Hemagglutination inhibition (HI) is a serological assay used to detect antibodies against specific viruses in horses. This method is based on the principle that certain viruses can cause red blood cells to agglutinate, or clump together, and the presence of specific antibodies can inhibit this process. In equine research, the HI test is often utilized to assess the immune response to viral infections such as equine influenza. The assay provides information on the antibody levels in a horse's serum, which can be indicative of exposure to the virus or the effectiveness of a vaccination. This page compiles peer-reviewed research studies and scholarly articles that explore the methodology, application, and implications of hemagglutination inhibition in the context of equine viral diseases.
Sentsui H, Kono Y.Six strains of equine infectious anemia (EIA) virus propagated in equine leukocyte cultures were found to agglutinate horse erythrocytes. Concentrated virus material containing about 20 units of complement fixation (CF) titer showed hemagglutinating (HA) titers ranging from 4 to 8 units. The HA activity remained stable after ether treatment and was reduced by trypsin, formaldehyde and KIO4. Cesium chloride equilibrium density gradient centrifugation revealed two populations of hemagglutinin, one in the density range of 1.15-1.16 g/ml coinciding with a peak of CF antigen and the other at round ...
McFarlane BL, Embree JE, Embil JA, Rozee KR, Weste JB, Artsob H, Spence L.A large number of North American equine samples were tested for the California group of arboviruses (CAL). Of 861 equine sera tested by hemagglutination inhibition using the snowshoe hare virus as an antigen, 106 (12.3%) were positive. Neutralization tests confirmed antibodies to this virus in 72 of the positive sera. This study provides evidence of CAL activity in the domestic animal population of Nova Scotia.
Ferguson JA, Reeves WC, Milby MM, Hardy JL.Of 359 horses vaccinated with attenuated Venezuelan equine encephalomyelitis (VEE) vaccine (strain TC-83), 87% developed hemagglutination-inhibition (HI) antibodies to VEE virus within 1 month. Blood from a subsample of 101 of the 359 horses was obtained over a 1-year period. Within 1 month after vaccination, 84% of the 101 horses had developed VEE HI antibodies, 87% had developed VEE-neutralizing (Nt) antibodies, and 78% had developed VEE complement-fixing (CF) antibodies. One year after vaccination, 58% of the horses had VEE HI antibodies and 73% had VEE Nt antibodies. The percentage of hors...
Goto H, Shimizu K.The first outbreak of equine influenza (EI) infection in Japan was recognized during the period December 1971 to January 1972 [1, 6]. No evidence of the disease had been found before then [2,6]. The etiological agent of this epizootic was identified by hemagglutination-inhibition (HI) and neutralization tests with chicken or ferret antiserum as the subtype 2 of EI virus (6, 7). However, the isolate, A/equine/Tokyo/71 (Tokyo) strain, was not completely identical to the prototypic A/equine/Miami /63 (Miami) strain of the subtype 2, since antibody responses of convalescent horses were 2 to 16 tim...
Kucera CJ, Beckenhauer WH.An inactivated, aluminum hydroxide adjuvant equine influenza vaccine was tested in horses and guinea pigs to determine the levels of antigen that would elicit maximum serological responses. Vaccine containing serial twofold increments of A/Equi-1/Prague and A/Equi-2/Miami strains of equine influenza virus was administered to random groupings of both types of test animals. The hemagglutination inhibition antibody response for each group was then measured. Results in horses and guinea pigs were compared to determine if the equine serological values could be related to a potency test in laborator...
Sentsui H, Kono Y.Equine infectious anemia (EIA) virus which was propagated on an equine dermal cell line agglutinated guinea pig erythrocytes. Viral fluids containing about 10(7.5) mean tissue culture infective doses/ml showed hemagglutinating (HA) titers ranging from 16 to 32 units/0.05 ml. Results of cesium chloride equilibrium density gradient centrifugation revealed that the hemagglutinin was inseparable from the virus particles. The hemagglutination reaction persisted over a wide range of temperature and pH, and the absence of divalent cations did not decrease its activity. The HA activity was stable at 4...
Poskus E, Zakin MM, Fernámdez HN, Paladini AC.Peptide fragments, obtained from equine growth hormone by cyanogen bromide cleavage and further chemical treatment, were isolated and identified. Their immunological reactivities were tested by hemagglutination and complement fixation methods using rabbit antisera against native hormone. Antigenic determinants were detected in the fragments comprising amino acid sequences 5-72 and 73-123, this last one being predominant. Fragment 124-178 had very low reactivity. Nitration of peptide 73-123 did not modify its immunological properties,but oxidation diminished them. Comparison of the antigenicity...
Stellmann C, Petermann HG.After vaccination of chickens, rabbits, foals and horses, HI and SN antibody dose response curves were compared for A/Equi 1/Prague and A/Equi 2/Paris strains.
The two curves are parallel for a given strain and the relationship of HI and SN titres is constant, whatever the animal species.
The distribution of HI and SN titres varies for the two strains.
This variation, which is independent of animal species, may be related to the number of sites necessary for the antigenic-antibody response in vitro.
It is suggested that the testing of equine influenza vaccine be carried out in the ...
Hierholzer JC, Gamble WC, Quist KD, Chappell WA.Horses were immunized by a variety of inoculation procedures designed to determine the most efficient method of producing antisera to adenovirus types 25 to 31. The procedures evaluated included immunization by (i) direct intravenous (iv) injection, (ii) iv infusion, (iii) intramuscular (im) injection of virus with and without Freund's incomplete adjuvant, (iv) combined iv and im injections, and (v) combined iv infusion and im injection. The im schedule (no. 3) was superior to the others in terms of immunizing antigen and time required, and hemagglutination-inhibition (HI) and serum-neutralizi...
Anderson RS, Day NK, Good RA.Natural hemagglutinin activity against vertebrate erythrocytes is present in the hemolymph of the cockroach Blabarus craniifer. The hemagglutinin titer against rabbit erythrocytes is high, whereas sheep and horse red cells agglutinate weakly. Hemagglutinin activity was depressed by the complement inhibitor, cobra venom factor. Cockroach hemagglutinin is heat-labile; all activity is destroyed by heating at 56 C for 1 hr. A humoral factor similar to the complement component 3 proactivator is also present in cockroach hemolymph. The formation of the cobra venom factor-hemolymph "complex" is depen...
Tumova B, Easterday BC.This study demonstrates relationships in envelope antigens of 4 human influenza A2 strains isolated during the period 1957-68 (including A2/Hong Kong/68), 2 strains of A/Equi-2/63 and 7 avian influenza viruses isolated in Europe, North America, and the Ukraine in the years 1960-67.Antigenic relationships among the strains were determined on the basis of haemagglutination-inhibition, virus-neutralization, strain-specific complement-fixation, and neuraminidase-inhibition tests.North American avian influenza strains, Turkey/California/64, Turkey/Massachusetts/65, Turkey/Wisconsin/66, Turkey/Ontar...
Favaro PF, Reischak D, Brandao PE, Villalobos EMC, Cunha EMS, Lara MCC, Benvenga GU, Dias RA, Mori E, Richtzenhain LJ.The equine influenza virus (EIV) H3N8 subtype is responsible for all EIV outbreaks worldwide while the H7N7 subtype is less pathogenic and is considered extinct as it has not been confirmed in outbreaks since 1980. Although EIV is enzootic in Brazil, few reports describe the actual EIV antibody status in the country. The aims of this study were: - to evaluate the efficiency of different serum treatments described by the World Organisation for Animal Health (OIE) and the World Health Organization (WHO) to remove non-specific haemagglutination inhibitors for the haemagglutination inhibition (HI)...
Thorsen J, Willoughby RA, McDonell W, Valli VE, Viel L, Bignell W.Samples of mucus from the lower trachea were collected from 53 horses with chronic obstructive pulmonary disease and from 24 clinically normal horses. Serum samples were collected from 35 of the horses with chronic obstructive pulmonary disease and from the 24 normal horses. Samples were tested for inhibition of hemagglutination by influenza A equine 1 and 2 viruses. There were high levels of hemagglutination inhibiting activity against influenza A equine 1 in mucus samples from horses with chronic obstructive pulmonary disease.
Bogdan JR, Morley PS, Townsend HG, Haines DM.This study has tested the effect of using homologous or heterologous equine influenza A virus isolates to evaluate serum antibody levels to influenza A virus in vaccinated and naturally-infected horses. In addition, the potential effect of antigenic selection of virus variants in egg versus tissue culture propagation systems was studied. Serum antibody levels in samples from horses recently infected with a local influenza A virus isolate (A/equine 2/Saskatoon/1/90) or recently vaccinated with a prototype isolate (A/equine 2/Miami/1/63) were assessed by hemagglutination inhibition and by single...
McFarlane BL, Embree JE, Embil JA, Rozee KR, Weste JB, Artsob H, Spence L.A large number of North American equine samples were tested for the California group of arboviruses (CAL). Of 861 equine sera tested by hemagglutination inhibition using the snowshoe hare virus as an antigen, 106 (12.3%) were positive. Neutralization tests confirmed antibodies to this virus in 72 of the positive sera. This study provides evidence of CAL activity in the domestic animal population of Nova Scotia.
Brundage-Anguish LJ, Holmes DF, Hosier NT, Murphy BR, Massicott JG, Appleyard G, Coggins L.Temperature-sensitive (ts) reassortants of an equine influenza virus, subtype A-1, were produced by mating a human influenza ts donor virus with an equine influenza A/Cornell/16/74 wild-type virus and by isolating a ts reassortant virus possessing the equine hemagglutinin and neuraminidase surface antigens. Two equine its reassortant clones, 8B1 and 71A1, were produced which had an in vitro shutoff temperature for plaque formation of 38 and 37 C, respectively. The human ts donor virus had ts mutation(s) on the polymerase 3 (P3) and nucleoprotein genes so that a ts equine reassortant virus coul...
Stellmann C, Petermann HG.After vaccination of chickens, rabbits, foals and horses, HI and SN antibody dose response curves were compared for A/Equi 1/Prague and A/Equi 2/Paris strains.
The two curves are parallel for a given strain and the relationship of HI and SN titres is constant, whatever the animal species.
The distribution of HI and SN titres varies for the two strains.
This variation, which is independent of animal species, may be related to the number of sites necessary for the antigenic-antibody response in vitro.
It is suggested that the testing of equine influenza vaccine be carried out in the ...
Sguazza GH, Fuentealba NA, Tizzano MA, Galosi CM, Pecoraro MR.Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for prod...
Dilai M, Piro M, Fougerolle S, El Harrak M, Mahir W, El Mourid R, Legrand L, Paillot R, Fassi Fihri O.In order to evaluate the vaccination status against equine influenza (EI) in Moroccan racehorses, a serological investigation was carried out on 509 racehorses using three serological tests: an Enzyme-Linked Immunosorbent Assay (ELISA), the Hemagglutination Inhibition (HI) test and the Single Radial Haemolysis (SRH) assay. The serological analysis showed 56% of seropositivity by ELISA, 67% by HI and 89.4% by SRH (with 69.9% above the clinical protection threshold). Using the Kappa test, the SRH and HI assays showed a strong agreement, the SRH and ELISA assays had a moderate agreement and the H...
Casseb Ado R, Nunes MR, Rodrigues SG, Travassos da Rosa ES, Casseb LM, Casseb SM, da Silva SP, Rodrigues ED, Vasconcelos PF.The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family. Results: Sensitivity...
Ryder E, Ryder S.Venezuelan equine encephalitis (VEE) virus has been implicated as producing alterations in glucose metabolism in animals. We performed oral glucose tolerance tests and measured serum immunoreactive insulin responses in 13 patients who were infected by VEE virus during an epidemic in 1969, in Zulia State, Venezuela. No significant alterations in the glucose tolerance test were found. Sera of 86 diabetic outpatients and 98 control individuals with normal glycemia at a local hospital were tested for antibodies to VEE virus by hemagglutination inhibition. No statistically significant difference wa...
Arthropod-borne viruses (arboviruses) cause morbidity and mortality in humans and domestic animals worldwide. The percentage of population immunity or susceptibility to these viruses in Ecuador is unknown. To investigate the proportion of Ecuadorian populations with IgG antibodies (Abs) (past exposure/immunity) and IgM Abs (current exposure) against flaviviruses and alphaviruses and to study the activity of these viruses in Ecuador. During 2009-2011, we conducted a serosurvey for selected arboviruses in humans (n=1,842), equines (n=149), and sentinel hamsters (n=84) at two coastal locations an...
Boussetta M, Chabchoub A, Ghram A, Jomaa I, Ghorbel A, Aouina T, Ben Amor H.Using the haemagglutination inhibition and immunodiffusion tests, a national serological survey was carried out to detect antibodies to equine influenza (EI) (A/equi/1/Prague 56 and A/equi/2/Miami 63) and equine infectious anaemia (EIA) in horse sera collected in northeastern Tunisia. 533 samples were analysed for EIA antibodies. All were negative. 13.6% of 433 equine sera tested for EI antibodies were positive. These results are discussed and compared with others obtained in Tunisia and bordering countries.
Lim SI, Kim MJ, Kim MJ, Lee SK, Yang HS, Kwon M, Lim EH, Ouh IO, Kim EJ, Hyun BH, Lee YH.Equine influenza virus (EIV) causes acute respiratory disease in horses and belongs to the influenza A virus family , genus . This virus may have severe financial implications for the horse industry owing to its highly contagious nature and rapid transmission. In the Republic of Korea, vaccination against EIV has been practiced with the active involvement of the Korea Racing Authority since 1974. In this study, we monitored the viral RNA for EIV using PCR, as well as the antibody levels against 'A/equine/South Africa/4/03 (H3N8, clade 1)', from 2020 to 2022. EIV was not detected using RT-PCR. ...
Kreutzfeldt N, Chambers TM, Reedy S, Spann KM, Pusterla N.Dexamethasone is routinely administered to horses but its effect on the antibody response to a commercial EIV/EHV vaccine is unclear. Objective: Horses receiving dexamethasone will have lower postvaccination antibody levels against EIV and EHV-1 than vaccinated controls. Methods: Fifty-five healthy adult research horses. Methods: Randomized cohort study. Control (no vaccine, group 1), vaccination only (EIV/EHV-1/EHV-4, Prestige 2, Merck Animal Health, group 2), vaccination and concurrent single intravenous dose of dexamethasone (approximately .05 mg/kg, group 3), vaccination and 3 intravenou...
