Backyard pigs are a reservoir of zoonotic hepatitis E virus in southern Brazil.

Background: Hepatitis E virus (HEV) is the causative agent of acute hepatitis worldwide. There is no seroprevalence study in backyard farms, which are characterized by suboptimal hygienic conditions in Brazil. We aimed to determine the seroprevalence and genetic diversity of HEV in backyard pigs in Brazil. Methods: Swine serum samples collected in 2012 (n=731) and 2014 (n=713) were analysed. The presence of anti-HEV immunoglobulin G in pig serum was evaluated by indirect enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction was performed and phylogenetic analyses were carried out based on the partial ORF1 and ORF2 coding regions. Results: Anti-HEV antibodies were detected in 77.6% (567/731; 95% confidence interval [CI] 74.5 to 90.6%) of serum samples in 2012 and 65.5% (467/713; 95% CI 62.0 to 69.0%) in 2014. The herd seroprevalence was 91.7% (187/204; 95% CI 91% to 99%) in 2012 and 83.7% (164/196; 95% CI 78% to 89%) in 2014. Further, HEV RNA was detected in 0.8% (6/713) of samples from 2014. Phylogenetic analysis showed three different genotype 3 subtypes with high similarity to human HEV strains. Conclusions: This study showed that backyard pigs are a reservoir of HEV and alerts us to the need to control infection and spillover from backyard farms. GenBank accession numbers: MF438128-MF438135.

Prevalence of Rhodococcus equi from the nasal cavity of 1010 apparently healthy horses.

BACKGROUND: Rhodococcus equi is an important cause of foal pneumonia. While its isolation from different sources has been widely evaluated, there is a need to better understand the R. equi epidemiology from samples of the nasal cavity of healthy horses. OBJECTIVES: To determine the prevalence of R. equi from the nasal cavity of healthy horses, along with its virulence profile, antimicrobial susceptibility and environmental variables associated. STUDY DESIGN: Cross-sectional study. METHODS: Swabs from the nasal cavity of 1010 apparently healthy horses from 341 farms were submitted for bacteriological analyses. The identity and virulence profile of the R. equi isolates were assessed by multiplex PCR; antimicrobial susceptibility was determined by the disk-diffusion method. The occurrence of R. equi was calculated at the level of both animal and farm. The association of seven specific environmental factors with R. equi isolation was assessed using logistic regression and by a spatial scan statistical method to determine the presence of local clusters. RESULTS: Antimicrobial-sensitive R. equi was isolated from 10 (1%) of 1010 horses ranging between 3 and 29 years old. Ten farms (3%) had at least one positive horse. Only one R. equi isolate (10%) was classified as virulent. Red-Yellow Argisol (PVA/PV) soils were significantly associated with R. equi isolation (odds ratio (OR) 8.02; CI95% , 1.98-32.50, P = 0.01), and areas with well-drained soil were less likely to be test positive (OR 0.85; CI95% , 0.76-0.96, P = 0.03). MAIN LIMITATIONS: The use of culture-based method instead of PCR-based assay and the lack of soil sampling. CONCLUSIONS: Antimicrobial-sensitive R. equi may be considered a minor part of the normal bacterial flora in the nasal cavity of healthy and immunologically functional horses breeding on pasture. Further studies are warranted to determine if soils rich in iron and well-drained are, in fact, associated with the occurrence of R. equi.

Bovine Viral Diarrhoea Virus (BVDV) in Dairy Cattle: A Matched Case-Control Study.

Bovine viral diarrhoea virus (BVDV) causes one of the most important diseases of cattle in terms of economic costs and welfare. The aims were to estimate herd prevalence and to investigate the factors associated with antibodies in bulk tank milk (BTM) in dairy herds through a matched case-control study. To estimate herd prevalence, BTM samples were randomly selected (n = 314) from a population (N = 1604). The true prevalence of BVDV was 24.3% (CI 95% = 20.1-29.3%). For the case-control study, BVDV antibody-positive herds (high antibody titres) were classified as cases (n = 21) and matched (n = 63) by milk production with herds presenting low antibody titres (ratio of 1 : 3). Three multivariable models were built: 1) full model, holding all 21 variables, and two models divided according to empirical knowledge and similarity among variables; 2) animal factor model; and 3) biosecurity model. The full model (model 1) identified: age as a culling criteria (OR = 0.10; CI 95% = 0.02-0.39; P < 0.01); farms that provided milk to other industries previously (OR = 4.13; CI 95% = 1.17-14.49; P = 0.02); and isolation paddocks for ill animals (OR = 0.14; CI 95% = 0.01-0.26; P = 0.02). The biosecurity model revealed a significant association with the use of natural mating (OR = 9.03; CI 95% = 2.14-38.03; P < 0.01); isolation paddocks for ill animals (OR = 0.06; CI 95% = 0.05-0.83; P = 0.03); years providing milk for the same industry (OR = 0.94; CI 95% = 0.91-0.97; P = 0.02); and direct contact over fences among cattle of neighbouring farms (OR = 5.78; CI 95% = 1.41-23.67; P = 0.04). We recommend the application of grouping predictors as a good choice for model building because it could lead to a better understanding of disease-exposure associations.

Seroprevalence of Brucella ovis in rams and associated flock level risk factors in the state of Rio Grande do Sul, Brazil.

A cross-sectional study based on a planned probabilistic sampling was carried out to estimate animal and flock prevalence of Brucella ovis in rams, as well as to determine risk factors at the flock level. Data regarding the flocks were collected by means of a questionnaire applied on 705 farms in the state of Rio Grande do Sul, Brazil, using one-stage cluster sampling. From the 705 flocks, 20 (2.5%, CI95%: 2.0-3.1%) had at least one positive ram. At the animal level, out of 1800 rams, 52 were positive (2.89%, CI95%: 0.4-5.3%). Statistical analysis identified the following as risk factors: average age of rams in the flocks (PR: 1.99, CI95%: 1.19-3.32); farms larger than 5 km(2) (500 ha) on extension area (PR: 7.46CI95%: 2.03-27.43); and the lack of lambing paddocks (PR: 5.56, CI95%: 1.70-18.11). This study provided relevant information for authorities to elaborate plans for the first Brazilian state based B. ovis disease control and eradication program. To the authors' knowledge, this is the first study that shows the importance of lambing paddocks in order to keep pre-lambing and lambing ewes away from the rest of the flock, the lack of this infrastructure was considered an important risk factor for B. ovis.

High frequency of bovine viral diarrhea virus type 2 in Southern Brazil.

Ruminant pestiviruses can infect cattle populations worldwide and cause significant economic losses due to their impact on productivity and health. Knowledge of pestivirus diversity is important for control programs and vaccine development and for determining probable sources of infection. In this paper, we describe a search for ruminant pestiviruses with RT-PCR in sera of 9078 calves from 6 to 12 months of age. The calves were first analyzed in pools and then analyzed individually. Thirty-three RT-PCR positive animals were detected (0.36%) from 6.9% (24) of the 346 herds. The sequencing analysis of the 5' non-coding region and N terminal autoprotease showed the presence of BVDV-1a (15 isolates), -1b (3), -1d (1) and -2b (14), with a higher frequency (42.4%) of BVDV-2 in comparison with other countries. The presence of sheep was significantly associated with BVDV infection. Our results also suggested that a BVDV control program based only on the investigation of cattle would not be successful, especially in regions with farms harboring multiple animal species. This study may also serve as a reference for future control programs in Southern Brazil because it reports the prevalence of cattle with active infections and the genetic background of the circulating strains.

Frequency of antibodies against Besnoitia besnoiti in Brazilian cattle.

Besnoitia besnoiti is a cyst-forming parasite that has been associated with economic losses in Africa and Europe. Besnoitiosis is considered as a re-emergent disease in the European continent. It is unknown whether cattle are exposed to B. besnoiti in the Americas, thus the aim of this study was to serologically investigate antibodies against B. besnoiti in a total of 2014 cattle serum samples from two states from Brazil. All samples were evaluated by IFAT and part of the positive sera was tested by Western blot (WB) using tachyzoites extracts under non-reducing condition. A total of 3.48% (70/2014) of the tested sera reacted positively by IFAT with titers of 200 (85.7%), 400 (10%) and 800 (4.3%). When 47 positive samples were assessed by WB a range of antigens from 7 to 206 kDa was recognized by the IFAT-positive sera. The results are suggestive of exposure of Brazilian cattle to B. besnoiti due to the titers (≥ 200) observed for some sera using IFAT. However, the antigens recognized by the IFAT-positive animals did not completely match with the WB patterns previously described by other working groups. It is possible that Brazilian cattle are exposed to B. besnoiti strains with different antigenic composition of those described in the European and African continent. Further studies are needed to confirm the presence of B. besnoiti or other Besnoitia species in Brazilian cattle.

Herd-level risk factors for bovine viral diarrhea virus infection in dairy herds from Southern Brazil.

A cross-sectional study was carried out to identify risk factors for bovine viral diarrhea virus (BVDV) infection in 300 randomly selected dairy herds which were tested for antibodies in bulk tank milk (BTM) using a commercial indirect ELISA kit (SVANOVA). Results from the analysis were interpreted according to the Swedish BVDV control scheme. The testing revealed 129 (43%) BTM BVDV antibody-positive herds. Use of artificial insemination (AI) and herd size were significantly associated with BVDV serological status (P<0.05). Dairy herds that use AI had 2.82 increased odds of BVDV-seropositivity (95% CI: 1.02-7.24). Since the semen used in the studied population come from known selected sires, it was hypothesized that AI technicians should represent an important risk factor because the increasing number of visitors in the farm can introduce the virus through the clothes, shoes and contaminated equipment.

Combination of monoclonal antibodies improves immunohistochemical diagnosis of Neospora caninum.

Histological analysis is commonly used for a conclusive diagnosis of neosporosis. Immunohistochemistry (IHC) using monoclonal (mAb) and polyclonal (pAb) antibodies can improve diagnosis; however, the use of pAb may induce cross-reactivity with other related parasites. The aims of this study were to compare the performance of mAbs and their combinations with that of pAb in IHC and evaluate the usefulness of mAb to identify Neospora caninum infection in aborted bovine fetal tissues. For this purpose, mAbs targeting NcSRS2 (4.15.15) or NcGRA7 (4.11.5 and 1/24-12) and one pAb collected from a rabbit inoculated with N. caninum tachyzoites were tested by IHC. Artificial standardized tissue sections were prepared as positive controls using homogenized bovine brain spiked with cultured tachyzoites of N. caninum. The numbers of labeled parasites were counted in each positive control section. In addition, four equal proportional combinations of the mAbs were also analyzed in the IHC. Finally, the pAb and the best combination of mAbs obtained in the positive control experiments were tested with tissue sections of naturally-infected cattle. To confirm analytical specificity, mAbs and a pAb were tested with Toxoplasma gondii and Besnoitia besnoiti positive control slides and tissues sections from naturally infected cattle containing Sarcocystis spp. and B. besnoiti antigens. The mAb 4.15.15 detected 57% of the total parasites in sections while 4.11.5 and 1/24-12 were able to detect 49% and 41%, respectively. For the mAb combinations (I: 1/24-12+4.11.5, II: 1/24-12+4.15.15, III: 4.15.15+4.11.5, IV: 1/24-12+4.11.5+4.15.15), the detection capacity was 32.4%, 79.4%, 66.6% and 60.7% for each combination, respectively. The best mAb combination (1/24-12 and 4.15.15) and the pAb serum detected 100% (18/18) of naturally-infected animals. Sarcocystis spp. or B. besnoiti were not detected by mAb combinations in IHC, however the pAb cross-reacted with Sarcocystis spp. cysts. These results confirm the usefulness of mAb application in IHC to N. caninum.

Molecular frequency and isolation of cyst-forming coccidia from free ranging chickens in Bahia State, Brazil.

The Toxoplasmatinae parasites Toxoplasma gondii, Neospora caninum and Hammondia spp. have carnivores as definitive hosts that shed the parasite oocysts in their feces. Birds that feed directly from the soil, such as chickens, are exposed to infection and may serve as indicators of the presence of the parasite in the environment and as a source of infection for other animals. The aims of this study were to determine the frequency of infection by these parasites in free ranging chickens, to test whether chickens are intermediate hosts of Hammondia spp., and to isolate N. caninum from chickens. One hundred chickens, which were raised in contact to cattle and dogs, were bought in five towns located in Bahia, Brazil. Blood and tissues (brain and heart) were used for serology, molecular tests and bioassay in mice for parasite isolation. T. gondii DNA was detected in 29 chickens, and N. caninum DNA was observed in six animals. Hammondia spp. DNA was not detected in tissues from any chicken. Tissues from eight N. caninum seropositive chickens were bioassayed in interferon-gamma gene knockout mice, but the mice did not become infected; T. gondii was isolated from six of 14 seropositive chickens after bioassay in outbreed Swiss mice. The authors concluded that: chickens seem to be better hosts for T. gondii when compared to N. caninum, based on the molecular and bioassay results; Hammondia spp. probably does not infect chickens or is rarely found in this animal species.

Risk assessment of the introduction of H5N1 highly pathogenic avian influenza as a tool to be applied in prevention strategy plan.

Risks of the introduction of highly pathogenic avian influenza (HPAI) H5N1 through migratory birds to the main wintering site for wild birds in southern Brazil and its consequences were assessed. Likelihoods were estimated by a qualitative scale ranging from negligible to high. Northern migrants that breed in Alaska and regularly migrate to South America (primary Charadriiformes) can have contact with birds from affected areas in Asia. The likelihood of the introduction of HPAI H5N1 through migratory birds was found to be very low as it is a probability conditioned to successful transmission in breeding areas and the probabilities of an infected bird migrating and shedding the virus as far as southern Brazil. The probability of wild species becoming exposed to H5N1-infected birds is high as they nest with northern migrants from Alaska, whereas for backyard poultry it is moderate to high depending on proximity to wetlands and the presence of species that could increase the likelihood of contact with wild birds such as domestic duck. The magnitude of the biological and economic consequences of successful transmission to poultry or wild birds would be low to severe depending on the probability of the occurrence of outbreak scenarios described. As a result, the risk estimate is greater than negligible, and HPAI H5N1 prevention strategy in the region should always be carefully considered by the veterinary services in Brazil.

Thyroid surgery with harmonic focus, ligasure precise and conventional technique: a retrospective case-matched study.

BACKGROUND: New surgical technologies have been introduced in thyroid surgery, to achieve vessel sealing and hemostasis. The aim of the study was to examine their effectiveness and compare the outcome of total thyroidectomy using the Harmonic Focus™, the LigaSure® Precise and the conventional technique of suture ligation. METHODS: A retrospective case-matched study of all total thyroidectomies between October 2008 through May 2010 was conducted. Patients (n=240) underwent total thyroidectomy using three different methods; the Harmonic Focus™ (Group F, n=80), the LigaSure Precise® (Group L, n=80) and the conventional technique of suture ligation (Group C, n=80). RESULTS: No significant differences were identified between the 3 groups in terms of demographics, thyroid gland weight, pathologic diagnosis, preoperative and postoperative calcium levels, postoperative complications, duration of hospital stay, and final outcome. The operative time was shorter with Harmonic Focus® by about 15%. Statistical analysis revealed a statistically significant difference between operations with F (62.7±14.1 minutes) and C (72.7±13.6minutes) with (p=0.019). CONCLUSION: Both devices were safe and efficient. A significant reduction of the operative time was found with Harmonic Focus® compared to the other two techniques with no statistically significant differences in postoperative complications in the 3 groups.

Análise de série temporal para avaliação do perfil sorológico da leptospirose bovina no estado do Rio Grande do Sul de 1996 a 2006 / Time-series analysis for evaluating serological pattern of bovine leptospirosis in the state of Rio Grande do Sul from 1996 to 2006

A leptospirose é uma das principais enfermidades responsáveis pela baixa produtividade nos sistemas pecuários bovinos, sendo associada à redução na produção de leite, infertilidade, aborto, natimortalidade, além de aumento nos custos com despesas de assistência veterinária, vacinas e testes laboratoriais. Este estudo teve como objetivo analisar dados laboratoriais de sorologia para leptospirose bovina no Estado do Rio Grande do Sul, durante um período de 1996 a 2006, visando detectar tendências na frequência de reagentes e gerar hipóteses sobre a variação da frequência de soropositivos ao longo deste período. Análises de regressão linear e modelo linear generalizado foram realizadas para detectar tendências e verificar fatores que influenciam na frequência de soropositivos, respectivamente. Durante o período de 11 anos, foram analisadas 18.806 amostras de soro, sendo que 9.385 resultaram positivas (49,9%). A análise da série temporal por sorovar revelou uma tendência linear crescente estatisticamente significativa na frequência dos seguintes sorovares: Australis, Autumnalis, Bratislava, Copenhageni, Grippotyphosa, Pyrogenes e Tarassovi (p < 0,01). Por outro lado, foi observada uma tendência decrescente estatisticamente significativa na frequência dos sorovares Hardjo e Wolffi (p < 0,01) ao longo do período avaliado. Houve uma associação estatisticamente significativa entre índices pluviométricos (mm) e o número de soropositivos para Leptospira spp. (p = 0,01, OR = 1,003).

Leptospirosis is an infectious disease that causes losses of productivity in cattle due to reduction of milk production, reproductive disorders and calf mortality, while also increasing costs associated with veterinary care, vaccines and diagnostic tests. The objective of this study was to analyze serological findings for bovine leptospirosis in the State of Rio Grande do Sul, Brazil, in order to detect temporal trends in the frequency of seropositive animals and to generate hypothesis about the fluctuation in seroprevalence along the period from 1996 to 2006. Simple linear regression and generalized linear models were used to analyze trends and verify factors influencing the frequency of seropositive animals, respectively. During 11 years, 18,806 serum samples were analyzed, in which 9,385 were positive (49.9%). The time-series analysis by serovars revealed a statistically significant increasing trend in the frequency of the following serovars: Australis, Autumnalis, Bratislava, Copenhageni, Grippotyphosa, Pyrogenes and Tarassovi (p < 0.01). Conversely, a statistically significant decreasing trend was observed for the serovars Hardjo and Wolffi (p < 0.01). There was a significant association between rain precipitation (mm) and the number of samples seropositive for leptospirosis (p = 0.01, OR = 1,003).

[Comparison of conventional technique, Ligasure Precise and Harmonic Focus in total thyroidectomy]. / Studio comparativo tra tecnica tradizionale, Ligasure Precise ed Harmonic Focus nella tiroidectomia totale.

The aim of this study was to compare the results obtained using an electrothermal bipolar vessel sealing system (Ligasure Precise), a harmonic curved shears (Harmonic Focus) and traditional technique in total thyroidectomy. We have enrolled 93 patients and assigned randomly to three groups of 31 pt: groups L (Ligasure Precise), F (Harmonic Focus) and C (traditional thecnique). Recorded data were demographics, preoperative serum calcium levels, operation time, length of hospital stay, weight of exported gland and pathology, postoperative calcemia at one and two days and recurrent laryngeal nerve paralysis. The three groups did not present statistically significant differences in term of age, gender and pathology classification. No postoperative haemorrhages were observed. The overall incidence of hypocalcemia was 38.9% (36 pt) and the mean days of hospitalization were 2.3 days without statistically significant differences between the three groups. Only one patient (group F) presented temporary recurrent laryngeal nerve paralysis. Mean operation time (minutes) was significantly reduced by approximately 15% in group F (62.7+/-14.1) compared with group C (72.7+/-13.6; Kruskal-Wallis test: p<0.05). Both devices resulted safe and efficient. The only advantage observed was a significant reduction operation time when using Harmonic Foscus curved shears compared to the other techniques.

Why is there recurrence after transcystic laparoscopic bile duct clearance? Risk factor analysis.

AIM: Incidence of common bile duct stones (CBDS) is approximately 10% in patients with symptomatic gallstones undergoing laparoscopic cholecystectomy. Transcystic laparoscopic common bile duct exploration (TC-CBDE) is safe and efficient in achieving bile duct clearance from stones, with a success rate of between 85% and 95%. The aim of this retrospective study is the evaluation of risk factors and recurrence in patients treated with TC-CBDS. METHODS: From October 2003 until September 2007, 110 consecutive patients with common bile duct stones and gallbladder were included in the study. Average age was 64 years (range 21-82 years). The procedure was completed laparoscopically in 106 patients (96.4%): with TC-CBDE in 90 patients (85%), who are the object of this study; and with TC-CBDE + perioperative guide wire papillotomy ("rendezvous") in 16 cases (15%). In the 90 patients treated with TC-CBDE which we examined, the risk factors were: preoperative liver function tests, diameter of the common bile duct (normal 8 mm or less), number of stones (3 with biliary sludge predicted a recurrence (p < 0.05) while the other factors did not show important clinical variables. CONCLUSION: TC-CBDE is safe and effective in the majority of cases of CBDS. The incidence of recurrence is low but there are some risk factors, such as number of stones >3 with biliary sludge, which do not favor the successful outcome of the procedure. In such cases, it is essential that the TC-CBDE is integrated with other procedures which, although more complex, assure the clearance of the bile duct.

[Preliminary results in the use of a new composite prosthesis in incisional hernia repair]. / Risultati preliminari sull'utilizzo di una nuova protesi intraperitoneale nel trattamento del laparocele.

The repair of incisional hernias with the use of prosthetic biomaterials is the standard of care today. There are different prosthetic biomaterials that can be used to repair incisional hernias. These materials can be divided into products that are single component or a combination. Incisional hernia repair using the intraperitoneal implantation of a prosthesis requires mesh with impervious properties. This is preliminary study with a new composite non resorbable mesh in polyethylene terephthalate-polyurethane (HI-TEX PARP MP) used for incisional hernia repair in intraperitoneal implantation. This mesh has one permeable side in polyethylene terephthalate (PET) for rapid tissue fixation and another side in polyurethane (PEU), hydrophobic in order to avoid cell penetration. This is a preliminary study of medical records of 24 patients (17 women and 7 men) in whom intraperitoneal placement of composite prosthetics in polyethylene terephthalate-polyurethane (HI-TEX PARP MP) was used between September 2004 and September 2006. The incisional hernias were recurrent in 8 patients. The underside of the mesh was placed in direct contact with the visceral peritoneum, whereas the upper side made contact with the subcutaneous tissue. No intraoperative complications occurred. Postoperatively, 1 had seromas, 1 had phlegmon of the wound without removing prosthetics. There was 1 death but not dependent of the surgical performance. The follow-up, was 12 months (range 1 month-2 years); none had discomfort; only one patient had recurrence. Intraperitoneal placement of HI-TEX PARP MP has several advantages over other techniques including minimal adhesions, a decreased risk of infection and recurrences. In addition this mesh is more economics than the other prosthetics in use.

Histopathological and immunohistochemical aspects of Neospora caninum diagnosis in bovine aborted fetuses.

Lesions suggestive of protozoal infection such as nonsuppurative myositis, nonsuppurative myocarditis, nonsuppurative pneumonia associated with focal cellular necrosis and multifocal necrotizing nonsuppurative encephalitis were observed in 89 (34%) out of 258 aborted bovine fetuses. Immunohistochemistry (IHC) revealed tachyzoites and cysts of Neospora caninum in 55 (61.8%) of these 89 fetuses. Several fetal variables were tested by multivariate logistic regression, in which increasing odds for positivity in IHC were associated with the presence of lesion in the lung (OR=12.11) and less autolytic brain tissue (brain consistency, OR=5.73). However, autolysis in the brain precluded presumptive diagnosis made by histopathology in 53% of the fetuses examined. These results indicate that, when fetal brain is autolyzed, the lung may be adequately used for the presumptive diagnosis of N. caninum infection. The results also confirmed that, even autolyzed, the brain is the most suitable organ for anti-N. caninum IHC.

Otimização da imunoistoquímica para detecção de herpesvírus bovino tipo 5 (BHV-5) em tecidos do sistema nervoso central fixados com formaldeído / Optimization of immunohistochemistry for Bovine Herpesvirus 5 (BHV-5) detection on formalin-fixed tissues of the central nervous system

Com o objetivo de otimizar a técnica de imunoistoquímica para detecção de herpesvírus bovino tipo 5 (BHV-5) em tecidos do sistema nervoso central fixado em formaldeído, foram avaliados diferentes métodos de digestão enzimática, diferentes anticorpos e reagentes para bloqueio de reações inespecíficas. As reações apresentaram a máxima intensidade de coloração específica e a quantidade mínima de coloração de fundo quando foram usadas protease de Streptomyces griseus (0,1%) ou proteinase K de Tritirachium album limber (0,05%), mediante incubação durante 15 minutos a 37ºC. Entre os anticorpos monoclonais analisados, dois foram capazes de detectar BHV-5. As reações inespecíficas foram bloqueadas mais efetivamente pela incubação do tecido com caseína (0,5%), durante cinco minutos, ou leite em pó (2,5%), durante 60 minutos, ou soro eqüino (2,5%), durante 60 minutos. Com a técnica otimizada foi possível a detecção de BHV-5 em material de arquivo.