increased capillary perfusion time, enlarged regional lymph nodes, increased respiratory and cardiac frequencies, decreased peripheric arterial pulse frequency, elevated rectal temperature and decreased of ruminal movements. Bleeding, necrotic point and increase (P< 0.05) diameter and skin fold of the foreleg were identified on the inoculated site, confirming local edema. All the animals were evaluated for Bothrops alternatus venom specific IgG on the 17th, 24th, 31st, 45th, 60th and 180th day. Only the animals receiving crude venom produced IgG specific until the 45th day. The animals inoculated with iodinated venom showed general alterations and minor local effects and did not produce specific IgG, indicating that the iodination decreased both toxicity and immunogenicity response.

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.