[Postvaccinal fatal Streptococcus zooepidemicus necrotizing fasciitis in a young dog: a case report]. / Fatale necrotiserende fasciitis door Strepto- coccus zooepidemicus na vaccinatie bij een jonge hond, een case report.

A 2.5-years-old female mongrel dog was routinely subcutaneously vaccinated. A few hours later mental dullness was noticed by the owner progressing into stupor the next day and resulting in a comatose state and death within 48 hours after vaccination. At post mortem examination, which was extended with histology and bacteriology, a necrotizing fasciitis and bacteremia caused by Streptococcus equi subsp. zooepidemicus were established. In the isolated Streptococcus strain four different superantigens were demonstrated that appeared to be able to produce exotoxins in vitro. Therefore, it is concluded that the minor skin trauma caused by vaccination enabled this strain to gain access to the subcutaneous tissue and to induce a necrotizing fasciitis. This process was complicated with a bacterial septicemia leading to death of the dog within 48 hours.

Establishment of a candidate equine influenza Florida Clade 2 strain A/eq/Richmond/1/07 horse antiserum as Ph. Eur. Biological Reference Preparation/OIE International Reference Reagent.

Equine influenza (EI) is an important respiratory disease of horses, with welfare and economic consequences. Vaccination remains one of the most efficient prevention methods available. Equine influenza virus (EIV) is constantly evolving and consequently EI vaccines need to be updated on a regular basis. In 2010, the World Organisation for Animal Health (OIE) Expert Surveillance Panel (ESP) on EI provided a new recommendation for EI vaccine strain composition, including the incorporation of representative EIV strains of both Florida Clade 1 and Clade 2 sub-lineages (FC1 and FC2, respectively). In this context, the European Pharmacopoeia (Ph. Eur.) - OIE reference panel for EI had to be complemented by an antiserum raised in horses against the FC2 representative EIV strain A/eq/Richmond/1/07. An international collaborative study was organised and managed by the European Directorate for the Quality of Medicines and HealthCare (EDQM) within the framework of its Biological Standardisation Programme (BSP). The study aimed at evaluating a new candidate reference for use as a common OIE International Standard/Ph. Eur. Biological Reference Preparation (BRP) horse antiserum to FC2 EIV A/equine/Richmond/1/07. The standard was to be established using the SRH and HI tests for subsequent use in immunogenicity, efficacy and batch potency assay of EI vaccines as a Ph. Eur. BRP (Ph. Eur. monograph 0249) and for use in clinical diagnostic tests as an OIE-approved International Standard Reagent (OIE chapter 3.5.7). The collaborative study confirmed the suitability of the candidate and an SRH titre was assigned. The candidate was adopted as a BRP by the Ph. Eur. Commission and approved by the OIE Biological Standards Commission as an International Standard Serum in November 2017 and February 2018, respectively.

Immunosuppressive properties of methotrexate: apoptosis and clonal deletion of activated peripheral T cells.

The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.

Equine herpesvirus-1-specific interferon gamma (IFNgamma) synthesis by peripheral blood mononuclear cells in thoroughbred horses.

REASONS FOR PERFORMING STUDY: An assay has been developed that measures EHV-1 specific interferon gamma synthesis (IFNgamma), a cytokine produced following the activation of memory T lymphocytes and therefore a measure of cell mediated immunity. The method requires validation in the field. OBJECTIVES: To measure the frequency of EHV-1 specific, IFNgamma synthesising peripheral blood mononuclear cells (PBMC) in a population of Thoroughbred horses, and examine its relationship with age, gender, premises and history of vaccination or field infection with EHV-1. METHODS: Lymphocytes from 200 Thoroughbred horses were stimulated with EHV-1 in vitro, and IFNgamma detected using a monoclonal antibody and indirect immunofluorescence. Percent positive cells were enumerated by flow cytometric analysis and the results described and compared statistically between groups. RESULTS: The frequency of IFNgamma+ PBMC was significantly higher in animals age >5 years compared with 2-4 years, in females vs. males, on stud farms vs. training yards and following vaccination of 2-year-olds with inactivated virus compared with nonvaccinates. Age strongly confounded all these associations and care must therefore be taken interpreting these results. Mares exposed to a field infection with EHV-1 also had higher frequencies of IFNgamma+ PBMC than other vaccinated horses. CONCLUSIONS: The frequency of EHV-1 specific, IFNgama+ PBMC among the sample Thoroughbred population was diverse but lowest in young, unvaccinated horses-in-training. POTENTIAL RELEVANCE: The frequency of EHV-1 specific lymphocytes synthesising IFNgamma in this population may be associated with its susceptibility to infection with this virus. This easy technique may be applied to monitor the antigenicity of vaccines and their effectiveness at stimulating cellular immunity.

Clinical, immunophenotypic and functional characterisation of T-cell leukaemia in six horses.

REASON FOR PERFORMING STUDY: Lymphoid leukaemia (LL) is rare in equids. In man, immunophenotypic classification identifies distinct leukaemic types with different treatment strategies. Improved understanding and classification of equine LL may allow similar advances. OBJECTIVES: To document the clinical, immunophenotypic and functional characteristics in 6 cases of equine LL of T-cell origin. METHODS: The clinical records and pathological findings from 6 cases of equine LL were analysed. Immunohistochemistry to identify T or B lymphocytes was performed on paraffin embedded tissues in 4 cases. Peripheral blood mononuclear cells (PBMC) were phenotyped for expression of CD4, CD8, MHC class I and II and B-cell antigens in 4 cases using monoclonal antibodies (mAbs) and flow cytometry. Neoplastic lymphocytes from 4 horses were stimulated with mitogens. RESULTS AND CONCLUSIONS: Six horses of various breeds were identified with LL of T-cell origin. The clinical course and presenting signs varied. Neoplastic lymphocytes were identified in peripheral blood samples from all horses and tissue invasion was confirmed at examination post mortem in 4 horses. Immunophenotyping identified a predominance of CD3+ T-cells in lymphoid tissues and CD4+ T-cells in circulating peripheral blood mononuclear cells (PBMC) in the affected horses. Neoplastic lymphocytes from the 4 cases that were tested failed to proliferate in response to mitogens. POTENTIAL RELEVANCE: Characterisation of the clinical, pathological and immunological findings in 6 horses with LL has added to reports of this rare condition, characterised it in greater detail and therefore provides a starting point for further investigations.

Immunogenicity of phospholipase A2 toxins and their role in Streptococcus equi pathogenicity.

Streptococcus equi subsp. equi (S. equi) is the causative agent of strangles, one of the most frequently diagnosed infectious diseases of horses worldwide. Phospholipase A2 toxins (PLA2) cleave phospholipid molecules at position sn-2 contributing to the production of leukotrienes that are important inflammatory mediators. Two homologous phospholipases, SlaA and SlaB are encoded by the S. equi genome suggesting that PLA2 toxins may contribute to its pathogenicity. Here we report the immunogenicity and role of PLA2 toxins during natural and experimental infection of horses with S. equi. The levels of anti-PLA2 specific antibodies in serum from horses naturally exposed to S. equi or without exposure were measured by indirect ELISA. Furthermore, the importance of PLA2 was determined during experimental infection of Welsh Mountain ponies with a mutant strain of S. equi lacking slaA and slaB. Our results show that PLA2 toxins are immunogenic, which supports their production during natural S. equi infection, but that these toxins are not essential for the development of strangles in a susceptible natural host.

Antibody and IFN-gamma responses induced by a recombinant canarypox vaccine and challenge infection with equine influenza virus.

In horses, equine influenza virus (EIV) is a leading cause of respiratory disease. Conventional inactivated vaccines induce a short-lived immune response. By comparison, natural infection confers a long-term immunity to re-infection. An aim of new equine influenza vaccines is to more closely mimic natural infection in order to achieve a better quality of immunity. A new live recombinant vaccine derived from the canarypox virus vector and expressing haemagglutinin genes of EIV (subtype H3N8) has been developed. Stimulation of the immune system was studied after immunisation with this canarypox-based vaccine and challenge infection by exposure to a nebulised aerosol of EIV. The humoral immune response was evaluated by measuring serum antibody levels using the single radial haemolysis (SRH) assay. The cellular immune response was assessed by the measurement of interferon gamma (IFN-gamma) synthesis in peripheral blood mononuclear cells (PBMC). Clinical signs of the disease (temperature, coughing, nasal discharge, dyspnoea, depression and anorexia) and virus excretion were monitored after challenge infection. Clinical signs and virus shedding were significantly reduced in vaccinates compared with unvaccinated controls. EIV-specific immunity was stimulated by vaccination with a recombinant vaccine as serological responses were detected after immunisation. This study also provided the first evidence for increased IFN-gamma protein synthesis in vaccinated ponies following challenge infection with EIV compared with control ponies.

Toward a detailed characterization of feline immunodeficiency virus-specific T cell immune responses and mediated immune disorders.

Infection of domestic cats with feline immunodeficiency virus (FIV) is associated with the development of an acquired immunodeficiency syndrome (AIDS). The pathogenesis of FIV is not fully understood but it has been reported that the immune system is progressively impaired during disease progression. As a result, anti-FIV specific immune response will usually not clear the virus and the acute stage is followed by a chronic asymptomatic phase. The overall objective of this study was to characterized FIV-induced immune cellular responses and -mediated immune disorder following the first weeks post-infection. Using both cytokine ELISpot and intracellular staining assays, FIV-specific T cells were monitored at 6, 9 and 12 weeks post-infection. We demonstrated that both IFNgamma(+) and, CD4 and CD8 TNFalpha(+) T cells specifically respond to FIV antigens. These responses were found to reach a peak at 9 weeks post-infection. It was further shown that the TNFalpha(+)CD8(+) responding T cells were contained within a CD8beta(low)CD62L(-) T cell subpopulation, expanded in FIV-infected cats. This T cell subpopulation which present features of activated CD8 T cells was further shown to be susceptible to spontaneous apoptosis following a short-term in vitro culture. Moreover, it was observed that cell death by apoptosis of this T cell subset was increased following FIV antigen-recognition. Therefore, FIV might alter immune homeostasis in inducing chronic activation of TNFalpha(+)CD8(+) T cells which eventually will die following antigen contact while deleting CD4(+) T cells. Interestingly, this study confirmed the strong similarity between FIV and HIV pathogenesis.

Localised mitogenic activity in horses following infection with Streptococcus equi.

Streptococcus equi subspecies equi (S. equi) is the causative agent of strangles, a highly contagious upper respiratory disease of equids. Streptococcus equi produces superantigens (sAgs), which are thought to contribute to strangles pathogenicity through non-specific T-cell activation and pro-inflammatory response. Streptococcus equi infection induces abscesses in the lymph nodes of the head and neck. In some individuals, some abscess material remains into the guttural pouch and inspissates over time to form chondroids which can harbour live S. equi. The aim of this study was to determine the sites of sAg production during infection and therefore improve our understanding of their role. Abscess material, chondroids and serum collected from Equidae with signs of strangles were tested in mitogenic assays. Mitogenic sAg activity was only detected in abscess material and chondroids. Our data support the localised in vivo activity of sAg during both acute and carrier phases of S. equi infection.

Quantitative analysis of the antigen-specific IFNgamma+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNgamma+ CD8+ T cell response.

It is now well established that antigen-specific CD8(+) T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNgamma-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNgamma(+) T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNgamma(+) T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNgamma(+) responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8alpha(bright) pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.

Prevalence and disease associations of superantigens szeF, szeN and szeP in the S. zooepidemicus population and possible functional redundancy of szeF.

Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) causes a variety of infections in a broad range of species. This study broadens prevalence data for three recently identified novel superantigens (szeF, szeN, and szeP) to define links between their presence and disease type. Screening of 437 strains across 190 sequence types (STs) revealed that 50% of strains contained superantigen genes. Results confirmed that the presence of S. zooepidemicus superantigen genes is significantly associated with non-Strangles lymph node abscessation in the horse (p-value = 0.003) and their absence is associated with uterine infection/abortion (p-value = 0.006). This study also investigated the lack of mitogenicity observed in szeF only. Results show that szeF is polymorphic, with 23 different alleles, and mutations altering the protein sequence. Gene expression differences are not responsible for lack of mitogenic activity in these strains. Taken together, these findings suggest that superantigens are important for S. zooepidemicus pathogenesis but SzeF probably has little involvement.

A systematic review of the immune-modulators Parapoxvirus ovis and Propionibacterium acnes for the prevention of respiratory disease and other infections in the horse.

Inactivated Parapoxvirus ovis (iPPVO) and Propionibacterium acnes (P. acnes) are currently used in equine medicine as immune-modulators for prophylactic treatment or adjunct to conventional therapy in order to improve immune defences, to prevent or treat infectious diseases. Their mode of action relies on a non-antigen specific interaction with the innate and/or adaptive immune responses. iPPVO stimulates and regulates cytokine secretion by leucocytes, while P. acnes acts primarily through the activation of macrophages. This report aims to describe their activity as immune-modulators and to summarise the scientific literature and reports available about their use in horses, particularly in the prevention or treatment of equine respiratory diseases. This systematic review regroups articles published in peer-review journals, clinical trials reports, conference proceedings and other information made available in the last 2 decades.

Whole inactivated equine influenza vaccine: Efficacy against a representative clade 2 equine influenza virus, IFNgamma synthesis and duration of humoral immunity.

Equine influenza (EI) is a serious respiratory disease of horses induced by the equine influenza virus (EIV). Surveillance, quarantine procedures and vaccination are widely used to prevent or to contain the disease. This study aimed to further characterise the immune response induced by a non-updated inactivated EI and tetanus vaccine, including protection against a representative EIV isolate of the Florida clade 2 sublineage. Seven ponies were vaccinated twice with Duvaxyn IE-T Plus at an interval of four weeks. Five ponies remained unvaccinated. All ponies were experimentally infected with the EIV strain A/eq/Richmond/1/07 two weeks after the second vaccination. Clinical signs of disease were recorded and virus shedding was measured after experimental infection. Antibody response and EIV-specific IFNgamma synthesis, a marker of cell-mediated immunity, were measured at different time points of the study. Vaccination resulted in significant protection against clinical signs of disease induced by A/eq/Richmond/1/07 and reduced virus shedding when challenged at the peak of immunity. Antigenic drift has been shown to reduce protection against EIV infection. Inclusion of a more recent and representative EIV vaccine strain, as recommended by the OIE expert surveillance panel on equine influenza vaccine, may maximise field protection. In addition, significant levels of EIV-specific IFNgamma synthesis by peripheral blood lymphocytes were detected in immunised ponies, which provided a first evidence of CMI stimulation after vaccination with a whole inactivated EIV. Duration of humoral response was also retrospectively investigated in 14 horses vaccinated under field condition and following the appropriate immunisation schedule, up to 599 days after first immunisation. This study revealed that most immunised horses maintained significant levels of cross-reactive SRH antibody for a prolonged period of time, but individual monitoring may be beneficial to identify poor vaccine responders.

Duration of equine influenza virus shedding and infectivity in immunised horses after experimental infection with EIV A/eq2/Richmond/1/07.

Equine influenza (EI) is a major respiratory disease of horses. Recent outbreaks of EI have demonstrated the ease with which EI virus (EIV) can be transmitted internationally. This study aimed to improve our understanding of EIV shedding after infection of vaccinated horses, which would inform possible changes to current quarantine requirements. Our objectives were to compare commonly used diagnostic tests and to evaluate the relative merits of nasal and nasopharyngeal swabs for detection of EIV in vaccinated and unvaccinated ponies following EIV infection and to use these data to inform optimal quarantine procedures for the safe international movement of horses. Five ponies vaccinated against EI were infected experimentally with A/eq/Richmond/1/07 (Florida clade 2), 11 weeks after V2. Nasal and nasopharyngeal swabs were taken daily for 14 days and every 2 days for another 2 weeks. The 5 vaccinates were introduced sequentially for 48h to 3 groups of 2 naïve sentinel ponies each on days 2, 4 and 6 post-challenge respectively. Clinical signs of disease and EIV shedding were monitored for 14 days after co-mingling. EIV was detected by 3 different methods of detection (EIV nucleoprotein ELISA, EIV nucleoprotein qRT-PCR and isolation/titration in embryonated hens' eggs). Directigen™ EZ Flu A+B tests were also performed on samples from the vaccinated ponies for 6 days after infection. Results show that nasopharyngeal swabs were superior to nasal swabs, with increased frequency and amount of virus detected. The average mean duration of shedding was 6-8 days in naïve animals. All 3 sentinel groups were infected successfully with EIV after commingling with vaccinates, indicating up to 6 days of transmission. EI protection induced by vaccination is a dynamic process, naturally fluctuating and dependent on the time since last immunisation, with periods of high immunity (peak of immunity shortly after boost immunisation) and periods of susceptibility to EIV infection. This result indicates that vaccinated horses may actively transmit EIV if the immunity gap (a usual period of susceptibility between V2 and V3) is not adequately closed by immunisation. In infected sentinels EIV was detectable up to 12 days after commingling. Results also suggest that tests such as qRT-PCR may be a suitable substitute for time spent in pre-export quarantine.

ISCOM-matrix-based equine influenza (EIV) vaccine stimulates cell-mediated immunity in the horse.

The humoral immune response induced by ISCOM-matrix (Immuno Stimulating COMplex-Matrix)-adjuvanted equine influenza virus (EIV) vaccine is well documented in horses. ISCOM-matrix adjuvanted vaccines against human influenza are strong inducers of cell-mediated immunity (CMI), including T cell proliferation and virus-specific cytotoxic T cell. In the horse, the CMI response to equine influenza vaccination is less well characterised. An ISCOM-based vaccine has been shown to induce interferon gamma (IFN-γ) synthesis, a CMI marker, in the horse, but this has not been shown for the ISCOM-matrix vaccine, which is a different formulation. The objective of this study was to measure EIV-specific IFN-γ synthesis after vaccination with an ISCOM-matrix-adjuvanted EIV vaccine. Equilis Prequenza is a commercialised inactivated EIV vaccine containing purified haemagglutinin (HA) and neuraminidase (NA) subunits adjuvanted with ISCOM-matrix. Six influenza-naïve Welsh mountain ponies were vaccinated twice with Equilis Prequenza at an interval of four weeks. Six control ponies received a placebo of physiological water. EIV-specific IFN-γ synthesis by peripheral blood lymphocytes and the antibody response to a panel of representative EIV isolates were measured prior to and after both injections. Immunisation with the ISCOM-matrix-based EIV vaccine stimulated significant EIV-specific IFN-γ synthesis and EIV-specific single radial haemolysis (SRH) antibody. In conclusion, EIV vaccine adjuvanted with ISCOM-matrix stimulates both antibody and a cellular immune response in the horse.

Efficacy of a whole inactivated EI vaccine against a recent EIV outbreak isolate and comparative detection of virus shedding.

An outbreak of H3N8 Equine Influenza virus (EIV) that occurred in vaccinated horses in Japan was caused by a genetically divergent EIV isolate of the Florida clade 1 sub-lineage. This virus subsequently entered Australia where it infected thousands of immunologically naïve horses. The objective of this study was to evaluate the ability of a non-updated whole inactivated equine influenza (EI) vaccine to protect if used in the face of an outbreak induced by a virus similar to the ones circulating in Japan and Australia in 2007. Seven naïve Welsh mountain ponies were immunised twice with the commercially available vaccine Duvaxyn IE-T Plus and experimentally infected with A/eq2/Sydney/2888-8/07. Five ponies remained unvaccinated as controls. The ponies were challenged in an ACDP (Advisory Committee on Dangerous Pathogens) Category III containment facility by exposure to a nebulised aerosol of A/eq2/Sydney/2888-8/07 two weeks after the second vaccination. Clinical signs and virus shedding were monitored for 14 days post-challenge infection. After challenge infection, all control ponies developed clinical signs of disease with coughing being particularly noteworthy when compared with vaccinated ponies. Only 3 out of 5 controls developed pyrexia for up to 3 days, and 1 out of 7 vaccinates was pyretic for 1 day. Nasal discharge was evident in both control and vaccinated ponies with no significant difference between groups. Three different methods were used to measure virus shedding in nasal secretions (i.e. titration in embryonated hens' eggs, EIV NP ELISA and EIV NP qRT-PCR). The intensity and duration of EIV shedding significantly decreased in the vaccinated group when compared with the control ponies. All control ponies seroconverted after experimental infection with A/eq2/Sydney/2888-8/07 whereas only 1 out of 7 vaccinated ponies had a significant increase in antibody. Duvaxyn IE-T Plus therefore reduced clinical signs and virus shedding when ponies were challenged with A/eq2/Sydney/2888-8/07 (H3N8), 2 weeks after a second dose of vaccine.

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.