The length of BTV-8 viraemia in cattle according to infection doses and diagnostic techniques

Four groups of BTV free Frisian and cross bred calves were used to determine the length of viraemia following infection with different doses of BTV-8 Italian isolate. The first group of five animals was infected with 10 TCID₅₀ of BTV-8, the second group of four animals with 10³ TCID₅₀ and the third group, which also included four animals, was infected with 10⁶ TCID₅₀. A placebo containing uninfected tissue culture medium was given to the four animals of the fourth group. The viraemia was evaluated by real time RT-PCR and virus isolation. In all infected groups, virus isolation was able to detect infectious virus up to 39 days post infection (dpi) while RT-PCR was positive up to 151–157 dpi. Infectious dose did influence neither the length nor the pattern of BTV-8 viraemia and confirmed that real time RT-PCR remains positive although no circulating virus is detectable in the peripheral circulation.     

Establishment of inflammatory model induced by Pseudorabies virus infection in mice

BackgroundPseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear.ObjectivesOur study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection.MethodsKunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi.ResultsAt 10⁵–10⁶ TCID₅₀ PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRV-infected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10² TCID₅₀ of PRV produced a significant inflammatory mediator increase.ConclusionsAn inflammatory model induced by PRV infection was established in mice, and 10² TCID₅₀ PRV was considered as the best concentration for the establishment of the model.     

Experimental inoculation study indicates swine as a potential host for Hendra virus

Hendra virus (HeV) is a zoonotic virus from the family Paramyxoviridae causing fatal disease in humans and horses. Five-week-old Landrace pigs and 5-month-old Gottingen minipigs were inoculated with approximately 10⁷ plaque forming units per animal. In addition to fever and depression exhibited in all infected pigs, one of the two Landrace pigs developed respiratory signs at 5 days post-inoculation (dpi) and one of the Gottingen minipigs developed respiratory signs at 5 dpi and mild neurological signs at 7 dpi. Virus was detected in all infected pigs at 2–5 dpi from oral, nasal, and rectal swabs and at 3–5 dpi from ocular swabs by real-time RT-PCR targeting the HeV M gene. Virus titers in nasal swab samples were as high as 10^4.6 TCID₅₀/mL. The viral RNA was mainly distributed in tissues from respiratory and lymphoid systems at an early stage of infection and the presence of virus was confirmed by virus isolation. Pathological changes and immunohistochemical staining for viral antigen were consistent with the tissue distribution of the virus. This new finding indicates that pigs are susceptible to HeV infections and could potentially play a role as an intermediate host in transmission to humans.     

Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV)

The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6 month-old lambs. Twelve groups of two 6 month-old lambs were inoculated intravenously (i.v.) with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titers ranging from 10^7.6 TCID₅₀/lamb down to 10^−3.4 TCID₅₀/lamb and were monitored for seroconversion using the OPPV agar gel immunodiffusion assay (AGID). Fifteen of the 16 lambs given equal or greater than 10^0.6 TCID₅₀ seroconverted, and virus could be isolated from peripheral blood leukocytes in 13 out of the 15 of these lambs. None of the eight lambs receiving less than 10^0.6 TCID₅₀ seroconverted during the 12 months. The results of this study indicated that 10^0.6 or 4 TCID₅₀/lamb given i.v. was capable of establishing infection.     

一例猪伪狂犬病病毒的分离鉴定

从临床疑似猪伪狂犬病发病仔猪的脑组织等病料中,经PCR扩增出大小为217 bp的伪狂犬病病毒gp50的基因片段,结果证实为猪伪狂犬病病毒(porcine pseudorabies virus,PRV)感染。随后采用BHK-21细胞进行猪伪狂犬病病毒的分离培养,该分离株经细胞传代培养5代后,能够产生典型的细胞病变,经PCR鉴定为伪狂犬病病毒,其病毒感染力达10^8.68 TCID₅₀/0.1mL。最后用10^7.0 TCID₅₀/mL病毒培养物接种家兔,48 h后注射部位出现典型瘙痒、皮肤破损等症状,于72 h后全部死亡。结果表明,该伪狂犬病病毒分离株对易感动物具有高致病性,为进一步开展该病毒流行病学、致病机理、疫苗免疫及诊断研究奠定了基础。     

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3⁺, CD4⁺ and γδ T-cells; whereas CD8⁺ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.     

Replication of neurovirulent equine herpesvirus type 1 (EHV-1) in CD172a+ monocytic cells

Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disorders, abortion and myeloencephalopathy (EHM) in horses. Two pathotypes of EHV-1 strains are circulating in the field: neurovirulent (N) and non-neurovirulent (NN). For both strains, CD172a⁺ monocytic cells are one of the main carrier cells of EHV-1 during primary infection, allowing the virus to invade the horse’s body. Recently, we showed that EHV-1 NN strains showed a restricted and delayed replication in CD172a⁺ cells. Here we characterize the in vitro replication kinetics of two EHV-1 N strains in CD172a⁺ cells and investigate if the replication of these strains is similarly silenced as shown for EHV-1 NN strains. We found that EHV-1 N replication was restricted to 7–8% in CD172a⁺ cells compared to 100% in control RK-13 cells. EHV-1 N replication was not delayed in CD172a⁺ cells but virus production was significant lower (10^3.0 TCID₅₀/10⁵ inoculated cells) than in RK-13 cells (10^8.5 TCID₅₀/10⁵ inoculated cells). Approximately 0.04% of CD172a⁺ cells produced and transmitted infectious EHV-1 to neighbour cells compared to 65% of RK-13 cells. Unlike what we observed for the NN strain, pretreatment of CD172a⁺ cells with histone deacetylases inhibitors (HDACi) did not influence the replication of EHV-1 N strains in these cells. Overall, these results show that the EHV-1 replication of N strains in CD172a⁺ cells differs from that observed for NN strains, which may contribute to their different pathogeneses in vivo.     

Influence of environmental temperature on experimental infection of redfin perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss) with epizootic haematopoietic necrosis virus, an Australian iridovirus

SUMMARY Experimental transmission of epizootic haematopoietic necrosis virus (EHNV) to adult redfin perch Perca fluviatilis and juvenile rainbow trout Oncorhynchus mykiss was undertaken at different water temperatures using intraperitoneal (IP) and bath inoculation. Redfin perch were highly susceptible to EHNV by both routes of infection. Bath inoculation with as few as 0.08 TCID₅₀. _mL^-1 was lethal. The incubation period in redfin perch was about 11 days at a water temperature of 19–21°C but was longer at colder temperatures and disease did not occur at temperatures below 12°C. The longest incubation period recorded in redfin perch was 28 days. Rainbow trout were not susceptible to infection by bath inoculation but the disease was reproduced after IP inoculation with 10^5.6 TCID₅₀ at water temperatures ranging from 8–21°C. The incubation period was 3–10 days at 19–21°C, but was up to 32 days at 8–10°C. Persistent infection with EHNV was detected by virus isolation in a clinically unaffected rainbow trout after 63 days. The implications of these findings in the understanding of the epidemiology of EHNV infection are discussed.     

Correlating the immune response with the clinical-pathological course of persistent mastitis experimentally induced by Mycoplasma agalactiae in dairy goats

To correlate the clinical course of mycoplasma mastitis with its immune response, right mammary glands of 15 lactating goats were inoculating with 10¹⁰ colony-forming units (cfu) of Mycoplasma agalactiae (Ma). Before sacrificing the animals at 5, 15 or 45 days post-inoculation (dpi), blood Ma antibody titres and milk mycoplasma colony and somatic cell counts were monitored. Ma colonised the mammary gland and milk counts increased to over 10¹² cfu/ml within 5 dpi. During this period, an innate immune response involving neutrophils and macrophages was observed, and Ma antigen appeared in the degenerated acinar epithelium. From 7 dpi, a specific antibody response coincided with reduced viable mycoplasmas in milk. The humoral immune response was limited; by 37 dpi, all animals scored negative for anti-Ma antibodies, and around 10⁸ cfu/ml were shed. Results indicate an early immune response to Ma inoculation unable to control mycoplasmal invasion. An ensuing humoral response, despite reducing the mycoplasma burden, leads to chronic, persistent infection.     

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15 days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5?×?10⁵ TCID₅₀ of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5′-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.     

用间接免疫荧光检测方法评价猪瘟兔化弱毒活疫苗效力的研究

为用体外试验方法评价猪瘟兔化弱毒活疫苗效力,以猪瘟病毒抗体（兔源）为一抗、荧光素标记羊抗兔IgG 为二抗,建立了CSFV兔化弱毒株的间接免疫荧光检测方法（ IFA）。特异性试验表明,用该IFA方法检测CSFV兔化弱毒株接种的RK细胞为阳性,而检测伪狂犬病毒、猪细小病毒病、牛病毒性腹泻/粘膜病毒接种的RK细胞均为阴性。 CSFV兔化弱毒株和活疫苗的兔体感染量（ RID）用兔体测定,半数组织感染量（ TCID50）用IFA测定,拟合RID与TCID50的线性回归方程,确定1RID/mL＝（ TCID50/0．1mL＋200）/12。     

Analytical sensitivity of air samplers based on uniform point-source exposure to airborne Porcine reproductive and respiratory syndrome virus and swine influenza virus

Research and surveillance activities involving airborne pathogens rely on the capture and enumeration of pathogens suspended in aerosols. The objective of this study was to estimate the analytical sensitivity (detection threshold) of each of 4 air samplers for Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV). In a 5-min sampling period under controlled conditions, the analytical sensitivity of the AGI-30 (Ace Glass, Vineland, New Jersey, USA), AGI-4 (Ace Glass), SKC BioSampler (SKC, Eighty Four, Pennsylvania, USA), and Midwest Micro-Tek sampler (Midwest Micro-Tek, Brookings, South Dakota, USA) was calculated at 1 × 10^1.1, 1 × 10^1.3, 1 × 10^1.1, and 1 × 10^1.2 median tissue culture infectious dose (TCID₅₀) equivalents for PRRSV and 1 × 10^1.4, 1 × 10^1.1, 1 × 10^1.6, and 1 × 10^1.2 TCID₅₀ equivalents for SIV [per 60 L (5-min sampling period)]. Despite marked differences in sampler design, no statistically significant difference in analytical sensitivity was detected between the samplers for collection of artificially produced aerosols containing cell-culture-propagated PRRSV or SIV.     

B亚群禽白血病病毒SDAU09C2株的TCID50与p27抗原之间的相关性研究

为了研究B亚群禽白血病病毒(ALV-B)的半数细胞培养物感染量(TCID₅₀)与p27抗原的酶联免疫吸附试验(ELISA)的S/P值之间的相关性,本试验将ALV-B SDAU09C2 株接种鸡胚成纤维细胞(CEF细胞),换维持液后连续10 d取样,检测10 d的TCID₅₀值与p27抗原的S/P值之间的相关性;同时,将该毒株在DF-1细胞系上传代至20代,取其中的第1、5、10、15和20代分别进行TCID₅₀滴度的测定和p27抗原检测。结果表明,在CEF细胞上接种的ALV-B SDAU09C2株连续10 d的TCID₅₀值与p27抗原之间存在显著的正相关(r=0.94002;P<0.0001);在DF-1细胞系上传的不同代数之间也呈显著正相关(r=0.96449;P=0.0080)。由此可推测ALV-B的TCID₅₀与p27抗原呈显著正相关,可以用ELISA法测得的p27抗原的S/P值来估测病毒的TCID₅₀值。     

Development of maternal and foetal immune responses in cattle following experimental challenge with Neospora caninum at day 210 of gestation

This study examined the immunological responses of pregnant cattle and their foetuses following an experimental challenge with live Neospora caninum tachyzoites at day 210 of gestation. Animals were bled prior to and weekly throughout the experiment and sacrificed at 14, 28, 42 and 56 days post inoculation (dpi). At post mortem examination, samples of lymph nodes and spleen were collected from both dam and foetus for immunological analysis. Subcutaneous (sc) inoculation over the left prefemoral (LPF) lymph node of pregnant cattle at day 210 of gestation, led to the vertical transmission of parasites by 14 dpi, however no foetal deaths were observed in the infected animals. Foetuses from infected dams mounted Neospora-specific humoral and cell-mediated immune (CMI) responses by 14 dpi. These responses involved anti-Neospora IgG, antigen-specific lymphocyte proliferation, and the production of the cytokines IFN–γ, interleukin (IL)-4 and IL-10. There was also evidence of innate immunity during the response against Neospora from infected dams, with statistically significant (p < 0.05) increases in mean expression of toll like receptors (TLR)-2 on 56 dpi in maternal spleen, LPF, right prefemoral (RPF), left uterine (LUL) and right uterine (RUL) lymph nodes and TLR-9 in retropharyngeal (RLN), LPF and RPF lymph nodes from 28 dpi. Statistically significant (p < 0.05) increases in mean TLR-9 were detected in spleen samples from foetuses of infected dams, compared to the foetuses from control animals. Our results show that vertical transmission of the parasite occurred in all infected dams, with their foetuses showing effective Neospora-specific cell mediated, humoral and innate immune responses.     

无血清全悬浮PK15细胞培养猪圆环病毒2型的研究

为改进猪圆环病毒2型的培养工艺,驯化了一株可无血清培养的全悬浮PK15细胞用于培养猪圆环病毒2型,并对病毒的敏感性、接毒时间、接毒量、收获方法进行了试验.结果表明,用该细胞培养猪圆环病毒2型,如果采用批次收获,接毒时细胞密度为0.5×106/mL,接毒量为0.1 MOI,接毒72 h后收毒,病毒滴度能达到106.4 T...     

Experimental infection of highly pathogenic avian influenza virus H5N1 in black-headed gulls (Chroicocephalus ridibundus)

Historically, highly pathogenic avian influenza viruses (HPAIV) rarely resulted in infection or clinical disease in wild birds. However, since 2002, disease and mortality from natural HPAIV H5N1 infection have been observed in wild birds including gulls. We performed an experimental HPAIV H5N1 infection of black-headed gulls (Chroicocephalus ridibundus) to determine their susceptibility to infection and disease from this virus, pattern of viral shedding, clinical signs, pathological changes and viral tissue distribution. We inoculated sixteen black-headed gulls with 1 × 10⁴ median tissue culture infectious dose HPAIV H5N1 (A/turkey/Turkey/1/2005) intratracheally and intraesophageally. Birds were monitored daily until 12 days post inoculation (dpi). Oropharyngeal and cloacal swabs were collected daily to detect viral shedding. Necropsies from birds were performed at 2, 4, 5, 6, 7, and 12 dpi. Sampling from selected tissues was done for histopathology, immunohistochemical detection of viral antigen, PCR, and viral isolation. Our study shows that all inoculated birds were productively infected, developed systemic disease, and had a high morbidity and mortality rate. Virus was detected mainly in the respiratory tract on the first days after inoculation, and then concentrated more in pancreas and central nervous system from 4 dpi onwards. Birds shed infectious virus until 7 dpi from the pharynx and 6 dpi from the cloaca. We conclude that black-headed gulls are highly susceptible to disease with a high mortality rate and are thus more likely to act as sentinel species for the presence of the virus than as long-distance carriers of the virus to new geographical areas.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0084-9) contains supplementary material, which is available to authorized users.     

Biological properties of viral haemorrhagic septicaemia (VHS) virus: Adsorption and replication

Adsorption and growth of VHS virus of trout were studied in cultures of RTG-2 and FHM cells.Within 1 h virus infectivity in the culture fluid decreased by 90%. No measurable adsorption took place after this time. A higher adsorption rate was achieved by pretreatment of cell cultures with 10 μg zinc sulphate and 10 or 50 μg diethyl-aminoethyl (DEAE) dextran.In both cell lines small amounts of viral antigen could be detected in the perinuclear region by the immunofluorescence technique as early as 3 h after inoculation. Infectivity titres of the culture supernatants started to rise 8–10 h post-infection and reached their maximum after 18 h. In both cell lines, 50% or more of the total infectivity remained cell associated, the yields of free virus obtained from FHM cells being 0.5 log₁₀ TCID₅₀ lower.     

单细胞克隆技术提纯分化的IBRS-2细胞研究

IBRS-2细胞出现分化变异影响使用,为不影响正常的科学研究,本研究采用单细胞克隆的方法重新提纯,用96孔板经过3周的独立培养后,将具有代表性的4株进行扩增,然后用已知毒价的口蹄疫病毒(foot and mouth disease virus,FMDV)弱毒分别对其进行接毒试验和TCID₅₀测定。结果表明,B1株克隆细胞的病变最为理想,其TCID₅₀为7.60,比提纯前的4.52更接近实际毒价7.85,且和实际毒价无显著差异。