Immunohistochemical localisation of rabbit haemorrhagic disease virus VP-60 antigen in early infection of young and adult rabbits

This study evaluated the time course distribution of rabbit haemorrhagic disease virus (RHDV) structural protein VP60 in tissues from experimentally infected rabbits from three different age groups. Viral VP60 antigen could not be detected in tissue samples from animals under four weeks, and only a few hepatocytes (0.01 to 0.2 per cent) were stained in the 6-week-old animals. A 6-week-old rabbit euthanised at 72 hpi showed VP60-labelling in hepatocytes and macrophages close to areas of inflammation. Viral VP60 antigen was detected as early as 12 hpi in a few hepatocytes (0.03 per cent) from adult animals. Within this age group, the extent of hepatocyte labelling considerably increased at 18 (3.0 per cent), 24 (25.5 per cent), 36 (50 per cent) and 48 (60 per cent) hpi. Extrahepatic viral VP60 antigen was also detected at 36 and 48 hpi in spleen macrophages and lymphocytes from adult rabbits. These findings support the hypothesis that the hepatocyte is the only cell type in the liver able to support RHDV replication almost immediately after viral infection.     

Effects of epidermal growth factor on atrophic enteritis in piglets induced by experimental porcine epidemic diarrhoea virus

Epidermal growth factor (EGF) promotes gastrointestinal mucosal recovery by stimulating the mitogenic activity of intestinal crypt epithelial cells. The aim of this study was to determine the effects of EGF on atrophic enteritis induced in piglets by experimental infection with porcine epidemic diarrhoea virus (PEDV) strain Dr13. Two groups of 12 conventional, colostrum-deprived, 1-day-old, large White-Duroc cross breed piglets were inoculated orally with PEDV (3 x 10(5) 50% tissue culture infective doses), with or without EGF (10 microg/kg/day, intraperitoneally once daily for 4 days after infection) and compared to 12 uninfected, untreated control piglets. PEDV+EGF piglets had less severe clinical signs than PEDV only piglets at 48 and 60 h post-infection (hpi). Histologically, the ratio of villous height:crypt depth of PEDV+EGF piglets was significantly higher than PEDV only piglets at 36 and 48 hpi. Immunohistochemistry for Ki67 demonstrated increased proliferation in intestinal crypt epithelial cells of PEDV+EGF piglets compared to PEDV only piglets at 36, 48 and 60 hpi. EGF stimulates proliferation of intestinal crypt epithelial cells and promotes recovery from atrophic enteritis in PEDV-infected piglets.     

Increased susceptibility of peripheral blood mononuclear cells to equine herpes virus type 1 infection upon mitogen stimulation: a role of the cell cycle and of cell-to-cell transmission of the virus

Equine herpesvirus-1 (EHV-1) is an important pathogen of horses, causing abortion and nervous system disorders, even in vaccinated animals. During the cell-associated viremia, EHV-1 is carried by peripheral blood mononuclear cells (PBMC), mainly lymphocytes. In vitro, monocytes are the most important fraction of PBMC in which EHV-1 replicates, however, mitogen stimulation prior to EHV-1 infection increases the percentage of infected lymphocytes. The role of the cell cycle in viral replication and the role of cluster formation in cell-to-cell transmission of the virus were examined in mitogen-stimulated PBMC. Involvement of the cell cycle was examined by stimulating PBMC with ionomycin/phorbol dibutyrate (IONO/PDB) during 0, 12, 24 and 36 h prior to inoculation. Cell cycle distribution at the moment of inoculation and the percentage of EHV-1 antigen-positive PBMC at 0, 12 and 24 hours post inoculation (hpi) were determined by flow cytometry and immunofluorescence microscopy, respectively. The role of clusters was examined by immunofluorescence staining within clusters of stimulated PBMC using antibodies against EHV-1. Significant correlations were found between the increase of cells in the S- or G2/M-phase after a certain time interval of prestimulation and the increase of EHV-1 antigen-positive cells. The percentage of clusters with adjacent infected cells significantly increased from 3.3% at 8 hpi to 23.7% at 24 hpi and the maximal number of adjacent infected cells increased from 2 to 7. Addition of anti-EHV-1 hyperimmune serum did not significantly alter these percentages. Mitogen stimulation favours EHV-1 infection in PBMC by: (i) initiating cell proliferation and (ii) inducing formation of clusters, thereby facilitating direct cell-associated transmission of virus.     

Detection of in ovo-inoculated infectious bronchitis virus by immunohistochemistry and in situ hybridization with a riboprobe in epithelial cells of the lung and cloacal bursa

In situ hybridization and immunohistochemistry were utilized to identify tissues infected in ovo with infectious bronchitis virus (IBV). Chicken embryos were inoculated in ovo (chorioallantoic sac) with the Arkansas (Ark) serotype of IBV at 18 days of age. At 24, 48, 72, and 120 hr postinfection (HPI), bursa, lung, spleen, heart, and thymus were collected, fixed in 10% neutral buffered formalin, and paraffin embedded. The digoxigenin-labeled antisense S1 riboprobe detected viral mRNA in the cytoplasm of respiratory epithelial cells in the primary bronchus at 24, 48, and 72 HPI. Viral mRNA was detected in bursa samples collected at 48 hr. Immunohistochemistry detected viral antigens in epithelial cells of the parabronchi and bursal tissues at 24 and 48 hr, respectively. No viral mRNA or antigen was detected by in situ hybridization or immunohistochemistry, respectively, in heart, thymus, or spleen at any time after inoculation. On the basis of these data, IBV apparently initially infects lung tissue, then migrates to and infects cells of the bursa. These results indicate that in situ hybridization can be useful in detection of IBV-infected chickens and in understanding the pathogenesis and virulence of IBV infection.     

Faecal shedding, alimentary clearance and intestinal spread of prions in hamsters fed with scrapie

Shedding of prions via faeces may be involved in the transmission of contagious prion diseases. Here, we fed hamsters 10mg of 263K scrapie brain homogenate and examined the faecal excretion of disease-associated prion protein (PrP(TSE)) during the course of infection. The intestinal fate of ingested PrP(TSE) was further investigated by monitoring the deposition of the protein in components of the gut wall using immunohistochemistry and paraffin-embedded tissue (PET) blotting. Western blotting of faecal extracts showed shedding of PrP(TSE) in the excrement at 24-72 h post infection (hpi), but not at 0-24 hpi or at later preclinical or clinical time points. About 5% of the ingested PrP(TSE) were excreted via the faeces. However, the bulk of PrP(TSE) was cleared from the alimentary canal, most probably by degradation, while an indiscernible proportion of the inoculum triggered intestinal infection. Components of the gut-associated lymphoid tissue (GALT) and the enteric nervous system (ENS) showed progressing accumulation of PrP(TSE) from 30 days post infection (dpi) and 60 dpi, respectively. At the clinical stage of disease, substantial deposits of PrP(TSE) were found in the GALT in close vicinity to the intestinal lumen. Despite an apparent possibility of shedding from Peyer's patches that may involve the follicle-associated epithelium (FAE), only small amounts of PrP(TSE) were detected in faeces from clinically infected animals by serial protein misfolding cyclic amplification (sPMCA). Although excrement may thus provide a vehicle for the release of endogenously formed PrP(TSE), intestinal clearance mechanisms seem to partially counteract such a mode of prion dissemination.     

Protection against bovine rotaviruses in newborn calves by continuous feeding of immune colostrum

Three pregnant cows were inoculated intramuscularly with inactivated vaccine to bovine rotavirus (BRV) serotype 1 (BRV-1) and serotype 2 (BRV-2). Serum neutralizing antibody (NA) titers against both serotypes increased significantly after immunization. NA titers of colostrum obtained from immunized cows against BRV-1 and BRV-2 were 29286 and 38109, respectively, which were significantly higher than those from non-immunized control cows. Nine and 6 colostrum deprived calves were orally challenged with BRV-1 and BRV-2, respectively, and monitored for clinical manifestation and viral shedding. Five calves of them, 3 with BRV-1 and 2 with BRV-2, received 2 l of milk replacer supplemented with 10% immune colostrum 2 hr before challenge and twice daily for the first 5 days after challenge. Other 10 calves, 6 with BRV-1 and 4 with BRV-2, were fed only milk replacer as controls. All control calves developed severe diarrhea and shed a large amount of BRV in feces, beginning from 24 to 48 hr after challenge inoculation. On the contrary, all calves but one fed colostrum supplement remained clinically healthy after challenge, and BRV was not detected in their feces during feeding immune colostrum. The possibility that continuous feeding of immune colostrum is capable of preventing newborn calves from diarrhea associated with BRV and viral shedding was suggested.     

Identification and characterization of duck enteritis virus dUTPase gene

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous and important enzyme that hydrolyzes dUTP to dUMP. Many viruses encode virus-specific dUTPase, which plays an essential role in maintaining the integrity of the viral DNA both by reducing the dUTP levels and by providing the substrate for the thymidylate synthase. A 1344-bp gene of duck enteritis virus (DEV) homologous to herpesviral dUTPase was first reported in this paper. The gene encodes a protein of 477 amino acids, with a predicted molecular mass of 49.7 kDa. Multiple sequence alignment suggested that DEV dUTPase was quite similar to other identified herpesviral dUTPase and functioned as a homotrimer. The five conserved motifs of DEV dUTPase with 3-1-2-4-5 arrangement have been recognized, and the phylogenetic analysis showed that DEV dUTPase was genetically close to the avian herpesvirus. Furthermore, RNA dot blot, western blot, and immunofluorescence analysis indicated that the enzyme was expressed at early and late stages after infection. Immunofluorescence also confirmed that DEV dUTPase localized in the cytoplasm of DEV-infected duck embryo fibroblasts as early as 4 hr postinfection (hpi). Later, the enzyme transferred from cytoplasm to nucleus at 8 hpi, and then reached its expression peak at 12 hpi, both in the cytoplasm and nucleus. The results suggested that the DEV dUTPase gene might be an early viral gene in DEV vitro infection and contribute to ensuring the fidelity of genome replication.     

Oxidized-low density lipoprotein inhibits cyclic AMP production by porcine luteal cells

Oxidized(OX)-low density lipoprotein (LDL) inhibits steroidogenesis by luteal cells (LC) from regressing porcine CL. The present study was designed to investigate the mechanism of inhibition by determining whether OX-LDL inhibits basal and agonist-stimulated cAMP production in regressing LC. Collagenase-dispersed porcine LC (n = 7 animals, estrous cycle Day 12-15) were cultured (2.5 x 10(5) cells/0.5 ml) in serum-free DMEM/Hams F-12 in duplicate wells at 37 degrees C. Approximately 18 hr after plating, media were replaced and LC were immediately treated with human LDL (0, 25, or 100 microg/ml) or OX-LDL (25 or 100 microg/ml). LC were incubated for 2 hr before addition of isobutylmethylxanthine (IBMX) to inhibit phosphodiesterase activity, immediately followed by hCG (100 ng/ml), cholera toxin (CT; 0.1 microM), forskolin (FS; 50 microM), or no further treatment (controls). LC were incubated for an additional 90 min. After removal of culture media, cells were extracted with 0.1 N HCl. Cell extracts were assayed for cAMP by enzyme immunoassay (EIA). HCG, CT, and FS increased (P < 0.05) cAMP production approximately four-, 10-, and 25-fold, respectively, relative to controls. OX-LDL (25 and 100 microg/ml) inhibited (P < 0.05) cAMP production by unstimulated, hCG-, and CT-stimulated LC, but not that by FS-stimulated LC. The highest concentration of OX-LDL (100 microg/ml) reduced cAMP formation by 39.8 +/- 6.6%, 44.7 +/- 10.5%, and 67.7 +/- 4.5% in unstimulated, hCG-, and CT-stimulated LC, respectively. In contrast, unmodified LDL (25 and 100 microg/ml) did not alter cAMP production. We conclude that OX-LDL can interfere with the cAMP signaling pathway in regressing luteal cells by acting at sites proximal to adenylate cyclase activation.     

Effect of topical ophthalmic application of cidofovir on experimentally induced primary ocular feline herpesvirus-1 infection in cats

OBJECTIVE: To evaluate the efficacy of twice-daily ophthalmic application of 0.5% cidofovir solution in cats with experimentally induced primary ocular feline herpesvirus-1 (FHV-1) infection. ANIMALS: Twelve 6-month-old sexually intact male cats. PROCEDURES: Cats were randomly assigned to either a treatment or control group. Ocular infection with FHV-1 was induced (day 0) in all cats via inoculation of both eyes with 10(4) plaque-forming units of a plaque-purified FHV-1 field strain. Twice daily for 10 days beginning on day 4 after virus inoculation, the treatment group received 1 drop of 0.5% cidofovir in 1% carboxymethylcellulose in both eyes, and the control group received 1 drop of 1% carboxymethylcellulose in both eyes. A standardized scoring method was used to evaluate clinical signs of FHV-1 infection in each cat once daily for 24 days. The amount of ocular viral shedding was assessed by use of a quantitative real-time PCR procedure every 3 days during the study period. Clinical scores and viral quantification were averaged over the pretreatment (days 0 to 3), treatment (days 4 to 14), and posttreatment (days 15 to 24) periods for each cat. RESULTS: During the treatment period, clinical scores and amount of viral ocular shedding were significantly lower in the treatment group, compared with findings in the control group. CONCLUSIONS AND CLINICAL RELEVANCE: Twice-daily application of 0.5% cidofovir solution in both eyes significantly decreased the amount of viral shedding and the severity of clinical disease in cats with experimentally induced ocular FHV-1 infection.     

猪宿主蛋白G3BP1对猪圆环病毒2型在PK15细胞上复制的影响

猪宿主蛋白G3BP1在宿主抵抗病毒感染过程中起着重要作用,然而还未有相关报道G3BP1与猪圆环病毒2型(PCV2)感染过程中的联系。本文构建了G3BP1真核表达载体和干扰RNA,实现在PK15细胞上G3BP1的过表达及敲低。结果发现,PCV2感染细胞24 h后病毒复制出现显著差异,感染48 h相比于感染24 h病毒复制差异更显著,表明G3BP1能显著促进PCV2复制。过表达或敲低G3BP1后,用干扰素刺激DNA(ISD)刺激12 h, G3BP1能促进ISD诱导IFN-βmRNA的表达;感染PCV2 12 h后,G3BP1也能正调控IFN-βmRNA的转录。本文揭示了宿主蛋白G3BP1显著促进PCV2的复制,很可能是通过上调IFN-β表达引起的。     

The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.     

Improvement of the developmental ability of nuclear transfer embryos by using blastomeres from in vitro fertilized embryos selected according to the early developmental stage and cell division status as donor cells in cattle

This study was conducted to improve the developmental ability of nuclear transfer (NT) embryos by using blastomeres from in vitro fertilized (IVF) embryos with high quality as donor cells. The IVF embryos selected at the 2-cell stage at 24-h postinsemination (hpi) and again at the ≥8-cell stage at 48 hpi (Selected-IVF-embryos) showed the highest blastocyst formation rate among embryos. When blastomeres from the Selected-IVF-embryos (Selected-NT group) or Nonselected-IVF-embryos (Non-selected-NT group) were used as donor cells for NT, the blastocyst formation rate in the Selected-NT group (25.6%) was significantly higher than that in the Non-selected-NT group (13.5%). When blastomeres from the Selected-IVF-embryos at 108 (contained many cells before cell division) and 126 hpi (contained many cells immediately after cell division) were used as donor cells for NT (108- and 126-NT groups, respectively), the 126-NT group showed a significantly higher blastocyst formation rate (32.1%) than the 108-NT group (16.8%). Embryo transfer of blastocysts in the 126-NT group showed that 11 of 23 recipients became pregnant; nine calves were obtained. For the NT embryos reconstructed using in vivo derived embryos, 9 of 20 recipients became pregnant; seven calves were obtained. These results indicate that the blastocyst formation rate of NT embryos can be improved by using blastomeres from IVF embryos selected at the early developmental stage, especially immediately after cell division, and that the resultant NT embryos have a high developmental ability to progress to term that is comparable to NT embryos reconstructed using in vivo derived embryos.     

Pharmacokinetics of orally administered ibuprofen in African and Asian elephants (Loxodonta africana and Elephas maximus).

The pharmacokinetic parameters of S(+) and R(-) ibuprofen were determined in 20 elephants after oral administration of preliminary 4-, 5-, and 6-mg/kg doses of racemic ibuprofen. Following administration of 4 mg/kg ibuprofen, serum concentrations of ibuprofen peaked at 5 hr at 3.9 +/- 2.07 microg/ml R(-) and 10.65 +/- 5.64 microg/ml S(+) (mean +/- SD) in African elephants (Loxodonta africana) and at 3 hr at 5.14 +/- 1.39 microg/ml R(-) and 13.77 +/- 3.75 microg/ml S(+) in Asian elephants (Elephas maximus), respectively. Six-milligram/kilogram dosages resulted in peak serum concentrations of 5.91 +/- 2.17 microg/ml R(-) and 14.82 +/- 9.71 microg/ml S(+) in African elephants, and 5.72 +/- 1.60 microg/ml R(-) and 18.32 +/- 10.35 microg/ml S(+) in Asian elephants. Ibuprofen was eliminated with first-order kinetics characteristic of a single-compartment model with a half-life of 2.2-2.4 hr R(-) and 4.5-5.1 hr S(+) in African elephants and 2.4-2.9 hr R(-) and 5.9-7.7 hr S(+) in Asian elephants. Serum concentrations of R(-) ibuprofen were undetectable at 24 hr, whereas S(+) ibuprofen decreased to below 5 microg/ml 24 hr postadministration in all elephants. The volume of distribution was estimated to be between 322 and 356 ml/kg R(-) and 133 and 173 ml/kg S(+) in Asian elephants and 360-431 ml/kg R(-) and 179-207 ml/kg S(+) in African elephants. Steady-state serum concentrations of ibuprofen ranged from 2.2 to 10.5 microg/ml R(-) and 5.5 to 32.0 microg/ml S(+) (mean: 5.17 +/- 0.7 R(-) and 13.95 +/- 0.9 S(+) microg/ml in African elephants and 5.0 +/- 1.09 microg/ml R(-) and 14.1 +/- 2.8 microg/ml S(+) in Asian elephants). Racemic ibuprofen administered at 6 mg/kg/12 hr for Asian elephants and at 7 mg/kg/12 hr for African elephants results in therapeutic serum concentrations of this antiinflammatory agent.     

不同来源传染性支气管炎病毒诱导SPF鸡发病的免疫机制研究

试验旨在探讨不同来源的传染性支气管炎病毒(Infectious bronchitis virus,IBV)诱导SPF鸡发病的免疫机制。选用140只1日龄SPF白来航鸡,随机分为4组,3组攻毒组通过滴鼻点眼途径分别接种鸡源IBV强毒株、鸡源IBV弱毒株和野鸡源IBV毒株3个毒株,对照组以同种方式接种等量灭菌的磷酸盐缓冲液。在感染后12 h、36 h、72 h、7 d和14 d,每组随机选取5只进行剖检,并分别采集法氏囊、肾脏和气管组织,剩余鸡用于观察临床症状、发病及死亡情况。应用实时荧光定量PCR检测攻毒后不同时间点采集的各组织中IBV的病毒载量、Toll样受体(Toll-like receptors,TLRs)及部分细胞因子(白细胞介素(interleukin,IL)和干扰素(interferon,IFN))表达量的变化。结果显示,感染不同来源IBV毒株之后仅鸡源IBV强毒株感染组SPF鸡出现抑郁、翅膀下垂、甩头等典型的临床症状,且在感染后5~10 d共有7只死亡,死亡率为20%。病理剖检发现,感染鸡源IBV强毒株的鸡肾脏肿大、尿酸盐沉积和有花斑样病变,而感染野鸡源IBV毒株、鸡源IBV弱毒株和对照组的鸡无明显的眼观病变。实时荧光定量PCR结果显示,在鸡源IBV强毒株组的法氏囊、肾脏和气管3个组织中均检测到病毒。对照组和野鸡源IBV毒株组中均未检测到病毒,鸡源IBV弱毒株组只在部分组织中检测到病毒。在感染后72 h,鸡源IBV强毒株组与其他各组相比,TLR1、TLR2、TLR3、TLR5、TLR7和TLR15基因在法氏囊中的表达量均显著升高(P<0.05),IL-6和IFN-β参与更强烈的抗病毒免疫反应;在感染后7 d,鸡源IBV弱毒株组与其他各组相比,肾脏中TLR2、TLR3、TLR15、TLR21、IL-6和IL-18基因表达量均显著升高(P<0.05)。野鸡源IBV感染后36 h法氏囊组织中IFN-γ基因表达量显著上调(P<0.05)。综上所述,3个IBV毒株中仅鸡源IBV强毒感染引起SPF鸡典型临床发病症状与可视组织病变,且可提高SPF鸡组织中免疫相关因子的基因表达量。本研究结果揭示,不同来源的IBV对SPF鸡的不同致病性与其感染诱导的免疫反应不同有关。     

Pharmacokinetics of piperacillan after intramuscular injection in red-tailed hawks (Buteo jamaicensis) and great horned owls (Bubo virginianus).

This study characterized and compared the pharmacokinetics of piperacillin after single 100 mg/kg i.m. injections in nine red-tailed hawks (Buteo jamaicensis) and five great horned owls (Bubo virginianus) over 48 hr by a modified agar well diffusion microbial inhibition assay. The mean maximum plasma piperacillin concentrations were 204 microg/ml and 221 microg/ml for the hawks and owls, respectively, and times of maximum concentrations were 15 min and 30 min, respectively. The calculated mean terminal elimination half-lives were 77 min in the hawks and 118 min in the owls. Area-under-the-curve values were 218 +/- 52 microg x hr/ml in the hawks and 444 +/- 104 microg x hr/ml in the owls. On the basis of the most common minimal inhibitory concentration (90%) for various bacterial isolates from clinical samples of 8 microg/ml, analysis of the data suggests that the maximum dosing interval for piperacillin at 100 mg/kg in medium sized raptors should be 4-6 hr.     

Use of in situ hybridization for the detection of foot-and-mouth disease virus in cell culture

Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.