Comparison of two modern vaccines and previous influenza infection against challenge with an equine influenza virus from the Australian 2007 outbreak

During 2007, large outbreaks of equine influenza (EI) caused by Florida sublineage Clade 1 viruses affected horse populations in Japan and Australia. The likely protection that would be provided by two modern vaccines commercially available in the European Union (an ISCOM-based and a canarypox-based vaccine) at the time of the outbreaks was determined. Vaccinated ponies were challenged with a representative outbreak isolate (A/eq/Sydney/2888-8/07) and levels of protection were compared. A group of ponies infected 18 months previously with a phylogenetically-related isolate from 2003 (A/eq/South Africa/4/03) was also challenged with the 2007 outbreak virus. After experimental infection with A/eq/Sydney/2888-8/07, unvaccinated control ponies all showed clinical signs of infection together with virus shedding. Protection achieved by both vaccination or long-term immunity induced by previous exposure to equine influenza virus (EIV) was characterised by minor signs of disease and reduced virus shedding when compared with unvaccinated control ponies. The three different methods of virus titration in embryonated hens’ eggs, EIV NP-ELISA and quantitative RT-PCR were used to monitor EIV shedding and results were compared. Though the majority of previously infected ponies had low antibody levels at the time of challenge, they demonstrated good clinical protection and limited virus shedding. In summary, we demonstrate that vaccination with current EIV vaccines would partially protect against infection with A/eq/Sydney/2888-8/07-like strains and would help to limit the spread of disease in our vaccinated horse population.     

Comparison of laboratory techniques for the evaluation of the antigenic potency of foot-and-mouth disease virus cultures and vaccines

Forty-seven suspensions of type A virus grown in surviving bovine tongue epithelium (Frenkel) cultures were compared in a quantitative complement fixation test (CFT) and an antibody combining test (ACT) to evaluate the antigenic mass of the 140 S component, and with three assay methods for infectivity titration.The cultures were then converted into vaccines and the potency of these was measured with a guinea-pig PD₅₀ test and again with the ACT. The relationship between the results obtained with different methods used for titration of infectivity, i.e. baby mice and plaque counting in BHK cells in monolayer (BHK-M) or suspended in agar (BHK-S) was poor and, with the exception of the BHK-S technique, showed little correlation with the results of the serological tests.The correlation coefficient between values obtained by the CFT and the ACT on virus cultures was 0.82 and that between CFT and ACT on vaccines 0.88. The guinea-pig PD₅₀'s of 19 vaccines were compared with the ACT and with the CFT and ACT of the corresponding cultures yielding r values of 0.74, 0.62 and 0.62 respectively. Regression lines are presented for the different relations.An accurate cattle PD₅₀ was determined for two vaccines of the series, using groups of 20 animals per vaccine dilution. The relationship between guinea-pig PD₅₀ (X) and cattle PD₅₀ (Y), expressed by the regression formula Y = 1.05 X + 0.84, showed that differences in cattle PD₅₀'s are of the same magnitude as those observed in the guinea-pig test, provided that sufficient numbers of animals are used for both tests.The results of the quantitative CFT and ACT were in good agreement with the guinea-pig test and as such these tests can provide valuable information on the quality of Frenkel cultures.     

Evidence for evolutionary stasis and genetic drift by genetic analysis of two equine influenza H3 viruses isolated in France

The amino acid sequences of the HA(1) portion of the haemagglutinin of two equine A(H3N8) influenza viruses isolated in France in 1993 and 1998 were analysed to determine their evolutionary relationship with 51 other HA(1) amino acid sequences available in databanks. Our data show that the French strain isolated in 1993 belongs to a group of phylogenetically related viruses branched on the main trunk, illustrating the main lineage of evolution of the equine-2 H3 sequences before its split into two distinct lineages in the late 1980s. By contrast, the 1998 French isolate appears to belong to the more recent 'Eurasian' lineage. These data suggest that equine-2 strains antigenically related to old prototype viruses may cocirculate with the more recent 'Eurasian' and 'American' lineages. In conclusion, it may be necessary to include both strains representative of recent equine influenza variants and an older prototype strain in the current equine influenza vaccines.     

Generation and evaluation of reassortant influenza vaccines made by reverse genetics for H9N2 avian influenza in Korea

The prevalence and continuous evolution of H9N2 avian influenza viruses in poultry have necessitated the use of vaccines in veterinary medicine. Because of the inadequate growth properties of some strains, additional steps are needed for producing vaccine seed virus. In this study, we generated three H9N2/PR8 reassortant viruses using a total cDNA plasmid-transfection system, as an alternative strategy for developing an avian influenza vaccine for animals. We investigated the vaccine potency of the reassortant viruses compared with the existing vaccine strain which was adapted by the 20th serial passages in embryonated eggs with A/Ck/Kor/01310/01 (H9N2). The H9N2/PR8 reassortant viruses, containing the internal genes of the high-yielding PR8 strain and the surface gene of the A/Ck/Kor/01310/01 strain, could be propagated in eggs to the same extent as existing vaccine strain without additional processing. Similar to vaccine strain, the H9N2/PR8 reassortant viruses induced hemagglutination-inhibiting antibodies in chickens and prevented virus shedding and replication in multiple organs in response to homologous infection. However, due to the continuing evolution and increasing biologic diversity of H9N2 influenza in Korea, the vaccine provided only partial protection against currently isolates. Taken together, our results suggest that the H9N2/PR8 reassortant virus can be used as a seed virus for avian influenza vaccines in poultry farm. Considering the constant genetic changes in H9 strains isolated in Korea, this reverse genetic system may offer a prompt and simple way to change the vaccine seed virus and mitigate the impact of unexpected influenza outbreaks.     

Viraemia and abortions are not prevented by two commercial equine herpesvirus-1 vaccines after experimental challenge of horses

Eighteen horses, vaccinated on a number of occasions over a period of 12 to 20 months with either a live equine herpesvirus-1 (EHV-1) or an inactivated EHV-1 vaccine, were challenged by the intranasal instillation of the subtype 1 virus isolated from the 1983 outbreak of abortion and paralytic disease at the Lipizzan Stud, Piber, Austria. The prechallenge serum titres of all vaccinated horses were remarkably low, although most horses had received their last vaccine dose only 3 weeks before test-infection. Higher titres were obtained with the inactivated product than with the live virus vaccine. However, no obvious differences were found between the two vaccines in their ability to prevent disease, in that all vaccinated and two 'sentinel' horses became infected and developed viraemia and some degree of clinical disease after challenge; five of the 10 in-foal mares aborted.     

Comparison of protection induced in lambs by Corynebacterium pseudotuberculosis whole cell and cell wall vaccines

Colostrum-deprived lambs were vaccinated IM with 10 mg (dry weight) of Corynebacterium pseudotuberculosis whole cells (WC) or cell walls (CW) and their immunity was challenged by IV injection of 3.1 X 10(4) colony-forming units of C pseudotuberculosis. Before challenge exposure, the logarithmic mean antibody titers were 2.0837 in lambs that were vaccinated with WC, 2.6858 in lambs that were vaccinated with CW, and 1.4214 in control lambs. Significant protection was demonstrated by fewer abscesses and organisms in the lungs of lambs vaccinated with WC or CW (P less than 0.05) than in control lambs. By the same criteria, more protection was provided to lambs vaccinated with CW than to lambs vaccinated with WC.     

Morphologic and ultrastructural evaluation of effect of ischemia and dimethyl sulfoxide on equine jejunum

Morphologic changes in equine jejunal segments subjected to 1 hour of ischemia and 1 hour of reperfusion, and protective effects of systemic administration of dimethyl sulfoxide (DMSO; 1 g/kg of body weight) were investigated in 18 ponies, using light microscopy and scanning and transmission electron microscopy. Ponies were allotted to 4 groups: group 1--control (n = 3); group 2--DMSO (n = 3); group 3--ischemia (n = 6); and group 4--ischemia and DMSO (n = 6). In each pony, 2 jejunal sections were evaluated. The first section was obtained prior to induction of ischemia, and the second was obtained 2 hours later after reperfusion. Mucosal lesions were graded from 0 (normal) to 5 (most severe). Combined ischemia and reperfusion of 2 hours' duration induced moderately severe mucosal injury to the equine jejunum (group 3; grade 1.5 to 2.5), characterized principally by disruption of enterocyte attachment from the basement membrane and lamina propria. Fluid accumulation disrupted enterocyte cell-to-cell adhesion toward cell bases, while apical tight junctions and desmosomal junctions toward the luminal surface remained intact. Intracytoplasmic organellar changes within enterocytes were not a prominent feature of the injury. The aforementioned processes were marked at the villus tip and progressed down the villus sides. These findings support the importance of mechanisms leading to early subepithelial fluid accumulation rather than that of direct severe enterocyte injury. Further, fluid accumulation does not appear to arise from intercellular migration from the luminal surface. In this model, a pathomechanical effect caused by vigorous villus retraction appears to exacerbate epithelial lifting toward the villus tip.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosecurity and vaccination strategies to minimise the effect of an equine influenza outbreak on racing and breeding

Three biosecurity and relief-and-recovery initiatives adopted by the NSW horse racing industries reduced the economic and social disruption caused by the disease and subsequent movement controls during the 2007 Australian equine influenza (EI) incursion. The first was the creation of biosecure horse training and racing precincts around the Sydney area to permit racing to continue with healthy horses. Infection was excluded for 3-5 weeks and race meetings were conducted safely during this period. The second was a vaccination program of racehorses at these and other precincts to maintain an ongoing healthy pool of racehorses. Vaccination commenced too late to enable viable racing to continue in Sydney in the short term, but assisted in managing an early return to racing throughout the state before EI-free status had been regained. The third was the establishment of approved quarantine stations to facilitate the movement of racing and breeding horses out of high-risk regions. The difficulties in establishing and managing these initiatives in the face of the EI incursion are discussed.     

A rapid and highly sensitive method for diagnosis of equine influenza by antigen detection using immuno-PCR

A rapid and highly sensitive method for diagnosis of influenza by detecting viral antigens using immuno-PCR has been developed. The sensitivity of the immuno-PCR for the detection of the nonstructural protein 1 (NS 1) antigenically common among influenza A viruses was 10(1.0-2.0) times higher than that of RT-PCR for the detection of the viral genome. For the detection of the hemagglutinin (HA) subtype-specific antigen, this assay using anti-HA monoclonal antibodies attained a sensitivity of up to 10(7.0-8.0) times higher than those by virus isolation or by RT-PCR.     

The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model

Vaccination of poultry with naked plasmid DNA has been successfully demonstrated with several different poultry pathogens, but the technology needs to be further developed before it can be practically implemented. Many different methods can conceivably enhance the efficacy of DNA vaccines, and this report examines the use of different eukaryotic expression vectors with different promoters and different adjuvants to express the influenza hemagglutinin protein. Four different promoters in five different plasmids were used to express the hemagglutinin protein of an H5 avian influenza virus, including two different immediate early cytomegaloviruses (CMVs), Rous sarcoma virus, chicken actin, and simian virus 40 promoters. All five constructs expressed detectable hemagglutinin protein in cell culture, but the pCI-neo HA plasmid with the CMV promoter provided the best response in chickens when vaccinated intramuscularly at 1 day of age on the basis of antibody titer and survivability after challenge with a highly pathogenic avian influenza virus at 6 wk postinoculation. A beneficial response was observed in birds boostered at 3 wk of age, in birds given larger amounts of DNA, and with the use of multiple injection sites to administer the vaccine. With the use of the pCI-neo construct, the effects of different adjuvants designed to increase the uptake of plasmid DNA, including 25% sucrose, diethylaminoethyl dextran, calcium phosphate, polybrene, and two different cationic liposomes, were examined. Both liposomes tested enhanced antibody titers as compared with the positive controls, but the other chemical adjuvants decreased the antibody response as compared with the control chickens that received just the plasmid alone. The results observed are promising for continued studies, but continued improvements in vaccine response and reduced costs are necessary before the technology can be commercially developed.     

The effect of heterotypic infections of older horses with equine influenza virus type-2 on some clinical and immunological parameters

Twelve horses, all of them 10 years old, were vaccinated intramuscularly on 0 and 28 days of the experiment with inactivated vaccine containing only antigens of A-equi-2/Miami/63. Another three unvaccinated horses, each at the age of 10 years, were the negative control group. One, ten-year-old horse was vaccinated with commercial inactivated vaccine containing both antigens of A-equi-2/Miami/63 as well as A-equi-1/Praha/56 as positive control. Three horses were challenged intranasally with homotypic strain of Miami/63, while six other were challenged with heterotypic strains--three with Suffolk/89 and three with Kentucky/86. Three horses vaccinated with vaccine containing only strain A-equi-2/Miami/63 were not challenged. In the group of three unvaccinated horses, each one was challenged intranasally with different strains studied in this experiment. The horse vaccinated with commercial vaccine was not challenged. Replication of each strain was done in chick embryos. During the experiment blood from horses was collected for hematological and immunological examinations (antigen-specific and antigen-nonspecific lymphocyte transformation tests, lymphocyte immunophenotyping, antigen-specific leukocyte migration inhibition test and hemagglutination inhibition test). The statistical analysis showed that the dynamics of lymphocyte immunological reactivity in horses vaccinated with inactivated vaccine containing antigens of A-equi-2/Miami/63 in response to further antigen stimulation (in vitro) was different comparing the homotypic or nearly homotypic challenging with Miami/63 and Suffolk/89 respectively, to the more heterotypic one with the strain Kentucky/86. In horses challenged with classical homotypic strain of Miami/63 no clinical signs were observed. These results confirm that the vaccine shall consist of the strains currently circulating in the horse population.     

Cloning and expression of recombinant equine interleukin-3 and its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes

Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL).Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culicoides nubeculosus extract with or without IL-3. Sulfidoleukotriene (sLT) production was measured in supernatants by ELISA and mRNA expression of IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) assessed in cell lysate by quantitative real-time PCR.Recombinant equine IL-3 (req-IL-3) had a dose dependent effect on sLT production by stimulated equine PBL and significantly increased IL-4, IL-13 and TSLP expression compared to non-primed cells.IL-3 priming significantly increased Culicoides-induced sLT production in IBH-affected but not in non-affected horses and was particularly effective in young IBH-affected horses (≤3 years).A functionally active recombinant equine IL-3 has been produced which will be useful for future immunological studies in horses. It will also allow improving the sensitivity of cellular in vitro tests for allergy diagnosis in horses.     

Studies on cross protection induced in calves by rotaviruses of calves, children and foals.

Inoculation at birth with a live attenuated strain of a bovine rotavirus isolated in the USA (scourvax-reo) induced protection in five gnotobiotic calves seven to 21 days later against a UK isolate of pathogenic bovine rotavirus. However, no protection was induced in three calves challenged three to five days after vaccination. There was a close antigenic relationship demonstrated between the two bovine rotavirus isolates. In contrast only one of three gnotobiotic calves inoculated with foal rotavirus, and one of three with human rotavirus, were protected against bovine rotavirus challenge. Protection in these two calves correlated with high heterologous immunofluorescent antibody titre (320 or greater), although the neutralising antibody titres was less than 20.     

An evaluation of the role of antibodies to Actinobacillus pleuropneumoniae serovar 1 and 15 in the protection provided by sub-unit and live streptomycin-dependent pleuropneumonia vaccines

OBJECTIVE: To evaluate the serological response of pigs receiving either the Porcilis APP vaccine or a modified live vaccine based on a streptomycin-dependent (SD) strain of Actinobacillus pleuropneumoniae, and then challenged with an Australian isolate of A. pleuropneumoniae of either serovar 1 or 15 as a means of understanding the protection provided by both vaccines against serovar 1 but not against serovar 15. DESIGN: The serological tests evaluated were serovar-specific polysaccharide ELISA tests (for serovar 1 and 15), ELISA tests for antibodies to three A. pleuropneumoniae toxins (ApxI, ApxII and ApxIII) as well as to a 42 kDa outer membrane protein (OMP), a haemolysin neutralisation (HN) assay and immunoblotting. The tests were used to detect antibodies in vaccinated pigs that had been shown to be protected against serovar 1 but not serovar 15. RESULTS: In the polysaccharide antigen ELISA assays, both vaccines resulted in a significant rise in the titre in the serovar 1 ELISA but not the serovar 15 ELISA. The Porcilis APP vaccinated pigs showed a significant response in the ApxI, ApxIII and 42 kDa OMP ELISA. In the ApxII ELISA, all pigs tested (the Porcilis APP vaccinates and the controls) were positive on entry to the trial. In the HN assay, the Porcilis APP vaccinated pigs showed a significant response after one dose while the SD vaccinated pigs required two doses of vaccine before a marked rise in titre was induced. Immunoblotting revealed that neither vaccine generated antibodies that recognised the ApxIII produced by serovar 15. CONCLUSIONS: The failure of these vaccines to provide protection against serovar 15 may be due to novel virulence factors possessed by serovar 15, significant differences between the ApxIII toxin of serovar 15 and those present in the Porcilis APP vaccine or failure by both vaccines to induce antibodies to the serovar 15 specific polysaccharide.     

Optimization of the protocols for double vaccination against Marek's disease by using commercially available vaccines: evaluation of protection, vaccine replication, and activation of T cells

Revaccination against Marek's disease is a widespread practice in some countries. The rationale of this practice is unknown, and there is no consensus in the protocols. Recently, we have demonstrated that administration of the first vaccine at 18 days of embryonation followed by a more protective second vaccine at hatch (18ED/1d) reproduced systematically the benefits of revaccination under laboratory conditions. Here, we have used the same model to optimize the revaccination protocols by using currently available vaccines and to determine whether two features associated with Marek's disease vaccine-induced protection (activation of T cells and replication of vaccine virus) are involved in the revaccination protocols. Protection conferred by three revaccination protocols (turkey herpesvirus [HVT] 18ED/HVT+SB-1 1d, HVT 18ED/CVI988 1d, and HVT+SB-1 18ED/ CVI988 1d) was evaluated. Revaccination protocols also were compared with single vaccination protocols (HVT 18ED, HVT+SB-1 18ED, HVT+SB-1 1d, CVI988 18ED, and CVI988 1d). Our results demonstrated that it is possible to improve efficacy of the currently available vaccines by using them in revaccination programs. Administration of HVT 18ED/CVI988 1d and HVT+SB-1 18ED/CVI988 1d were the two protocols that conferred the highest protection against a very early challenge (2 days of age) with very virulent plus Marek's disease virus strain 648A. In a separate experiment, we evaluated vaccine replication and activation of T cells in single and revaccination protocols. Our results demonstrated that replication of the second vaccine, although decreased compared with single vaccination, could be detected at 3 days (HVT, CVI988) or at 6 days (SB-1). Administration of the first vaccine (HVT) at 18ED resulted in a high percentage of activated T cells. Administration of a second vaccine (either HVT-SB-1 or CVI988) at 1d resulted in increased intensity of MHC-II stain in activated T cells.     

Further studies on the adjuvant effect of an interferon inducer (BRL 5907) on Newcastle disease and avian influenza inactivated vaccines.

Vaccination of fowls with inactivated Newcastle disease (ND) virus and avian influenza (AI) virus oil emulsion vaccines containing an interferon inducer (BRL 5907) produced an enhanced immunological response. The Newcastle disease vaccine containing BRL 5907 induced earlier protection to challenge than Newcastle disease vaccine by itself and also produced an increase immune response when administered to day-old maternally immune and susceptible chicks.     

2012年山东鸡传染性支气管炎病毒分离株遗传进化和免疫保护分析

从2012年山东省发病商品鸡群中分离鉴定了9株鸡传染性支气管炎病毒(IBV),并对其S1基因进行了序列测定和分析。遗传进化分析发现,9个IBV流行株和国内外参考毒株可分成4个基因型。其中8个IBV流行株属于以QX为代表的基因I型,而SDIB781/2012与其他8个流行株遗传距离较远,属于基因IV型。为明确现有弱毒活疫苗对IBV流行株的免疫保护效力,分别用491、2886、MA5和H120弱毒疫苗免疫3日龄SPF鸡,14d后用流行株SDIB821/2012进行攻毒。根据攻毒后试验鸡发病死亡情况,491免疫组保护率为90%;与对照组相比,2886、MA5和H120免疫组几乎没有保护效力。但是攻毒后3、5和7d各免疫组和对照组试验鸡气管、肺和肾脏组织病毒检出率没有明显差异,表明4种IB疫苗均不能有效抵抗病毒在试验鸡气管、肺脏和肾脏组织内复制。     

In vitro evaluation of the effect of the opioid antagonist N-methylnaltrexone on motility of the equine jejunum and pelvic flexure

REASONS FOR PERFORMING STUDY: Although potent analgesics, opioids decrease intestinal activity, leading to ileus in many species. N-methylnaltrexone (MNTX), an opioid antagonist which does not cross the blood-brain barrier and antagonises the morphine effect on the intestine, directly stimulates motility and restores function without affecting analgesic properties. While its use has been reported in human subjects, there is no information with regard to its usage in the horse. OBJECTIVES: To determine whether MNTX has an effect on contractile activity of the equine jejunum and pelvic flexure. METHODS: Using circular smooth muscle strips obtained from 8 mature horses, increasing concentrations of MNTX were added to tissue baths in the range of 1 x 10(-9) to 1 x 10(-5) mol/l, and contractile responses were recorded for 3 mins. Data were analysed using a repeated measures ANOVA to determine whether there was a significant drug effect compared to baseline activity. Data were analysed between the jejunum and pelvic flexure using a Mann-Whitney U test. Statistical significance was established as P < 0.05. RESULTS: The administration of MNTX significantly increased the contractile frequency and amplitude at all concentrations relative to baseline (P < 0.0001) for the jejunum. The response was greatest at 1 x 10(-7) mol/l (P = 0.0005), with a mean difference from baseline of 115.12 g/cm2. The highest concentration evaluated (1 x 10(-5) mol/l) had a mean contractile strength of 69.76 g/cm2, which was significantly greater than baseline activity (P = 0.04). A significant increase in contractile activity for the colon was detected at 3 x 10(-7) mol/l and all subsequent concentrations (P < 0.04). Unlike the jejunum, the contractile activity of the pelvic flexure increased progressively with the addition of each subsequent concentration. CONCLUSIONS: N-methylnaltrexone has a direct effect on circular smooth muscle of the equine jejunum and pelvic flexure resulting in an increase in contractile activity. POTENTIAL RELEVANCE: N-methylnaltrexone could potentially be used in conjunction with morphine to provide potent and effective analgesia without compromising intestinal function. Further in vivo investigations are required to determine whether this agent antagonises morphine's effect on motility.