Neutralizing antibodies to bovine and bubaline alphaherpesviruses in water buffaloes (Bubalus bubalis).

Water buffaloes (Bubalus bubalis) have been introduced in many regions of the world as a source of animal protein. In many instances, bubaline cattle are reared close to or mixed with bovine or zebuine cattle. However, little is known about infectious diseases of bubaline and the interactions that may arise involving the microbiota of those species. Alphaherpesviruses of ruminants (bovine alphaherpesviruses types 1 and 5, BoHV-1, BoHV-5; bubaline alphaherpesvirus 1, BuHV-1) are highly cross-reactive in serological assays performed with bovine or zebuine sera. However, the profile of reactivity of bubaline cattle sera to alphaherpesviruses remains unknown. As such, it is not known which virus strain (or strains) would be most appropriate to be used as the challenge virus in the laboratory in search for alphaherpesvirus-neutralizing antibodies. In this study, the profile of neutralizing antibodies to alphaherpesviruses in bubaline sera was determined against different types/subtypes of bovine and bubaline alphaherpesviruses. Sera (n=339) were screened in a 24-h serum neutralization test (SN) against 100 TCID50 of each of the challenge viruses. From those, 159 (46.9 %) neutralized at least one of the viruses assayed; 131 (38.6%) sera neutralized the three viral strains used for screening. The viral strain that was neutralized by the largest number of sera was BoHV-5b A663 (149/159; 93.7%). A few sera neutralized only one of the challenge viruses: four sera neutralized BoHV-1 LA only; another neutralized BoHV-5 A663 only and four others neutralized BuHV-1 b6 only. SN testing with two additional strains gave rise to similar results, where maximum sensitivity (defined here as the largest number of sera that neutralized the challenge viruses) was obtained by adding positive results attained with three of the challenge strains. Differences in neutralizing antibody titers were not significant to allow inferences on which would be the most likely virus that induced the antibody responses detected here.

An ELISA to Detect Antibodies to Bovine Alphaherpesviruses 1 and 5 and Bubaline Alphaherpesvirus 1 in Cattle Sera.

Bovine alphaherpesvirus 1 (subtypes 1.1, 1.2a, and 1.2b), type 5 (subtypes 5a, 5b, and 5c), and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity (κ = 0.93; PPV = 95.0%; NPV = 97.3%). The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses.

Molecular survey of porcine respiratory disease complex pathogens in Brazilian wild boars.

Wild boar (Sus scrofa) is an exotic invasive species in Brazil and may be a reservoir for several pathogens, including those related to the porcine respiratory disease complex (PRDC), a critical infectious disease in pig production. The objective of this study was to investigate viral and bacterial pathogens related to PRDC in free-living wild boars from Brazil. Eighty animals were examined in search of genomes of porcine circovirus 2 (PCV2), Torque teno Sus virus 1a (TTSuV1a) and 1b (TTSuV1b), Influenza A virus (IAV), Actinobacillus pleuropneumoniae, Glaesserella parasuis, Pasteurella multocida, and Mycoplasma hyopneumoniae. The results demonstrated that 57.5% (46/80) of the animals had at least one detected pathogen, and 11.3% of them (9/80) were co-infected. TTSuV1a was the most prevalent genome, for which risk factors were associated with increased contact between wild boars and other animals. The other pathogens were detected at much lower frequencies or not detected (M. hyopneumoniae and IAV). An additional IAV serology search identified H1N1pdm09 antibodies in 35.5% (16/45) of the wild boars, bringing concern related to public health. In conclusion, wild boars are infected with pathogens that cause swine diseases, so their eventual contact with domestic pigs might risk animal production in Brazil.

Investigation on porcine circovirus type 3 in serum of farrowing sows with stillbirths.

Since its first identification in 2016, porcine circovirus 3 (PCV3) has been detected in healthy and/or diseased swine in many countries worldwide. In a previous study by our group, PCV3 was detected in sera of sows which had at least one stillborn piglet in the last parturition. As such, it became important to investigate if the presence of PCV3 in sows' sera could be associated to the occurrence of stillbirths. With that aim, the frequency of PCV3 infections and viral DNA loads in sows' sera was investigated through a real-time quantitative PCR in 89 serum samples of just farrowed sows with or without stillbirths. PCV3 genomes were identified in most samples, with genome loads ranging between less than 10 to 200,000 copies per mL of serum. No significant differences were observed either in the frequency of infection or PCV3 viral loads in sows with or without stillbirths. Thus, no association could be established between PCV3 infection of sows at farrowing and stillbirths' occurrence.

Viral DNA genomes in sera of farrowing sows with or without stillbirths.

A study was conducted to investigate the serum virome of sows with and without stillbirths after farrowing. Sera from sows with at least one stillbirth or with normal litters were collected immediately after farrowing. Viral DNA was extracted from serum pools and submitted to high throughput sequencing. No differences in the proportion of virus-related reads were found in both groups (p > 0.05). A variety of viral DNA genomes were identified, mostly representative of three viral families: Anelloviridae, Circoviridae and Smacoviridae. Besides, a number of novel unclassified circular Rep-encoding single stranded DNA (CRESS DNA) viruses were also identified. These findings suggest that the presence of such viral genomes in sows' sera bears no correlation with stillbirths' occurrence; it seems likely that these constitute part of the normal serum microbiome of sows at farrowing.

Genome sequence of bubaline alphaherpesvirus 1 (BuHV1) isolated in Australia in 1972.

Bubaline alphaherpesvirus 1 (BuHV1) is a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. To date, no full genome sequence of BuHV has been published. Here, we report the complete genome sequence of bubaline alphaherpesvirus 1 (BuHV1) strain b6 (BuHV1-b6), isolated from a water buffalo (Bubalus bubalis) in 1972 in Australia. The virus was multiplied in MDBK cells, and the DNA was extracted and subjected to high-throughput sequencing. The reads were aligned and combined into a single genome sequence, with bovine alphaherpesvirus 5 (BoHV5) strain SV507/99 (accession number NC005261) as a reference. The BuHV1-b6 genome is a linear double-stranded DNA molecule, 137,452 bp long, with a GC content of 76.8%. The genome consists of two unique sequences: a long, or UL, sequence (103,818 bp) and a short, or US, sequence (9,586 bp), with the latter being flanked by inverted IR and TR elements of 12,024 bp each. The arrangement is typical of herpesvirus genomes of the D-type. The overall sequence has a 92.2% similarity at the nucleotide level to the reference BoHV5 strain. Our report provides a significant landmark in the history of herpesviruses, represented by the genome sequence of this 44-year-old virus isolate.

Ungulate copiparvovirus 1 (bovine parvovirus 2): characterization of a new genotype and associated viremia in different bovine age groups.

A novel bovine parvovirus 2 (BPV2) genotype comprising 5394 nt was identified by next generation sequencing from sera of healthy cattle at different age groups farmed in the state of Rio Grande do Sul, Brazil. The genome organization of new BPV2 genotype retains the two ORFs typical of members of the Parvovirinae with 86.4 % of overall nucleotide sequence identities in comparison to other members of the subfamily. Phylogenetic analysis revealed similar clustering with two previously described bovine BPV2 within the genus Copiparvovirus. No significant differences (P ≥ 0.05) were detected in the distribution of BPV2 infection in cattle at different age groups. This is the third complete or near complete genome sequence of BPV2 reported to date and may contribute to a better understanding of the biology of copiparvoviruses and its interactions with the host.

Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) viral loads in serum of postweaning multisystemic wasting syndrome (PMWS)-affected and healthy pigs in Brazil.

Associations between Torque teno sus viruses (TTSuVs) and the occurrence of postweaning multisystemic wasting syndrome (PMWS) have been reported with controversial results. Currently, no studies have been performed comparing simultaneously viral loads of TTSuVs and PCV2. To examine the role for TTSuVs in PMWS-affected animals, a SYBR Green-based quantitative PCR (qPCR) was designed to detect and quantify TTSuV1, TTSuV2 and PCV2 genomes in swine sera. TTSuV1 genome loads were significantly higher in healthy adults than in young and SPF animals (p<0.05) suggesting that the prevalence of TTSuV1 infection increases with age and bears no association with PMWS. Regarding TTSuV2, no significant variation was detected in viral loads within any of the groups. As expected, PCV2 genome loads were higher in PMWS-affected swine than in healthy or SPF animals (p<0.001). These findings provide clear evidence to indicate that neither TTSuV1 nor TTSuV2 viral loads have any correlation with the occurrence of PMWS.

Chicken anemia virus and avian gyrovirus 2 as contaminants in poultry vaccines.

This study focuses on the detection of chicken anemia virus (CAV) and avian gyrovirus 2 (AGV2) genomes in commercially available poultry vaccines. A duplex quantitative real-time PCR (dqPCR), capable of identifying genomes of both viruses in a single assay, was employed to determine the viral loads of these agents in commercially available vaccines. Thirty five vaccines from eight manufacturers (32 prepared with live and 3 with inactivated microorganisms) were examined. Genomes of CAV were detected as contaminants in 6/32 live vaccines and in 1/3 inactivated vaccines. The CAV genome loads ranged from 6.4 to 173.4 per 50 ng of vaccine DNA (equivalent to 0.07 to 0.69 genome copies per dose of vaccine). Likewise, AGV2 genomes were detected in 9/32 live vaccines, with viral loads ranging from 93 to 156,187 per 50 ng of vaccine DNA (equivalent to 0.28-9176 genome copies per dose of vaccine). These findings provide evidence for the possibility of contamination of poultry vaccines with CAV and AGV2 and they also emphasize the need of searching for these agents in vaccines in order to ensure the absence of such potential contaminants.

Full-Genome Sequence of a Reassortant H1N2 Influenza A Virus Isolated from Pigs in Brazil.

In this study, the full-genome sequence of a reassortant H1N2 swine influenza virus is reported. The isolate has the hemagglutinin (HA) and neuraminidase (NA) genes from human lineage (H1-δ cluster and N2), and the internal genes (polymerase basic 1 [PB1], polymerase basic 2 [PB2], polymerase acidic [PA], nucleoprotein [NP], matrix [M], and nonstructural [NS]) are derived from human 2009 pandemic H1N1 (H1N1pdm09) virus.

Partial sequence analysis of B2L gene of Brazilian orf viruses from sheep and goats.

We herein describe the partial nucleotide sequencing and phylogenetic analysis of the B2L gene of seventeen Brazilian orf viruses (ORFV). Seventeen viruses were recovered from outbreaks of contagious ecthyma in sheep and goats in four states in Southern and Northeast country, and three from commercial vaccines. Most analyzed viruses were associated with outbreaks of classical contagious ecthyma, with lip, nostrils and labial commissure involvement, yet udder/teat, feet, vulvar and disseminated lesions were also reported in some cases. Nucleotide sequence analysis revealed a high degree of B2L similarity among sheep sequences (>99%) regardless the geographic origin, and a remarkable high identity for the two goat isolates (>99.8%), with similarity dropping to below 99% when comparing viruses from the two species. A phylogenetic tree grouped most sheep and goat viruses on different branches. In addition, sequence alignment allowed the identification of up to six scattered nucleotide changes that were predominant and more consistent in goat isolates, including a number of sequences from other continents. Thus, in spite of the high nucleotide similarity, different degrees of similarity and discrete nucleotide changes in the B2L gene may help in grouping ORFV viruses according to host species.

Vaccinia viruses isolated from cutaneous disease in horses are highly virulent for rabbits.

Two genotypically distinct Vaccinia viruses (VACV), named P1V and P2V, were isolated from an outbreak of cutaneous disease in horses in Southern Brazil. We herein investigated the susceptibility of rabbits, a proposed animal model, to P1V and P2V infection. Groups of weanling rabbits were inoculated intranasally (IN) with P1V or P2V at low (10(2.5) TCID50), medium (10(4.5)TCID50), or high titer (10(6.5)TCID50). Rabbits inoculated with medium and high titers shed virus in nasal secretions and developed serous to hemorrhagic nasal discharge and severe respiratory distress, followed by progressive apathy and high lethality. Clinical signs appeared around days 3-6 post-inoculation (pi) and lasted up to the day of death or euthanasia (around days 5-10). Virus shedding and clinical signs were less frequent in rabbits inoculated with low virus titers. Viremia was detected in all groups, with different frequencies. Viral DNA was detected in the feces of a few animals inoculated with P1V and P2V, low titer, and with P2V at high titer. Gross necropsy findings and histological examination showed diffuse interstitial fibrousing pneumonia with necrosuppurative bronchopneumonia and intestinal liquid content. Neutralizing antibodies were detected in all inoculated animals surviving beyond day 9 pi. These results show that rabbits are highly susceptible to VACV isolated from horses, and develop severe respiratory and systemic disease upon IN inoculation. Thus, rabbits may be used to study selected aspects of VACV infection and disease.

Vaccinia viruses isolated from skin infection in horses produced cutaneous and systemic disease in experimentally infected rabbits.

The susceptibility of rabbits to two isolates of Vaccinia virus (VACV) recovered from cutaneous disease in horses in Southern Brazil was investigated. Rabbits were inoculated in the ear skin with both VACV isolates, either in single or mixed infection. All inoculated animals presented local skin lesions characterized by hyperaemia, papules, vesicles, pustules and ulcers. Infectious virus was detected in the lungs and intestine of rabbits that died during acute disease. Histological examination of the skin revealed changes characteristic of those associated with members of the genus Orthopoxvirus. These results demonstrate that rabbits develop skin disease accompanied by systemic signs upon intradermal inoculation of these two equine VACV isolates, either alone or in combination, opening the way for using rabbits to study selected aspects of the biology and pathogenesis of VACV infection.

Vacina experimental produzida em cultivo celular confere proteção parcial contra o ectima contagioso em ovinos / Experimental vaccine produced in tissue culture confers partial protection against contagious ecthyma in sheep

O ectima contagioso (também conhecido como orf), é uma doença debilitante de ovinos e caprinos causada pelo vírus do orf (ORFV). A vacinação tem sido usada com relativo sucesso no controle da doença. No entanto, as vacinas atuais contêm amostras virulentas do agente, são produzidas por escarificação cutânea de animais, e apresentam eficácia questionável. Assim, o presente trabalho teve como objetivo produzir e testar a eficácia de uma vacina experimental produzida em cultivo celular. A cepa IA-82 do ORFV foi submetida a 21 passagens em cultivo de células BHK-21 e usada para vacinar ovinos jovens (n=30), por escarificação cutânea na face interna da coxa. A vacinação produziu pústulas e crostas em 16 dos 30 ovinos vacinados, indicando imunização adequada. Noventa dias após a vacinação, ovinos vacinados (n=16) e controles (n=16) foram inoculados com uma cepa virulenta do ORFV (10(6,9)DICC50/mL) após escarificação na comissura labial. Todos os animais desenvolveram lesões típicas de ectima, incluindo hiperemia, vesículas, pústulas e crostas. No entanto, os animais vacinados desenvolveram lesões mais leves e passageiras do que os controles, e os escores clínicos foram estatisticamente diferentes (p<0,05) entre os dias 10 e 22 pós-desafio. Além disso, o tempo de duração da doença foi significativamente inferior (p<0,05) nos animais vacinados. Os animais vacinados também excretaram menor quantidade de vírus (p<0,05) e por um período significativamente mais curto do que os controles (13 dias versus 22 dias, p<0,001). Esses resultados demonstram a proteção parcial conferida pela vacina experimental e, dependendo da melhoria dos índices de imunização e proteção, são promissores no sentido da utilização de vacinas contra o ORFV produzidas em cultivo celular.

Contagious ecthyma, also known as orf, is a debilitating disease of sheep and goats caused by the parapoxvirus, orf virus (ORFV). Vaccination has been used with relative success to reduce the losses caused by the disease, yet the current vaccines contain virulent virus, are empirically produced through skin scarification of live lambs, and present questionable efficacy. Therefore, the present study aimed at developing and testing an experimental ORFV vaccine produced in tissue culture. The ORFV strain IA-82 was submitted to 21 passages in BHK-21 cells and then used to immunize lam bs (n=30) through skin scarification of the internal face of the hind limb. Vaccination produced localized pustules and scabs lesions in 16 out of 30 animals, indicating an adequate replication of the vaccine virus. Ninety days after vaccination, vaccinated (n=16) and control lambs (n=16) were inoculated with a virulent ORFV strain (10(6,9)TCID50/ml) in the labial commissure. Vaccinated and control lambs developed typical orf lesions, characterized by hyperemia, vesicles, pustules and scab formation. Nonetheless, vaccinated animals developed milder lesions compared to controls and the clinical scores were significantly lower (p<0.05) between days 10 and 22 post-challenge. In addition, the mean duration of clinical disease was significantly reduced in vaccinated animals (p<0.05). Furthermore, vaccinated animals excreted much less virus (p<0.05) and for a significantly shorter period of time than did the controls (13 days versus 22 days, p<0.001). These results demonstrate partial protection by the experimental vaccine and, upon improvement of immunization and protection indices, are promising towards the use of tissue culture-based ORFV vaccines.

Óleos essenciais como substituintes de antibióticos promotores de crescimento em frangos de corte: perfil de soroproteínas e peroxidação lipídica / Essential oils as substitutes for antibiotic growth promoters in broilers: soroproteins profile and lipid peroxidation

Os óleos essenciais são alternativas ao uso de promotores de crescimento antibióticos na avicultura, devido à sua ação antimicrobiana, além de possuírem propriedades antioxidante e imunomoduladora. Este estudo foi realizado com o objetivo de avaliar o efeito da suplementação dietética de três doses de óleos essenciais (OLES) de orégano (Origanum vulgare L.), sálvia (Salvia officinalis L.), alecrim (Rosmarinus officinalis L.) e extrato de pimenta (Capsicum frutescens L.) em frangos de corte, pela análise do perfil eletroforético de soroproteínas e da peroxidação lipídica plasmática. Os animais (n=910) foram alocados de forma aleatória em cinco tratamentos, com sete repetições de 26 aves cada: o grupo controle (Tc), que recebeu dieta basal sem aditivos; o grupo que recebeu promotor de crescimento antibiótico na dieta (Tatb); e os grupos T50, T100 e T150, alimentados com OLES na doses de 50, 100 e 150mg kg-1, respectivamente. Aos 42 dias de idade, sete animais foram aleatoriamente selecionados (um de cada repetição) para o estudo do perfil eletroforético de soroproteínas e para a avaliação da peroxidação plasmática de lipídeos, pelo teste de formação de substâncias reativas ao ácido tiobarbitúrico (TBARS). Houve diminuição na concentração de globulinas totais no T150 e na fração betaglobulina nos grupos Tatb e T150 em relação ao grupo controle e ao T50 (P<0,05). Além disso, os níveis de TBARS plasmático foram menores nos grupos que receberam OLES em comparação ao Tc (P<0,05). Dessa forma, pode-se inferir que o efeito dos OLES, na maior dose administrada, sugere menor estímulo ao sistema inume humoral de frangos de corte, assim como acontece com a suplementação de promotores de crescimento antibióticos. Adicionalmente, ocorre menor peroxidação plasmática de lipídios e, consequentemente, menos dano oxidativo em frangos de corte, em resposta ao uso dos OLES.

Essential oils are an alternative to growth promoters based on antibiotics used in animal diets, due to its antimicrobial potential, and immunomodulatory properties. Serum proteins electrophoresis and plasma lipid peroxidation were evaluated in broilers fed with diets supplemented with antibiotics or essential oils from oregano (Origanum vulgare L.), sage (Salvia officinalis L.), rosemary (Rosmarinus officinalis L.) and pepper (Capsicum frutescens L.) crude extract (OLES). The animals (n=910) were distributed within five treatment groups and seven replicates containing 26 birds each one: control group (diet without additives); the group receiving an antibiotic growth promoter diet (Tatb); and the groups T50, T100 and T150 (supplemented with 50, 100 and 150mg kg-1 of OLES, respectively). After 42 days, seven animals were randomly selected for serum proteins electrophoretic fractionation and plasma lipid peroxidation evaluation by thiobarbituric acid reactive species (TBARS) test. Total globulins (T150), betaglobulin fraction (Tatb and T150) and plasma TBARS levels in the groups that received OLES (P<0.05) presented a decrease in relation to the control group. These results suggests lower stimulus to the humoral immune response at the higher dose of OLES, as occurred in the antibiotic growth promoter group. Moreover, it suggests lower lipid peroxidation and, consequently, lower oxidative damage caused by OLES use in broiler chickens.