Alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses

Replicon particles derived from a vaccine strain of Venezuelan equine encephalitis (VEE) virus were used as vectors for expression in vivo of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). The immunogenicity of the different replicons was evaluated in horses, as was their ability to protectively immunize horses against intranasal and intrauterine challenge with a virulent strain of EAV (EAV KY84). Horses immunized with replicons that express both the G(L) and M proteins in heterodimer form developed neutralizing antibodies to EAV, shed little or no virus, and developed only mild or inapparent signs of equine viral arteritis (EVA) after challenge with EAV KY84. In contrast, unvaccinated horses and those immunized with replicons expressing individual EAV envelope proteins (M or G(L)) shed virus for 6-10 days in their nasal secretions and developed severe signs of EVA after challenge. These data confirm that replicons that co-express the G(L) and M envelope proteins effectively, induce EAV neutralizing antibodies and protective immunity in horses.     

Immunokinetics of equine herpesvirus 1 in donkey mares: suppression of secondary cell-mediated response.

To study the immunokinetics of equine herpesvirus 1 (EHV1), donkey mares were immunised with a laboratory strain of EHV1, or with recommended doses of Pneumabort-K vaccine (EHV1 Army 183 strain, formalin-inactivated, with an oil adjuvant) and a booster was given after three months. Humoral immune responses were studied by employing a virus neutralisation (VN) test. A leucocyte migration inhibition test (LMIT) was employed for the assay of cellular immune responses. The VN antibody titre reached 1:64 or 1:128 after primary immunisation and showed a marginal increase (1:256) after secondary immunisation with either of the immunogens. After the primary dose of immunogen, there was a gradual increase in host cellular response which persisted for up to three months. However, on secondary immunisation, cell-mediated immune response was short-lived and weak compared to the primary response with both immunogens. This could be one possible explanation for breakdown of anti-EHV1 immunity leading to abortion in immunised mares.     

The Clostridium perfringens enterotoxin from equine isolates; its characterization,sequence and role in foal diarrhoea.

During a survey of foal diarrhoea between 1991 and 1994, Clostridium perfringens was significantly associated with disease with 56% of cases infected [1]. The contribution of enterotoxigenic C. perfringens to this association, was assessed by use of the reverse passive latex agglutination test for enterotoxin (RPLA; Oxoid Unipath) and vero cell toxicity neutralized by antitoxin on stored faecal samples and sporulated faecal isolates of C. perfringens. Polymerase chain reaction (PCR1) based on the DNA sequence for the whole enterotoxin gene [2] yielded a fragment from an equine isolate of the anticipated size which, cloned into plasmid M13 phage, had a sequence essentially identical to the published sequence. Consequently, all faecal isolates were also tested by PCR1 and for a part of the enterotoxin gene (PCR2). Significant association with diarrhoea (controls not in contact with cases) was found with positive RPLA tests on faeces (OR = 13, P = 0.002) and isolates (OR = 4.57, P = < 0.0001), vero cell toxicity of isolates (OR = 1.78, P = 0.026), and PCR1 (OR = nd, P = 0.029) but not PCR2 or vero cell toxicity of faeces. Significant association with diarrhoea was also found for isolates negative by RPLA (OR = 3.91; CI 2.05-7.57; P < 0.0001) or PCR1 (OR = 4.81; CI 2.84-8.20; P < 0.0001). Many of the isolates from RPLA positive faeces and verotoxic isolates were PCR negative and no evidence could be found for the presence of the enterotoxin gene in a random selection of RPLA positive/PCR negative isolates by gene probe on chromosomal DNA and PCR reaction product or vero cell toxicity neutralized by specific antiserum. Failure of the vero cell toxicity on faeces to be associated with diarrhoea or for cytotoxicity of cultures and RPLA on cultures to agree with the PCRs was believed to be related to the presence of other cytotoxins, the inherent cytotoxicity of equine faeces and to the poor specificity of the commercial antiserum used in the test. Enterotoxigenic C. perfringens could not account for the overall association of C. perfringens with foal diarrhoea because (a) cultures positive by PCR, RPLA or cytotoxicity were not significantly more common amongst isolates from cases than controls; and (b) the proportion of isolates from cases positive by PCR (PCR1 or PCR2) was too small at 9.7%.     

Experiences with new generation vaccines against equine viral arteritis, West Nile disease and African horse sickness

Viral diseases constitute an ever growing threat to the horse industry worldwide because of the rapid movement of large numbers of horses for competition and breeding. A number of different types of vaccines are available for protective immunization of horses against viral diseases. Traditional inactivated and live-attenuated (modified live virus, MLV) virus vaccines remain popular and efficacious but recombinant vaccines are increasingly being developed and used, in part because of the perceived deficiencies of some existing products. New generation vaccines include MLVs with deletions and/or mutations of critical genes, subunit vaccines that incorporate immunogenic proteins (or portions thereof) or expression vectors that produce these proteins as immunogens, and DNA vaccines. New generation vaccines have been developed for several viral diseases of horses. We recently have developed an alphavirus replicon-vectored equine arteritis virus (EAV) vaccine, and evaluated a commercial canary pox virus-vectored vaccine for West Nile disease. The success of these new-generation vaccines has catalyzed efforts to develop improved vaccines for the prevention of African horse sickness, a disease of emerging global significance.     

Importance of equine piroplasmosis antibody presence in Spanish horses prior to export

Serological analysis of equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is included in the export testing requirements for most of the countries worldwide, thus involving a high economic impact on equine industry of EP-endemic countries, such as Spain. A total of 3368 serum samples from healthy horses collected prior to export between 2015 and 2018 in Spain were tested for antibodies against T. equi and B. caballi by using a competitive inhibition enzyme-linked immunosorbent assay (cELISA). The overall seroprevalence results in Spain revealed that almost a quarter of the horses analysed (24.1 %; 95% CI 22.6–25.5) could not be exported to countries free from EP. The implementation of prevention measures such as the use of acaricides and daily checks for ticks in horses, as well as regular serological screening of horses in Spain would aid to increase the number of horses exported to other countries.     

浙江地区幽门螺杆菌临床菌株空泡毒素基因的变异及其表达活性分析

目的 了解浙江地区消化性溃疡(PU)和慢性胃炎(CG)患者感染幽门螺杆菌(Hp)vacA基因的变异及其空泡毒素表达情况。方法 从70株Hp中选择部分菌株测定其vacA基因的s区和m区核苷酸序列。用细胞培养的方法检测Hp菌株的空泡毒性。结果 6株sla型Hp菌株的s区扩增产物与报道的sla型60190株核苷酸序列相似性为93．2％～98．3％，4株m2型Hp菌株的m区扩增产物与报道的m2型87—203株核苷酸序列之间相似性为93．8％～97．6％。1株m1b型Hp菌株的m区扩增产物与报道的m1型60190株相应序列相似性为87．3％，与m2型87—203株相应序列之间相似性为71．7％。所有5株sla／m1型Hp对HeLa、RK-13和SGC-7901三种细胞均有明显的空泡毒作用；仅27.9％(12／43)的sla／m2型菌株对HeLa细胞表现为空泡毒作用，但分别有65．1％(28／43)和62.8％(27／43)的菌株对RK-13细胞和SGC-7901细胞有空泡毒作用。所有sla／m1b和sla／m1b—m2型Hp菌株对三种细胞均有明显的空泡毒作用，以RK-13细胞计，81.0％(17／21)的sla／m1b型Hp菌株和1株sla／m1b—m2型有空泡毒性。PU组感染的Hp菌株空泡毒性(84．4％)强于CG组(60．5％)，差异有统计学意义(P＜0．05)。结论 浙江地区患者感染的Hp菌株vacA基因变异主要在于m区。vacA基因空泡毒性的表达在HeLa细胞中低毒性的sla／m2型菌株在RK-13和SGC-7901细胞中仍有较高的空泡毒性。m1b型Hp空泡毒性强度介于m1和m2型之间。PU患者感染的Hp菌株其空泡毒性明显强于CG组。     

Human heterophilic antibodies against equine immunoglobulins: assessment of their role in the early adverse reactions to antivenom administration

The presence of human heterophilic antibodies against horse immunoglobulins (HHA-HI) was determined by ELISA in sera from healthy volunteers and from patients who received equine antivenom for therapy of snake bite envenoming. These patients were selected from two independent clinical studies: one in Colombia in which patients received antivenom constituted by whole IgG (n=25); and the other in Brazil where an antivenom constituted by F(ab')(2) fragments was administered (n=31). Results show that healthy volunteers have antibodies, mainly of the IgG class, able to react with whole equine IgG. Additionally, patients have IgG antibodies that react both with whole equine IgG and F(ab')(2) fragments. In both clinical studies, no significant differences were observed in the HHA-HI titres between the patients who presented early adverse (anaphylactoid) reactions and those who did not develop them. In addition, no variation in titre was observed in samples collected before and after antivenom administration. These results do not support the hypothesis that the incidence of early adverse reactions to antivenom administration correlates with the titre of HHA-HI in the serum of patients. Nevertheless, participation of these antibodies as part of a multifactorial pathogenic mechanism associated with these reactions cannot be ruled out.     

Novel vaccination approaches against equine alphavirus encephalitides

The current production of inactivated vaccines for the prevention of equine alphavirus encephalitides caused by Eastern, Western and Venezuelan Equine Encephalitis viruses (EEEV, WEEV, VEEV) involves the manipulation of large quantities of infectious viral particles under biosafety level 3 containment laboratories with the potential risk of transmission to the operators. Moreover, these vaccines are not capable of inducing a long-lasting immunity. Modified live vaccines, which were also attempted, maintain residual virulence and neurotropism, causing disease in both horses and humans. Therefore, the production of an efficacious second generation vaccine which could be used in the prevention of alphavirus infection without the need to manipulate infectious viral particles under high biocontainment conditions could be of great benefit for the worldwide horse industry. Furthermore, equine alphaviruses are considered as biological threat agents. Subunit, chimeric, gene-deleted live mutants, DNA and adenovirus-vectored alphavirus vaccines have been evaluated; such approaches are reviewed in this work.     

Characterization of the major antigens of Haemophilus equigenitalis (contagious equine metritis organism).

Immunoelectrophoresis of ultrasonically disrupted Haemophilus equigenitalis (contagious equine metritis organism) cells against rabbit and equine antisera disclosed at least 11 precipitating antigens. Two of these, a polysaccharide and a lipopolysaccharide-protein complex, were of high molecular weight and located on the cell surface. The remaining antigens were intracellular and were small- to medium-sized proteins. The surface antigens were the most significant in relation to the serological response in infected horses. They also reacted with sera from apparently healthy cattle, but the reason for this was not determined. No serological cross-reaction between H. equigenitalis and species of Achromobacter and Moraxella was detected.     

Identification of the encephalitis equine virus, Parana, Brazil

INTRODUCTION: In the period of 1996-1999 some virus associated with encephalitis have been reported in horses from different regions of Paraná State, Brazil. To identify the etiologic agent associated with this illness, mosquitoes and serum samples were collected in the endemic area. METHODS: The study area corresponded to four municipalities of Paraná State, Brazil. Mosquitoes were captured in Shannon trap and human bait. After identification, they were processed for virus isolation. Blood of equines were collected in the municipalities of Querência do Norte and Colorado. Antibodies to different Alphavirus and Flavivirus were analyzed by hemagglutination inhibition test. Specific seroneutralization reactions were performed in those sera with a positive reaction in the hemagglutination test. RESULTS: The mosquitoes genus collected were: Culex, Aedes, Mansonia, Coquillettidia, Psorophora, Sabethes, Wyeomyia, and Limatus. Even thought no virus was isolated, serologic analyses showed hemagglutinazing antibodies to Eastern equine encephalitis, Mucambo, Pixuna, Maguari, and St Luis encephalitis viruses. The neutralization test showed specific reaction to Eastern equine encephalitis virus in 12 tested sera. CONCLUSIONS: Species of mosquitoes that could be potential vectors of encephalitis, buniavirus, and other arboviruses of epidemiological importance were collected. It is believed that Eastern equine encephalitis virus affected the equines populations in the study regions because of the symptoms and antibodies for the virus in the sera detected in these equines.     

Multivariate analysis as a method to evaluate antigenic relationships between BVDV vaccine and field strains

Bovine viral diarrhea virus (BVDV) is comprised of two species, BVDV-1 and BVDV-2, but given the genetic diversity among pestiviruses, at least 21 subgenotypes are described for BVDV-1 and 4 for BVDV-2. Genetic characterization can be achieved through complete or partial sequencing and phylogeny, but antigenic characterization can be difficult to determine due to the antigenic diversity and cross-neutralization that exists among isolates. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to determine antigenic difference can be unclear. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between vaccine strains and multiple field isolates. Polyclonal sera were generated against 6 BVDV strains currently contained in vaccine formulations, and each serum was used in VN’s to measure the neutralizing antibody titers against 15 BVDV field isolates characterized as prevalent and divergent subgenotypes in the USA. Principal component analysis (PCA) were performed on the VN assay datasets, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among isolates suggestive of antigenic differences. While expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. In addition, other BVDV isolates had distinct spatial patterns suggesting antigenically divergent isolates. This analysis provides an alternative and more efficient means to analyze large VN datasets to visualize antigenic relationships between pestivirus isolates. This analysis could be beneficial for vaccine development and evaluation of efficacy, since most vaccines cannot fully protect animals from the broad range diversity of BVDV viruses.     

A vectored equine herpesvirus type 1 (EHV-1) vaccine elicits protective immune responses against EHV-1 and H3N8 equine influenza virus

Vaccination is commonly used to control equine respiratory pathogens such as equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV). Here, we describe the generation and characterization of a recombinant EHV-1 modified live virus vaccine (MLV) based on a recent abortogenic EHV-1 strain, NY03. The immunogenicity and efficacy of the MLV was tested in horses in an EHV-1 vaccination/challenge experiment using the highly virulent neurovirulent EHV-1 strain OH03. Induction of a robust EHV-1-specific immune response was observed. Upon challenge infection, vaccinated horses were partially protected against disease as demonstrated by a significant reduction in clinical signs, nasal shedding and viremia levels. In addition, the NY03-based MLV was used to express the EIV H3 protein and immunogenicity was tested in horses. Expression of H3 was readily detected in NY03-H3-infected cells in vitro. Vaccination of horses resulted in the induction of a robust serological immune responses against two recent but genetically distinct EIV representatives, VA05 and NY-99, which were above the threshold predicted to be protective against development of clinical disease.     

The relationship of two equine mycoplasmas to Mycoplasma mycoides

Two unidentified mycoplasmas, N3 and N11, isolated from the respiratory tract of horses, were found to cross-react with strains of M. mycoides subsp. mycoides in indirect immunofluorescence tests, growth-inhibition tests carried out by the running drop/agar-well method, and in complement-fixation and double immunodiffusion tests. Serologically, the equine mycoplasmas were not completely identical with any of the reference strains of M. mycoides with which they were compared. Their cultural characteristics, ability to digest coagulated serum and casein, and survival at 45 degrees C, however, suggested that they were more closely related to strains of M. mycoides subsp. mycoides, such as Y-goat, which are found in goats, than to strains of that subspecies which are pathogenic for cattle.     

The relationship of two equine mycoplasmas to Mycoplasma mycoides.

Two unidentified mycoplasmas, N3 and N11, isolated from the respiratory tract of horses, were found to cross-react with strains of M. mycoides subsp. mycoides in indirect immunofluorescence tests, growth-inhibition tests carried out by the running drop/agar-well method, and in complement-fixation and double immunodiffusion tests. Serologically, the equine mycoplasmas were not completely identical with any of the reference strains of M. mycoides with which they were compared. Their cultural characteristics, ability to digest coagulated serum and casein, and survival at 45 degrees C, however, suggested that they were more closely related to strains of M. mycoides subsp. mycoides, such as Y-goat, which are found in goats, than to strains of that subspecies which are pathogenic for cattle.     

Antigenic variation of equine (Heq2Neq2) influenzavirus

Influenza equine (Heq2Neq2) strains isolated during the course of epizootics observed in Guanabara (Rio de Janeiro) and São Paulo, Brazil, in July—October 1969 were shown to differ antigenically from earlier strains of the same subtype (A/equine/Miami/1/63 (Heq2Neq2)). The difference could be clearly demonstrated in haemagglutination inhibition tests performed with postinfection horse or ferret sera but not with hyperimmune rooster sera. Antibody responses of diseased horses were higher and more frequent against current isolates than against strain equine/Miami/1/63. Some animals also showed antibody responses to the Hong Kong variant of human influenzavirus A.     

New assays to measure equine influenza virus-specific Type 1 immunity in horses

Equine influenza virus (EIV) is a leading cause of respiratory disease in horses. Equine influenza infection induces a long-term immunity to re-infection. Recent strategies of vaccination aim to mimic this immunity by stimulating both antibody and cellular immune responses. Cell-mediated immunity (CMI) to influenza is well defined in man, but little has been done to characterise the responses in the horse. Additionally, the development of reliable assays for the measurement of equine CMI has lagged behind serological methods and vaccine development. In this study, two methods of measuring EIV-specific T lymphocyte responses have been developed. An EIV 'bulk' cytotoxic T lymphocytes (CTL) assay using equine dermal fibroblasts as target cells has been adapted from a method used in the 1980s. This method was also complemented with a new EIV-specific IFNgamma synthesis assay. When compared with the measurement of EIV-specific IFNgamma synthesis previously described, this method required the amplification of EIV-specific lymphocytes by culture and was sensitive enough to detect stimulation of EIV-specific T lymphocytes induced by experimental infection with EIV or vaccination with recombinant canarypox viruses coding for EIV-HA molecules. This study provides the tools to characterise the stimulation of CMI by the new generation of vaccines against equine influenza.     

Trajectory analysis of winds and eastern equine encephalitis in USA, 1980-5

Backward trajectories of winds were determined to identify possible sources of eastern equine encephalitis virus associated with isolation of virus from mosquitoes or birds or outbreaks in horses between 1980 and 1985 in Maryland, New Jersey, New York and Michigan, USA. The results of the trajectory analyses suggested that eastern equine encephalitis virus could have been carried by infected mosquitoes on surface winds at temperatures 13 degrees C or higher from North Carolina north-eastwards along the Atlantic Coast to Maryland and New Jersey and thence to upstate New York and from western Kentucky to Michigan. Landing of mosquitoes was associated with the presence of a cold front and rain leading to variations in the location and timing of outbreaks from year to year. The mosquito responsible was most likely to have been Culiseta melanura, but Coquillettidia perturbans and Aedes sollicitans could also have been involved. There may be a continual cycle of eastern equine encephalitis virus in mosquitoes and birds in south-eastern USA, from where the virus could be distributed by infected mosquitoes on the wind along the Gulf and Atlantic Coasts and up the Mississippi Valley.