Oral Administration of Electron-Beam Inactivated Rhodococcus equi Failed to Protect Foals against Intrabronchial Infection with Live,Virulent R. equi

There is currently no licensed vaccine that protects foals against Rhodococcus equi–induced pneumonia. Oral administration of live, virulent R. equi to neonatal foals has been demonstrated to protect against subsequent intrabronchial challenge with virulent R. equi. Electron beam (eBeam)-inactivated R. equi are structurally intact and have been demonstrated to be immunogenic when administered orally to neonatal foals. Thus, we investigated whether eBeam inactivated R. equi could protect foals against developing pneumonia after experimental infection with live, virulent R. equi. Foals (n = 8) were vaccinated by gavaging with eBeam-inactivated R. equi at ages 2, 7, and 14 days, or gavaged with equal volume of saline solution (n = 4), and subsequently infected intrabronchially with live, virulent R. equi at age 21 days. The proportion of vaccinated foals that developed pneumonia following challenge was similar among the vaccinated (7/8; 88%) and unvaccinated foals (3/4; 75%). This vaccination regimen did not appear to be strongly immunogenic in foals. Alternative dosing regimens or routes of administration need further investigation and may prove to be immunogenic and protective.     

Effects of Administration of Live or Inactivated Virulent Rhodococccus equi and Age on the Fecal Microbiome of Neonatal Foals

Background

Rhodococcus equi is an important pathogen of foals. Enteral administration of live, virulent R. equi during early life has been documented to protect against subsequent intrabronchial challenge with R. equi, indicating that enteral mucosal immunization may be protective. Evidence exists that mucosal immune responses develop against both live and inactivated micro-organisms. The extent to which live or inactivated R. equi might alter the intestinal microbiome of foals is unknown. This is an important question because the intestinal microbiome of neonates of other species is known to change over time and to influence host development. To our knowledge, changes in the intestinal microbiome of foals during early life have not been reported. Thus, the purpose of this study was to determine whether age (during the first month of life) or administration of either live virulent R. equi (at a dose reported to protect foals against subsequent intrabronchial challenge, viz., 1×10¹⁰ colony forming units [CFU]) or inactivated virulent R. equi (at higher doses, viz., 2×10¹⁰ and 1×10¹¹ [CFU]) altered the fecal microbiome of foals.

Methodology/Principal Findings

Fecal swab samples from 42 healthy foals after vaccination with low-dose inactivated R. equi (n = 9), high-dose inactivated R. equi (n = 10), live R. equi (n = 6), control with cholera toxin B (CTB, n = 9), and control without CTB (n = 8) were evaluated by 454-pyrosequencing of the 16S rRNA gene and by qPCR. No impact of treatment was observed among vaccinated foals; however, marked and significant differences in microbial communities and diversity were observed between foals at 30 days of age relative to 2 days of age.

Conclusions

The results suggest age-related changes in the fecal microbial population of healthy foals do occur, however, mucosal vaccination does not result in major changes of the fecal microbiome in foals.     

Plasmid Profiles of Virulent Rhodococcus equi Strains Isolated from Infected Foals in Poland

Rhodococcus equi is an important bacterial pathogen in foals up to 6 months old, widespread in horse farms all over the world. It was found that only virulent R. equi strains expressing 15–17 kDa virulence-associated protein (VapA) and having large virulence plasmid of 85–90 kb containing vapA gene are pathogenic for horses. To date, 12 plasmid types have been reported in VapA positive strains from horses. There are no data concerning plasmid types of Polish field R. equi strains isolated from horses and horse farm environment. The aim of the study is to determine plasmid profiles of virulent R. equi strains isolated in Poland from dead foals as well as from soil samples taken from horse breeding farms. Plasmid profiles of 10 clinical strains derived from 8 farms and 11 environmental strains from 3 farms, confirmed as virulent by PCR, were compared with 12 reference strains containing the known plasmid size and type. Plasmid DNAs were analysed by digestion with the restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII for detailed comparison and estimation of plasmid sizes. The results of RFLP analysis revealed that all except one isolates used in the study are classified as VapA 85 kb type I plasmid. One strain harboured VapA 87 kb type I plasmid. This is the first report of plasmid types of Polish field R. equi strains. The results of our preliminary investigations on horse farms located in central and eastern Poland indicate that the virulent R. equi strains thus far isolated from diseased foals and horse farms environment represent a highly uniform plasmid pattern.     

An Adenoviral Vector Based Vaccine for Rhodococcus equi

Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals.     

Preclinical Evaluation of the Immunogenicity and Safety of an Inactivated Enterovirus 71 Candidate Vaccine

Human enterovirus 71 (EV71) is a significant cause of morbidity and mortality from Hand, Foot and Mouth Disease (HFMD) and neurological complications, particularly in young children in the Asia-Pacific region. There are no vaccines or antiviral therapies currently available for prevention or treatment of HFMD caused by EV71. Therefore, the development of therapeutic and preventive strategies against HFMD is of growing importance. We report the immunogenic and safety profile of inactivated, purified EV71 preparations formulated with aluminum hydroxide adjuvant in preclinical studies in mice and rabbits. In mice, the candidate vaccine formulations elicited high neutralizing antibody responses. A toxicology study of the vaccine formulations planned for human use performed in rabbits showed no vaccine-related pathological changes and all animals remained healthy. Based on these preclinical studies, Phase 1 clinical testing of the EV71 inactivated vaccine was initiated.     

Safety,Immunogenicity and Duration of Immunity Elicited by an Inactivated Bovine Ephemeral Fever Vaccine

Bovine ephemeral fever (BEF) is an economically important viral vector-borne cattle disease. Several live-attenuated, inactivated and recombinant vaccines have been tested, demonstrating varying efficacy. However, to the best of our knowledge, duration of immunity conferred by an inactivated vaccine has never been reported. In the last decade, Israel has faced an increasing number of BEF outbreaks. The need for an effective vaccine compatible with strains circulating in the Middle East region led to the development of a MONTANIDE™ ISA 206 VG (water-in-oil-in-water), inactivated vaccine based on a local strain. We tested the safety, immunogenicity and duration of immunity conferred by this vaccine. The induced neutralizing antibody (NA) response was followed for 493 days in 40 cows vaccinated by different protocols. The vaccine did not cause adverse reactions or a decrease in milk production. All cows [except 2 (6.7%) which did not respond to vaccination] showed a significant rise in NA titer of up to 1:256 following the second, third or fourth booster vaccination. Neutralizing antibody levels declined gradually to 1:16 up to 120 days post vaccination. This decline continued in cows vaccinated only twice, whereas cows vaccinated 3 or 4 times showed stable titers of approximately 1:16 for up to 267 days post vaccination. At least three vaccinations with the inactivated BEF vaccine were needed to confer long-lasting immunity. These results may have significant implications for the choice of vaccination protocol with inactivated BEF vaccines. Complementary challenge data should however be added to the above results in order to determine what is the minimal NA response conferring protection from clinical disease.     

各种类型流行性出血热灭活疫苗的免疫性比较研究

本文对三种不同类型的出血热疫苗的免疫性进行了比较研究,其中沙鼠肾细胞疫苗由β—丙内酯灭活不加任何佐剂,地鼠肾细胞和鼠脑提纯疫苗由福尔马林灭活加氢氧化铝佐剂。三种疫苗经抗原量测定结果只有沙鼠肾细胞疫苗能测出较高的血凝素。家兔经三种疫苗免疫二针后只有沙鼠肾细胞疫苗能测出血抑抗体。中和抗体测定的结果亦以该疫苗的抗体反应最好,全部阳转,滴度也最高(1:20—≥320)。对家兔和长爪沙鼠免疫后用野鼠型病毒攻击结果,三种疫苗均有一定的保护作用,但仍以沙鼠肾细胞疫苗的保护力最强。     

Serogrouping of Rhodococcus equi

The serological relationships among 27 isolates of Rhodococcus equi selected from a total of 1,195 isolates were investigated by cross-agglutination and absorption tests. The presence of capsular material was demonstrated in all the 27 isolates by electron microscopic observation. Antisera were prepared by employing formalized antigen of each isolate. In the cross-agglutination test with formalized antigen, 13 antisera reacted with homologous antigens alone, but the remaining 14 antisera reacted not only with homologous antigens but also with one to four heterologous antigens. When these 14 antisera possessing heterologous agglutinins were absorbed with each of the cross-reacting antigens, 14 specific antisera were obtained. The cross-agglutination test with these 27 antisera proved the 27 strains examined to be serologically distinct from one another. These strains were designated serogroups 1 to 27. Thus the same number of grouping antisera were prepared. The distribution of each serogroup among the 1,195 isolates and 15 reference strains was investigated by the slide agglutination test. All the strains were found to be groupable. Most of them belonged to serogroups 1 to 4, 7 to 9, 11, 14, and 15. Of the serogroups designated, 4, 16, 2, 12, 21, 1, and 9 were identical with Prescott's serovars 1, 2, 3, 4, 5, 6, and 7, respectively.     

EV71灭活疫苗（人二倍体细胞）在恒河猴婴猴模型中的免疫保护性分析

肠道病毒71型作为引起儿童群体常见传染性手足口病（HFMD）的主要病原,具有导致少量感染个体出现脑炎等神经系统病变以及相关心肺功能衰竭的病理学特性．因此其预防性疫苗的研发具有重要的公共卫生意义．在前期工作的基础上,一种EV71灭活病毒疫苗（人二倍体细胞）在本研究中基于恒河猴婴猴模型进行了相应的免疫保护性分析．以160EU剂量对2～3月龄婴猴进行0,4周免疫后,动物在第4周接受了剂量为10。～CCID50的病毒经呼吸道的攻击．对病毒攻击后动物在14天内的临床症状、血液生物学、器官病原学分布以及病理学检测的动态观察表明,经疫苗免疫的动物未出现对照动物所具有的特征性临床表现,其血液生物学及病理学检测均无异常．同时,器官病原学分布亦呈阴性．结合动物中和抗体的明确增长及对照动物的综合表现分析,本文的工作证实了该EV71灭活病毒疫苗（人二倍体细胞）在恒河猴婴猴体内的免疫保护性．     

人轮状病毒ZTR-5株灭活疫苗的制备及小鼠血清免疫学评价

目的: 研究人轮状病毒ZTR-5株灭活疫苗的制备及在实验小鼠中的免疫原性评价。方法: 轮状病毒ZTR-5株在MA104细胞上经蚀斑筛选纯化后,获得单一克隆接种至Vero细胞上适应性培养,免疫荧光定量检测病毒的感染性滴度,对收获的病毒液进行离心、超滤、分子筛纯化,甲醛灭活,抗原定量检测Al(OH)₃吸附制备的实验性疫苗。使用不同剂量(8EU、32EU、128EU、256EU)经肌内注射免疫小鼠,共免疫三次,免疫间隔2周。采用间接ELISA法检测血清特异性抗体效价。 结果: 通过蚀斑纯化,筛选得到一株纯化的病毒株ZTR-5纯-1,在Vero细胞上适应性后感染性滴度达7.35logCCID₅₀/ml;大量培养收获的病毒原液滴度为7.57logCCID₅₀/ml,制备获得轮状病毒样品抗原含量为2 560EU/ml;经肌内注射,初次免疫后,所有剂量组动物均获得抗体阳转,阳转率为100%;第一次加强免疫后,各组血清特异性抗体水平均明显增高,免疫剂量为128EU和256EU的两组小鼠血清抗体效价均达1∶10 240;第二次加强免疫后,各剂量组(8EU、32EU、128EU、256EU)血清抗体效价依次达1∶5 120,1∶7 456,1∶14 481.54,1∶14 481.54。 结论:人轮状病毒ZTR-5株可在Vero细胞上稳定增殖,所制备的疫苗具良好免疫原性,用128EU/2次免疫即可获得良好的免疫效果。     

Study of Lysozyme Resistance in Rhodococcus equi

Lysozyme is an important and widespread component of the innate immune response that constitutes the first line of defense against bacterial pathogens. The bactericidal effect of this enzyme relies on its capacity to hydrolyze the bacterial cell wall and also on a nonenzymatic mechanism involving its cationic antimicrobial peptide (CAMP) properties, which leads to membrane permeabilization. In this paper, we report our findings on the lysozyme resistance ability of Rhodococcus equi, a pulmonary pathogen of young foals and, more recently, of immunocompromised patients, whose pathogenic capacity is conferred by a large virulence plasmid. Our results show that (i) R. equi can be considered to be moderately resistant to lysozyme, (ii) the activity of lysozyme largely depends on its muramidase action rather than on its CAMP activity, and (iii) the virulence plasmid confers part of its lysozyme resistance capacity to R. equi. This study is the first one to demonstrate the influence of the virulence plasmid on the stress resistance capacity of R. equi and improves our understanding of the mechanisms enabling R. equi to resist the host defenses.     

Composition and Diversity of the Fecal Microbiome and Inferred Fecal Metagenome Does Not Predict Subsequent Pneumonia Caused by Rhodococcus equi in Foals

In equids, susceptibility to disease caused by Rhodococcus equi occurs almost exclusively in foals. This distribution might be attributable to the age-dependent maturation of immunity following birth undergone by mammalian neonates that renders them especially susceptible to infectious diseases. Expansion and diversification of the neonatal microbiome contribute to development of immunity in the gut. Moreover, diminished diversity of the gastrointestinal microbiome has been associated with risk of infections and immune dysregulation. We thus hypothesized that varying composition or reduced diversity of the intestinal microbiome of neonatal foals would contribute to increased susceptibility of their developing R. equi pneumonia. The composition and diversity indices of the fecal microbiota at 3 and 5 weeks of age were compared among 3 groups of foals: 1) foals that subsequently developed R. equi pneumonia after sampling; 2) foals that subsequently developed ultrasonographic evidence of pulmonary abscess formation or consolidation but not clinical signs (subclinical group); and, 3) foals that developed neither clinical signs nor ultrasonographic evidence of pulmonary abscess formation or consolidation. No significant differences were found among groups at either sampling time, indicating absence of evidence of an influence of composition or diversity of the fecal microbiome, or predicted fecal metagenome, on susceptibility to subsequent R. equi pneumonia. A marked and significant difference identified between a relatively short interval of time appeared to reflect ongoing adaptation to transition from a milk diet to a diet including available forage (including hay) and access to concentrate fed to the mare.     

流感病毒灭活疫苗研究进展

流感病毒的不断变异是造成流感经常流行的主要原因．研究表明人流感病毒的来源与禽流感病毒的存在密切相关．最近出现的禽流感病毒跨种属感染人的事件,预示引起下一次爆发的流感病毒流行可能直接来源于禽流感病毒．因人类对新出现的病毒缺乏免疫力,开发有效疫苗仍然是预防流感流行的关键．对流感灭活疫苗包括灭活疫苗有效成分的改良,H5N1、H9N2型人．禽流感疫苗研究和应用反向遗传技术制备流感灭活疫苗等方面的研究进展进行了探讨．     

VapA of Rhodococcus equi binds phosphatidic acid

Rhodococcus equi is a multihost, facultative intracellular bacterial pathogen that primarily causes pneumonia in foals less than six months in age and immunocompromised people. Previous studies determined that the major virulence determinant of R. equi is the surface bound virulence associated protein A (VapA). The presence of VapA inhibits the maturation of R. equi‐containing phagosomes and promotes intracellular bacterial survival, as determined by the inability of vapA deletion mutants to replicate in host macrophages. While the mechanism of action of VapA remains elusive, we show that soluble recombinant VapA^32‐189 both rescues the intramacrophage replication defect of a wild type R. equi strain lacking the vapA gene and enhances the persistence of nonpathogenic Escherichia coli in macrophages. During macrophage infection, VapA was observed at both the bacterial surface and at the membrane of the host‐derived R. equi containing vacuole, thus providing an opportunity for VapA to interact with host constituents and promote alterations in phagolysosomal function. In support of the observed host membrane binding activity of VapA, we also found that rVapA^32‐189 interacted specifically with liposomes containing phosphatidic acid in vitro. Collectively, these data demonstrate a lipid binding property of VapA, which may be required for its function during intracellular infection.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

Numerical classification of Rhodococcus equi and related actinomycetes

Eighty-three strains received as Corynebacterium or Rhodococcus equi and marker cultures of Corynebacterium and Rhodococcus species were subjected to numerical phenetic analyses using 112 unit characters. The data were examined using the simple matching ( S _SM) and Jaccard ( S _J) coefficients and clustering was achieved using the average linkage algorithm. Cluster composition was not markedly affected by the coefficient used or by test error, estimated at 2.9%. Most of the strains received as R. (C.) equi formed a distinct and homogeneous cluster in the aggregate Rhodococcus taxon, the strains of which were sharply separated from marker cultures of C. pseudotuberculosis and C. renale. The taxon R. equi was redescribed and Nocardia calcarea Metcalf & Brown reduced to a subjective synonym of R. erythropolis (Gray & Thornton) Goodfellow & Alderson.     

Biotransformations of aromatic diniitriles using Rhodococcus equi cells

Short incubation (1 – 5 h) of aromatic dinitriles with resting cells of Rhodococcus equi A4 produces the corresponding cyano amides in 48 – 81% yield. The substrate reactivity decreases in order 1,4- 1,3- > 1,2-disubstitution. Prolonged treatment (20 – 50 h) is needed to achieve the conversion to acid or for transformation of the second cyano group.     

Safety and Immunogenicity in Neonatal Mice of a Hyperattenuated Listeria Vaccine Directed against Human Immunodeficiency Virus

CD8(+) T cells are a major component of the adaptive response of a host to infections by viruses and other intracellular pathogenic agents. However, because of the intrinsic immaturity of the immune system of neonatal animals, neonates are highly sensitive to a variety of pathogens and may be unable to respond in a protective manner. Here we explore whether a hyperattenuated strain of Listeria monocytogenes that can be used as a live vaccine vector in adults is safe and able to induce an effective response in neonates. We answer both questions affirmatively.