Validation of an immunoblot assay employing an objective reading system and used as a confirmatory test in equine infectious anaemia surveillance programs.

Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As for the in-house competitive ELISA (c-ELISA) and the AGIDT, the Italian National Reference Laboratory for EIA (NRL) validated the IB according to the OIE guidelines, employing eight panels containing positive sera, including those from EIA virus (EIAV) proven infected horses, and negative horse, mule and donkey sera collected from different geographical areas. In addition, two international reference image panels were employed for the optimization and the validation of the digital image reading system adopted that allows an impartial measurement of the serum reactivity in the IB assay. The immunological reactivity to EIAV antigens, p26, gp45 and gp90 adsorbed on the IB membrane, determines the serological status of the animal and for EIA, a p26 positive band together with at least one of the other antigen defines a subject as serologically positive for EIAV. For validation, the parameters assessed were threshold values, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility. These parameters were evaluated for each antigen as well as in combination, according to the diagnostic algorithm established above. The validation data defined the IB as having a satisfactory sensitivity, specificity, repeatability and reproducibility for all antigens and species tested. An instrumental recording of the results improves the confidence in using IB as a confirmatory test for EIAV, differently from the AGIDT that is read by an operator. The advantages of using the IB are its higher sensitivity, to that of the AGIDT, which allows an earlier detection of infection that reduces the risk of transmission and therefore the incidence of the EIA, and its higher specificity to that of the ELISA which is based on the discrimination of subjects reacting only against the p26, the antigen used by all ELISAs available, which are not considered as infected by EIAV. In particular, when this assay is used in outbreaks it can detect new cases earlier than the AGIDT, and therefore reduce the restriction period with an economic benefit for the animal owners and the public veterinary sanitary system.

Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions.

BACKGROUND: Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. OBJECTIVES: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 5' untranslated region (5' UTR)/exon 1 of the tat gene of EIAV. STUDY DESIGN: The analytical and clinical performance of the newly developed EIAV RT-iiPCR was evaluated by comparison with a EIAV real-time RT-PCR (RT-qPCR) along with the AGID test. METHODS: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 5' UTR/tat gene and samples from two EIAV-positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT-qPCR and AGID using samples derived from 196 inapparent EIAV carrier horses. RESULTS: EIAV RT-iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight genome equivalents), with a concordance of 95.41% to conventional EIAV RT-qPCR. However, the RT-qPCR and RT-iiPCR had sensitivities of 43.75 and 50.00%, respectively, when compared to the AGID test. MAIN LIMITATIONS: Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT-PCR-based tests. CONCLUSIONS: Although EIAV RT-iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently exposed or "serologically silent" equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore, the relatively low cost and field-deployable design enable utilisation of EIAV RT-iiPCR even in remote regions.

Equine infectious anemia prevalence in feral donkeys from Northeast Brazil.

Equine infectious anemia virus (EIAV) is an important cause of morbidity and mortality throughout the world. Although the virus infects all members of the Equidae the vast majority of studies have been conducted in horses (Equus caballus) with comparatively little information available for other equid species. Brazil has one of the most abundant donkey (E. asinus) populations of any nation although the economic importance of these animals is declining as transportation becomes increasingly mechanized. As a result, considerable numbers of donkeys especially in the Northeast of the country have been released and allowed pursue an almost feral existence. Consequently, this large and growing population constitutes a significant risk as a reservoir for the maintenance and transmission of important equine infectious diseases such as glanders and equine arteritis virus in addition to EIAV. This study examines the prevalence of EIA in a semi-wild donkey population from Mossoró city, in Northeast Brazil, using AGID followed by cELISA, rgp90 ELISA and immunoblot (IB). Serum samples were collected from 367 donkeys without obvious EIA clinical signs. Subsequent testing revealed seropositive rates of 1.6% (6/367) in officially approved AGID tests, 3.3% (12/367) in cELISA and 14.4% (53/367) in the rgp90 ELISA. However, 88.7% (47/53) of the rgp90 ELISA positive samples were almost certainly false reactions because they failed to react with two or more antigens in IB. Consequently, the rpg90 ELISA has a similar sensitivity to AGID with donkey serum samples. Such high false positive rates have not been observed previously with serum samples from horses. Another highly significant finding is that 56.9% (33/58) of the donkey serum samples tested in IB had reactivity to EIAV p26 only. Although this could result from recent infection with the virus, it has been found that in some equids p26 only reactivity persists for extensive periods of time suggesting exposure to antigens possessing cross-reactive determinants or EIAV strains with envelope glycoproteins that are different from any that have been previously characterized and so undetectable by current IB techniques.

Serologically silent, occult equine infectious anemia virus (EIAV) infections in horses.

Molecular and serological techniques for Equine Infectious Anemia Virus (EIAV) diagnosis were compared using samples from 59 clinically normal horses stabled on five farms in the Santa Fe Province of Argentina. Of these 26 (44.1%) were positive in official AGID tests and/or gp45/gp90-based ELISA. Surprisingly 18 of the 33 seronegative horses were positive in a PCR against viral sequences encoding gp45 (PCR-positive/AGID-negative) with all but one remaining EIAV-antibody negative throughout a two year observation period. The gp45 PCR results are supported by fact that 7/18 of these horses were positive in the Office International des Epizooties (OIE) recommended EIAV gag gene specific PCR plus 2 of this 7 also reacted in a PCR directed predominantly against the 5' untranslated region of the viral genome. Furthermore sufficient quantities of serum were available from 8 of these horses to verify their seronegative status in sensitive Western Blot tests and demonstrate by ELISA the absence of EIAV-specific antibodies was not attributable to abnormalities in total IgG concentration. Studies involving 7 of the PCR-positive/AGID-negative horses to measure lymphocyte proliferation in the presence of PHA showed no significant differences between this group and control animals. In addition, lymphocytes from 2 of these 7 horses responded to peptides derived from gp90 and gp45. Together these results demonstrate that apparently clinically normal horses with no gross signs of immunodeficiency in terms of total IgG concentration or T helper-cell function can remain seronegative for at least 24 months while harboring EIAV specific nucleic acid sequences.

Prevalence and risk factors for Equine Infectious Anemia in Poconé municipality, northern Brazilian Pantanal.

Serum samples collected from 547 equids in the Pantanal region of Brazil were evaluated for antibodies to Equine Infectious Anemia Virus (EIAV) by the agar gel immunodiffusion test. Risk factors associated with EIAV seropositivity were evaluated and spatial dependence investigated using a Spatial Lag Model. EIAV prevalence on farms in the Pantanal was 52.0% (13/25) with adjusted prevalence between equids of 31.5% (17.4-48.8% 95% CI). Intra-herd prevalence ranged from 5.0 to 77.0%. Statistical analysis demonstrated that farms and animals in regularly flooded areas had respectively 60 and 146 fold higher chance to be sero-positive than farms and animals located in non-flooded areas. Spatial Lag Model results were generally consistent with this conclusion although there was a negative spatial correlation between farms located within in regularly inundated regions, suggesting that other factors, such as management practices, probably play a significant role in transmission of EIAV. Equids with clinical signs were 3.74-fold more likely to be sero-positive than those without clinical signs. The results of this work reveal a high prevalence of EIAV in the Pantanal area of Brazil demonstrating that equids reared in this region are at great risk of infection.

Challenges and proposed solutions for more accurate serological diagnosis of equine infectious anaemia.

Serological diagnosis of equine infectious anaemia virus (EIAV) infections has depended mainly on the agar gel immunodiffusion test (AGIDT). This study documents the presence of EIAV genetic sequences in a number of persistently infected horses and mules whose serums were interpreted as negative/equivocal on AGIDT, but positive on more than one ELISA test and in immunoblot tests. Strategies designed to take advantage of the combined strengths of the ELISA and AGIDT are shown effective in a national surveillance program for EIA in Italy where 17 per cent (25/149) of the equids considered to be infected with EIAV on combined/comparative serological data had reactions in the AGIDT that were interpreted as negative or equivocal. These data document the benefits of using a three-tiered laboratory system for the diagnosis of EIA. Although the ELISA-first strategy introduces some confusing results, the discovery of up to 20 per cent more cases of EIA makes it compelling. In our opinion, it is better and more defensible to find two samples in 1000 with resolvable but falsely positive ELISA tests for EIA than to release two to three horses in 10,000 with falsely negative test results for EIA (the rates seen in the Italian surveillance presented here).

Selection of peptides for serological detection of equine infectious anemia.

Equine infectious anemia caused by equine infectious anemia virus is an important disease due to its high severity and incidence in animals. We used a phage display library to isolate peptides that can be considered potential markers for equine infectious anemia diagnosis. We selected peptides using IgG purified from a pool comprised of 20 sera from animals naturally infected with equine infectious anemia virus. The diagnostic potential of these peptides was investigated by ELISA, Western blot and dot blot with purified IgG and serum samples. Based on the results, we chose a peptide mimetic for glycoprotein gp45 epitopes of equine infectious anemia virus, with potential for use as an antigen in indirect diagnostic assays. Synthesis of this peptide has possible applications for the development of new diagnostic tools for this disease.

Oxidant-antioxidant imbalance in horses infected with equine infectious anaemia virus.

This study assesses the impact of equine infectious anaemia virus (EIAV) infection on the oxidant/antioxidant equilibrium of horses. Blood samples from 96 Romanian horses aged 1-25 years, were divided into different groups according to their EIAV-infection status, age, and time post-seroconversion. The effect of infection on oxidative stress was estimated by measuring enzymatic antioxidants (superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase), non-enzymatic antioxidants (uric acid and carotenoids), and lipid peroxidation (malondialdehyde [MDA]). Infection modified the oxidant/antioxidant equilibrium in the horses, influencing GPx and uric acid levels (P<0.05). Time post-seroconversion also contributed to oxidative stress imbalance, exhibiting a significant influence on both SOD and MDA concentrations in the blood (P<0.05). Animal age did not have a significant influence on oxidative stress. Recently infected horses (<1 year following seroconversion), and horses >5 years old, represented the most vulnerable category in terms of oxidative stress, followed by recently infected animals <5 years old. The results of this study are novel in implicating EIAV infection in the development of oxidative stress in horses.

A pilot study on an attenuated Chinese EIAV vaccine inducing broadly neutralizing antibodies.

The attenuated Chinese equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. In this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated Chinese vaccine strain EIAVFDDV and then observed the evolution of neutralizing antibodies against different EIAV strains. During the vaccination phase, all vaccinees rapidly developed high levels of neutralizing antibodies against the homologous vaccine strain (pLGFD3V), and 3 out of 4 horses showed a gradual increase in serum neutralizing activity against two relatively heterologous virulent variants of the challenge strain (pLGFD3Mu12V and DLV34). After challenge, the three horses that had developed high levels of neutralizing antibodies against pLGFD3Mu12V and DLV34 did not show signs of infection, which was demonstrated by immune suppression, while the one horse producing serum that could only neutralize pLGFD3V developed a febrile episode during the 8-month observation period. To assess whether the broadly neutralizing activity is associated with immune protection, sera drawn on the day of challenge from these four vaccinees and an additional four EIAVFDDV-vaccinated horses were analyzed for neutralizing antibodies against pLGFD3V, pLGFD3Mu12V and DLV34. Although there was no significant correlation between protection from infection and serum neutralizing activity against any of these three viral strains, protection from infection was observed to correlate better with serum neutralizing activity against the two heterologous virulent strains than against the homologous vaccine strain. These data indicate that EIAVFDDV induced broadly neutralizing antibodies, which might confer enhanced protection of vaccinees from infection by the challenge virus.

[Study of the correlation between the plasma viral load and protective immunity induced by the equine infectious anemia attenuated vaccine and its parental virulent strain].

The threshold hypothesis of attenuated lentiviral vaccine considers that the type of host response to infections of lentiviruses depends on the viral load. To evaluate the correlation between viral loads of the attenuated vaccine strain of equine infectious anemia virus (EIAV) and their effects to induce protective immunity, longitudinal plasma viral loads in groups of horses inoculated with either an attenuated EIAV vaccine strain (EIAV(DLV125)) or sub-lethal dose of an EIAV virulent strain (EIAV(LN40)) were compared. Similar levels of plasma viral loads ranging from 10(3)-10(5) copies/mL were detected from samples of these two groups of animals (P > 0.05) during 23 weeks post the inoculation. However, different responses to the challenge performed thereafter with lethal dose of the EIAV virulent strain were observed from the groups of horses inoculated with either EIAV(DLV125) or sub-lethal dose of EIAV(LN40). The protective efficiency was 67% (3 of 4 cases) and 0 (none of 2 cases), respectively. Our results implicate that the viral load of EIAV attenuated vaccine is not the primary factor, or at least not the solo primary factor, to determine the establishment of immune protection.

[Serological and clinical proof of freedom from Equine Infectious Anemia (EIA) in imported and domestic horses in Switzerland]. / Serologischer und klinischer Nachweis der Freiheit von Equiner Infektiöser Anämie (EIA) bei importierten und einheimischen Pferden in der Schweiz.

Since 1991, no cases of Equine Infectious Anemia (EIA) have been reported in Switzerland. Risk factors for introduction of the virus into Switzerland are still present or have even increased as frequent inapparent infections, large numbers of imported horses, (since 2003) absence of compulsory testing prior to importation, EIA cases in surrounding Europe, possible illegal importation of horses, frequent short-term stays, poor knowledge of the disease among horse owners and even veterinarians. The aim of this study was to provide evidence of freedom from EIA in imported and domestic horses in Switzerland. The serum samples from 434 horses imported since 2003 as well as from 232 domestic horses fifteen years of age or older (since older horses have naturally had a longer time of being exposed to the risk of infection) were analysed using a commercially available ELISA test. All samples were seronegative, indicating that the maximum possible prevalence that could have been missed with this sample was 0.5% (95% confidence).

Characterization of an equine macrophage cell line: application to studies of EIAV infection.

EIAV is a monocyte/macrophage tropic virus. To date, even though EIAV has been under investigation for numerous years, very few details have been elucidated about EIAV/macrophage interactions. This is largely due to the absence of an equine macrophage cell line that would support viral replication. Herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (EML) cell line with the functional and immunophenotype characteristics of differentiated equine monocyte derived macrophage(s) (eMDM(s)). These cells possess strong non-specific esterase (NSE) activity, are able to phagocytose fluorescent bioparticles, and produce nitrites in response to LPS. The EML-3C cell line expresses the EIAV receptor for cellular entry (ELR1) and supports replication of the virulent EIAV(PV) biological clone. Thus, EML-3C cells provide a useful cell line possessing equine macrophage related properties for the growth and study of EIAV infection as well as of other equine macrophage tropic viruses.

Specificity of serum neutralizing antibodies induced by transient immune suppression of inapparent carrier ponies infected with a neutralization-resistant equine infectious anemia virus envelope strain.

It has been previously reported that transient corticosteroid immune suppression of ponies experimentally infected with a highly neutralization resistant envelope variant of equine infectious anemia virus (EIAV), designated EIAV(DeltaPND), resulted in the appearance of type-specific serum antibodies to the infecting EIAV(DeltaPND) virus. The current study was designed to determine if this induction of serum neutralizing antibodies was associated with changes in the specificity of envelope determinants targeted by serum antibodies or caused by changes in the nature of the antibodies targeted to previously defined surface envelope gp90 V3 and V4 neutralization determinants. To address this question, the envelope determinants of neutralization by post-immune suppression serum were mapped. The results demonstrated that the neutralization sensitivity to post-immune suppression serum antibodies mapped specifically to the surface envelope gp90 V3 and V4 domains, individually or in combination. Thus, these data indicate that the development of serum neutralizing antibodies to the resistant EIAV(DeltaPND) was due to an enhancement of host antibody responses caused by transient immune suppression and the associated increase in virus replication.

Response of ELA-A1 horses immunized with lipopeptide containing an equine infectious anemia virus ELA-A1-restricted CTL epitope to virus challenge.

Lipopeptide containing an ELA-A1-restricted cytotoxic T lymphocyte (CTL) epitope from the envelope surface unit (SU) protein of the EIAV(WSU5) strain was used to immunize three horses having the ELA-A1 haplotype. Peptide-specific ELA-A1-restricted CTL were induced in all three horses, although these were present transiently in PBMC. These horses were further immunized with lipopeptide containing the corresponding CTL epitope from the EIAV(PV) strain. Then, the three immunized horses and three non-immunized horses were challenged by intravenous inoculation with 300 TCID(50) EIAV(PV). All horses developed cell free viremia, fever and thrombocytopenia. However, there was a statistically lower fever and thrombocytopenia severity score in the immunized group. Shorter duration of plasma viral load in two of the three immunized horses likely explains the less severe clinical disease in this group. Results indicate that lipopeptide immunization had a protective effect against development of clinical disease following virus challenge.

Effects of long terminal repeat sequence variation on equine infectious anemia virus replication in vitro and in vivo.

The long terminal repeat (LTR) is reported to be one of the most variable portions of the equine infectious anemia virus (EIAV) genome. To date, however, no information is available on the effects of observed sequence variations on viral replication properties, despite a widespread assumption of the biological importance of EIAV LTR variation. EIAV LTR sequence variability is confined mostly to a small portion of the enhancer within the U3 segment of the LTR. Analysis of published EIAV LTR sequences revealed six different types of LTR based on the pattern of putative transcription factor motifs within the variable region of the enhancer. To test directly the significance of LTR variation, the in vitro and in vivo replication properties of two variant LTR species were investigated using two isogenic viruses, EIAV(19-2) and EIAV(19-2-6A), differing only within the enhancer region. The results of these studies demonstrated that the two variants replicated with similar kinetics and to equal levels in cultured equine fibroblasts or in equine macrophage, the natural target cell of EIAV, even after prolonged serial passage in the latter cell type. Furthermore, EIAV(19-2) and EIAV(19-2-6A) variants demonstrated similar replication levels in experimentally infected ponies. However, ponies infected with EIAV(19-2-6A) exhibited a rapid switch in the prevalent LTR type, such that by 112 days postinfection, no original-LTR-type viruses were evident. This specific and rapid shift in LTR quasispecies indicates an in vivo selection that is not reflected in simple in vitro replication rates, suggesting undefined selection pressures in vivo that drive LTR variation during persistent EIAV infection.

Platelets from thrombocytopenic ponies acutely infected with equine infectious anemia virus are activated in vivo and hypofunctional.

Thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus. Multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of EIAV-associated thrombocytopenia. This study was undertaken to investigate whether regenerative thrombopoiesis and platelet destruction occurred in ponies acutely infected with EIAV. Circulating large, immature platelets were increased in ponies acutely infected with EIAV late in the infection when platelet count was at a nadir. Morphometric analysis of bone marrow from acutely infected ponies revealed significant increased in megakaryocyte area and megakaryocyte nuclear area. A trend toward increased numbers of megakaryocytes was also observed. Platelets from acutely infected ponies had increased surface-bound fibrinogen and ultrastructural changes consistent with in vivo platelet activation. Platelets also had hypofunctional aggregation responses to three agonists in vitro. We conclude that thrombocytopenia in ponies acutely infected with EIAV is regenerative and suggest that bone marrow platelet production is not severely compromised in these ponies. Our findings reveal that in vivo platelet activation occurs in ponies acutely infected with EIAV, and as a result platelets are hypofunctional in vitro. Activation of platelets in vivo may cause platelet degranulation or formation of platelet aggregates, which would result in removal of these damages platelets from circulation. This may represent a form of nonimmune-mediated platelet destruction in ponies acutely infected with EIAV.

Increased interleukin-6 activity in the serum of ponies acutely infected with equine infectious anaemia virus.

Seven ponies were infected with the virulent wild-type Wyoming strain of equine infectious anaemia virus (EIAV). Infection status was monitored by serum reverse transcriptase activity, rectal temperature, and complete blood count. Preinfection serum and serum obtained during the initial febrile episode following infection were assayed for interleukin 6 (IL-6) activity. Postinfection IL-6 activity was significantly increased as compared to preinfection values. The magnitude of increase in IL-6 was positively correlated with reverse transcriptase activity (an indirect measure of viraemia) but was not correlated with rectal temperature. IL-6 production in response to EIAV infection may play a role in pathogenesis of disease, especially the hyperglobulinaemia and apparent polyclonal B cell activation in these horses.

Flow cytometric method for detecting thiazole orange-positive (reticulated) platelets in thrombocytopenic horses.

OBJECTIVE: To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. ANIMALS: 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-negative horses. PROCEDURE: Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms. RESULTS: Healthy ponies (n = 9) had 1.28 to 2.83% (mean +/- SD, 2.03 +/- 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 +/- 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41% (26.51 +/- 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 +/- 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 +/- 20,500 and 39,300 +/- 13,500 platelets/microliters, respectively (reference range, 94,000 to 232,000 platelets/ microliters). CONCLUSION: Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses. CLINICAL RELEVANCE: Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses.

Suppression of megakaryocyte colony growth by plasma from foals infected with equine infectious anemia virus.

Foals infected with equine infectious anemia virus become thrombocytopenic 7 to 20 days after virus inoculation, and within a few days following the onset of detectable viremia. The thrombocytopenia is associated with suppression of platelet production. Possible mediators of suppression of thrombopoiesis include tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), cytokines that are released during inflammation. To assess effects of plasma or serum from infected foals on megakaryocyte (MK) growth and maturation in vitro, equine low-density bone marrow cells were cultured for clonogenic and ploidy assays. Neutralizing antibodies to TNF-alpha and TGF-beta were added to cultures to determine the contribution of these cytokines to suppression of thrombopoiesis. Plasma from the immediately pre-thrombocytopenia (Pre-Tp) period significantly reduced MK colony numbers. This suppression was partially reversed upon antibody neutralization of plasma TNF-alpha, TGF-beta, or both. There were no differences in ploidy distribution of MK grown in the presence of preinfection serum compared with those grown in the presence of Pre-Tp serum. These results indicate that TNF-alpha and TGF-beta may contribute to suppression of MK proliferation and represent likely factors in the pathogenesis of thrombocytopenia.

Control of equine infectious anemia virus is not dependent on ADCC mediating antibodies.

Horses infected with equine infectious anemia virus (EIAV) have recurrent episodes of viremia which are eventually controlled, but the immune mechanisms have not been identified. Antibodies were detected to the surface of EIAV-infected cells within 1 month postinfection and remained for at least 3.5 years postinfection. These antibodies recognized cell surface-exposed envelope (Env) glycoproteins, but could not mediate antibody dependent cellular cytotoxicity (ADCC) using EIAV-WSU5-infected equine kidney (EK) cells as targets and peripheral blood mononuclear cells (PBMC) or polymorphonuclear cells (PMN) as effector cells. Furthermore, purified IgG antibodies from horses infected with either EIAV-WSU5 or EIAV-Wyo did not mediate ADCC of infected target cells. Armed effector cells could not be detected in infected horse blood nor could effector cells be prearmed by incubation with serum antibodies to cell surface antigens. The use of EIAV-WSU5-infected equine macrophages as target cells did not result in ADCC. In contrast, serum antibody from EHV-1 vaccinated horses and PBMC or PMN as effector cells caused ADCC of EHV-1-infected EK cells. These results indicate that ADCC is not involved in the control of EIAV in carrier horses.