Identification of a novel herpesvirus from a California desert tortoise (Gopherus agassizii)

Herpesviruses are significant pathogens of tortoises, causing upper respiratory tract disease and necrotizing stomatitis, with infections often associated with high mortality rates. Herpesvirus infection in a captive California desert tortoise (Gopherus agassizii) was detected by light microscopic observation of intranuclear inclusion bodies in various tissues followed by transmission electron microscopic observation of herpesvirus-like particles, and amplification of herpesvirus nucleic acid sequences using polymerase chain reaction. Using an indirect enzyme linked immunosorbent assay, anti-tortoise herpesvirus antibodies were detected one month after initial onset of clinical signs. This novel herpesvirus is distinct from the previously described tortoise herpesvirus (tortoise herpesvirus-1, THV-1) sharing 83% sequence identity of 60 amino acids of a portion of the DNA polymerase gene and 79% sequence identity across 120 amino acids of a portion of the ribonucleotide reductase gene. Similar to THV-1, this novel herpesvirus, tortoise herpesvirus-2 (THV-2), also clusters with the alphaherpesviruses.     

Seroprevalence of Toxoplasma gondii antibodies in clinically ill cats in the United States

OBJECTIVE: To determine regional seroprevalence estimates of Toxoplasma gondi-specific IgM and IgG in clinically ill cats throughout the United States. Sample Population-Sera from 12,628 clinically ill, client-owned cats. PROCEDURE: Toxoplasma gondii-specific IgM and IgG antibodies were detected by use of ELISAs. Sera from clinically ill cats previously submitted for T. gondii antibody testing were sequentially selected from our serum bank and the sample submission paperwork reviewed. The country was divided into 12 geographic regions. Overall prevalence as well as prevalence for each region, age group, season, sex (male vs female), and breed (domestic shorthair vs other) was calculated. Data were analyzed by logistic regression analysis. RESULTS: Overall, 31.6% of the cats were seropositive for T. gondii-specific IgM, IgG, or both. Percentage of cats seropositive for T. gondii antibodies ranged from 16.1% (southwestern United States) to 43.5% (northeastern United States). As age increased, odds of positive T. gondii antibody assay results (IgM alone, IgG alone, and any combination of IgM or IgG) increased. Males were more likely than females to be seropositive for T. gondii antibodies (IgG alone and any combination of IgM or IgG). Domestic shorthair cats were more likely than other breeds to be seropositive for T. gondii antibodies (IgM alone, IgG alone, and any combination of IgM or IgG). CONCLUSIONS AND CLINICAL RELEVANCE: Toxoplasma gondii-specific antibodies are common in serum samples of clinically ill cats from all regions of the United States. Seroprevalence increases as cats age and is higher in male and domestic shorthair cats, compared with females and other breeds.     

Monoclonal antibodies against porcine immunoglobulin isotypes

Monoclonal antibodies (MCAs) against porcine immunoglobulin isotypes* G, G1, G2, M and A have been produced and characterized in detail. Epitope analysis using a competitive direct enzyme-linked immunosorbent assay (ELISA) indicated that the MCAs recognized 3 class-specific epitopes of IgG, 4 epitopes specific for IgG1, 3 epitopes specific for IgG2, 2 epitopes of IgM and 2 epitopes of IgA. Two MCAs against IgG2 were shown to react with an allotypic determinant (B2) and one MCA against IgM is probably allotype specific. The production of MCAs specific for IgG and for its subclasses G1 and G2 and, in addition, the one-step isolation of nearly pure IgG1 and IgG2 preparations by immunoaffinity chromatography using MCA 34.1.1a (anti-IgG2) confirmed the existence of at least two subclasses of IgG. Preliminary results further suggested the existence of a subpopulation of IgG1 which could be eluted selectively from Protein A-Sepharose columns at pH 5.0. MCA 34.17.2a appeared to react preferentially with this IgG1 subpopulation and could be used to isolate a similar IgG1 subpopulation by immuno-affinity chromatography. Several of the MCAs have been successfully applied for the detection of porcine immunoglobulin isotypes by a double antibody sandwich ELISA and for the (isotype-specific) detection of antibodies against various porcine viruses. The availability of a full set of MCAs against porcine immunoglobulin isotypes will stimulate and facilitate the further study of the porcine immune system.     

Reliable ELISAs showing differences between resistant and susceptible lines in hens orally inoculated with Salmonella Enteritidis

Reliable ELISAs were investigated with the aim to select hen lines resistant to Salmonella Enteritidis and producing high levels of antibodies. In the first experiment, the relation between the humoral response and the bacteriological results was assessed on hens from the Y11 resistant line and the L2 susceptible line, orally inoculated with 10(8) CFU S. Enteritidis per animal. Anti-lipopolysaccharide (LPS) IgG titres were higher but the liver and spleen were less contaminated in hens from the Y11 line than in hens from the L2 line (p = 0.013, 0.031 and 0.026 respectively). In the second experiment, the hens were inoculated orally with 1.7 x 10(8) CFU S. Enteritidis per animal in order to select the ELISA methods showing the more significant differences. ELISAs were based on LPS, flagella, LPS from rough (LPS-R) and smooth strains (LPS-S) and detected IgG and IgM antibodies from sera and yolks. No between-line host response variation was observed in the yolk, with LPS-S and R antigens nor with anti-LPS IgM in the sera. Otherwise, significant differences were encountered between hen lines with the ELISAs performed on the sera detecting anti-LPS IgG, anti-flagella IgG or IgM (p = 0.017, 0.017 and p < 0.001 respectively). When comparing the kinetics of the selected ELISAs, the IgG antibodies against LPS detected between-line variations as early as 1 to 4 weeks pi, whereas with IgG against flagella, the differences were only detected at 1 and 2 weeks pi and with IgM against flagella, the differences were significant at 1, 2, 4 and 8 weeks pi. In conclusion, resistant hen lines producing higher levels of antibodies than the susceptible hen lines may be selected with these ELISAs.     

Immune responses in chickens against Salmonella typhimurium monitored with egg antibodies

Three mature hens were immunized with an Aro- mutant of Salmonella typhimurium beginning with a subcutaneous dose in adjuvant followed by two oral boosters. Isotype-specific antibodies were measured in the white and yolk eggs collected weekly over a period of 230 days. Two hens showed a memory response to the first oral booster, with large increases in egg yolk IgG and smaller increases in IgA and IgM antibodies in egg whites. Smaller amounts of IgA and IgM antibodies were found in egg yolks, and a slight increase in IgG occurred in the whites. One hen showed an increase in serum titers of all isotypes against S. typhimurium. The second hen had high serum titers before immunization was started which did not change. The third hen had a high level of IgM in the white of eggs before immunization was started. This hen showed erratic responses in egg white antibodies following immunization, no increase in IgA or IgM in yolks and only a slight increase in IgG, no increase in serum IgG, and was the only hen with a high level of IgM antibody against S. typhimurium in the bile, conditions reflecting a state of oral tolerance. With the exception of this hen, the results showed that IgA and IgM antibodies were aroused in hens by immunization with an avirulent mutant of S. typhimurium, and that these antibodies were present in the white of eggs from immunized hens.     

Generation and characterisation of murine monoclonal antibodies specific for cervine immunoglobulin light chain, IgM and IgG.

Monoclonal antibodies (mAb) which react with cervine immunoglobulin (Ig) light chain, IgM and IgG were produced using conventional cell fusion technology. Hybridoma supernatants were initially screened for specificity against cervine Ig using an enzyme-linked immunosorbent assay (ELISA). The specificity of supernatants against size-fractionated cervine Ig was further determined. Supernatants were characterised using western blotting and autoradiographic techniques. The mAb OU1G, OU2G and OU3G were specific for cervine gamma-chain of IgG, whereas OU1L was specific for light chain of Ig. A further mAb (OU1M) bound IgM and not IgG. These mAb were found to have varying cross-reactivity against Ig from other species.     

The effect of adjuvants on active and passive immunity in pregnant deer and their offspring.

Experiments were carried out to determine the effects of a range of adjuvants on immunoglobulin M (IgM) and immunoglobulin G (IgG) serum concentrations to a protein antigen administered subcutaneously to farmed female deer following mating. The antibody responses of animals immunised with keyhole limpet hemocyanin (KLH) in Freund's Incomplete Adjuvant (FIA), diethylaminoethyl dextran (DEAE-dextran) and aluminium hydroxide (alum) were compared with the response to antigen administered in the absence of adjuvants. Animals were subsequently challenged with a subcutaneous immunisation of the antigen in saline. Following parturition, the concentration of passively transferred antigen-specific antibody was measured in the serum of the offspring. The polyionic adjuvant, DEAE-dextran, produced the greatest enhancement of both primary and secondary IgG responses to KLH. Offspring suckling from mothers immunised with antigen in DEAE-dextran consequently had higher concentrations of specific antibodies in their serum than other fawns in the experiment. The adjuvants FIA and alum were approximately 20-fold less effective in enhancing antigen-specific IgG than DEAE-dextran but induced greater amounts of antigen-specific IgM. From the results presented in this paper, there is evidence that immunisation of deer during pregnancy may be an effective way of reducing morbidity in both mothers and offspring.     

Evanescent-wave biosensor for field serodiagnosis of tortoise mycoplasmosis

Disease has become an increasingly important issue for wildlife management over the past two decades. Adequate surveillance is fundamental for disease prevention and control, thus there is an increasing need for diagnostic assays for wildlife management. The objective of this study was to evaluate the performance of a field-portable biosensor adapted for rapid detection of specific antibodies in tortoise plasma that reflect a history of exposure to Mycoplasma agassizii, which is an agent of tortoise upper respiratory tract disease. Banked plasma samples were tested in two blinded trials, and the parameters that define the reliability of a diagnostic test were estimated based on externally validated tortoise plasma controls. The mean sensitivity of the biosensor (ability to identify exposed tortoises in the group of all exposed individuals) was 78%; the mean specificity (unexposed individuals with negative test result, out of all unexposed individuals tested) was 73%; the mean positive predictive value (exposed individuals with positive test, out of all individuals with positive test) was 82%; the mean negative predictive value (unexposed individuals with negative test, out of all individuals with negative test) was 68%. In a 15-min field-portable format, the biosensor was able to discriminate between true seropositive (n=34) and true seronegative (n=23) tortoise plasma with overall accuracy of 84%. The goals established for the tortoise population can help managers decide whether potential diagnostic errors should impact management decision-making, and whether the benefits of the field-portable format of the biosensor assay outweigh any potential disadvantages.     

Stimulation and analysis of the immune response in calves from vaccinated pregnant cows

The effect of vaccinating pregnant cows with an inactivated vaccine against Mannheimia haemolytica, BRSV and PI3V infections on selected immune responses in their offspring was examined. Blood samples were collected weekly for 12 weeks from six newborn calves from each of vaccinated (experimental) and unvaccinated (control) dams. Specific antibodies to M. haemolytica, BRSV and PI3V and mean values of IgA, IgG concentrations were significantly higher in the experimental calves compared with the controls. However, specific antibody titres to adenovirus type 3, BHV1 and BVDV in the experimental calves had constant levels while the control group levels changed. The IgM, Hp and SAA concentrations generally increased until week 8 in the experimental group, but the control group titres became higher after week 9. This study demonstrates that specific immunisation of cows pre-partum significantly stimulated parameters associated with immunity and it also controlled the acute phase response intensity in their offspring. Therefore the vaccination of dams may provide additional antibody protection against infection to their offspring.     

Immunoglobulins in cattle vaccinated with leptospiral bacterins.

The growth-inhibition test was used to detect specific antibodies against Leptospira interrogans serotype hardjo in isolated immunoglobulin (IgG and IgM) fractions of serums from cattle vaccinated with leptospiral bacterins. The growth-inhibiting antibodies were detected mainly in the IgG class. Agglutinated clumps also occurred with the IgM fraction. The serums collected from cattle 4 months after vaccination were negative in the microscopic agglutination test.     

Specific antibodies to Besnoitia besnoiti precipitated from serum of cattle by live parasites and by soluble antigen

Specific precipitable antibodies of both IgG and IgM classes were detected in sera of cattle naturally infected with B. besnoiti. The amount of specific antibodies of the IgG class precipitated by soluble antigen was in the range of 17-50 micrograms/ml serum while that of the IgM class ranged between 5 and 24 micrograms/ml serum. Specific antibodies precipitated by live B. besnoiti parasites were in amounts of 10 to 22 micrograms/ml serum for IgG and 4 to 26 micrograms/ml serum for IgM. Different ratios of IgG/IgM were obtained by the two methods of precipitation. This might indicate that antibodies to B. besnoiti of the IgM class can be precipitated and detected in sera of naturally infected cattle in similar amounts either by live parasites or by solubilized antigen, whereas antibodies of the IgG class can be preferentially detected when solubilized antigen is used for precipitation.     

Enzyme-linked immunosorbent assay for detection of antibody to Pseudomonas aeruginosa and measurement of antibody titer in horse serum

Enzyme-linked immunosorbent assay (ELISA) was used for detection of immunoglobulin (Ig) M and IgG antibodies against a serologically common antigen (original endotoxin protein), protease, and elastase of Pseudomonas aeruginosa. The P aeruginosa antibody in horse sera was measured, using ELISA. Horseradish peroxidase-labeled rabbit anti-horse IgM and IgG antibodies were used for enzyme-labeled antibody conjugate. 5-Aminosalicylic acid and H2O2 were used for substrate. Sera collected from a vaccinated horse, a newborn foal, and 72 healthy racehorses were investigated for antibodies against P aeruginosa by ELISA and passive hemagglutination procedure. Changes in IgM and IgG antibody titers with vaccination were clear by ELISA. In the newborn foal, significant amounts of IgM and IgG antibodies from colostrum were present on the 1st day after birth. It was shown by ELISA that the level of antibodies in the newborn decreased initially and then increased. Some antibodies against original endotoxin protein, protease, and elastase of P aeruginosa were detected in almost all the healthy racehorses investigated.     

Experimental induction of arthritis in LEW rats and antibody response to four Mycoplasma arthritidis strains

Four Mycoplasma arthritidis strains were examined for differences in virulence for LEW rats and elicitation of antibody responses in the immunoglobulin (Ig) M and G classes and in the four IgG subclasses. Two strains were highly arthritogenic and two were relatively avirulent. When the latter strains did induce arthritis, it was significantly less severe (P less than 0.05) and developed significantly later (P less than 0.001) than in rats injected with the two virulent strains, suggesting that the low-virulence organisms are able to persist asymptomatically in rats for several weeks. None of the M. arthritidis-injected rats developed metabolism-inhibiting (MI) antibodies at any time during the 6-week observation period. Responses to other M. arthritidis antigens from all four strains were measured by enzyme immunoassay (ELISA); they were similar qualitatively but differed quantitatively. Rats injected with the two avirulent strains showed significantly lower titers of IgM antibodies (P less than 0.01) throughout the 6-week observation period and significantly lower early titers of IgG antibodies (P less than 0.05) than rats injected with the two virulent strains. In addition, peak IgM antibody titers, IgM titers measured 1 and 6 weeks after injection and IgG antibody titers measured 1 week after injection all correlated significantly with peak arthritis scores (P less than 0.05). The IgG antibody response against all four strains appeared mostly in the IgG2a and IgG2b fractions, with very little in the IgG1 and IgG2c fractions. Using immunoblotting, the immunodominant antigens of the two virulent strains appeared very similar, but the avirulent strains differed slightly from each other and from the other two. This study indicates that immune responses of rats to virulent and avirulent strains are similar but not identical and that immunogenicity for LEW rats may be a strain-specific characteristic for M. arthritidis.     

Effect of dietary manipulation and vaccination of turkey breeder hens on immunoglobulin levels of yolk,yolk sac and neonate poults

Two hundred turkey breeder hens and 24 viable toms of 30–35 weeks age of small white variety were distributed into two treatment groups having four replicates of 25 hens and three toms in each treatment. First four replicates were offered a turkey breeder diet (Diet A) (Nutrient requirements of poultry, 1994, National Academic Press, Washington, DC) and the rest four replicates were maintained on a higher plane of nutrition (Diet B) for 8‐week duration. After 6 weeks of experimental feeding, two replicates from each treatment groups were vaccinated with ND (R₂B) vaccine. Yolk sac of embryo from birds fed Diet B had a significantly higher (p < .05) IgG, IgM level and HI titre (log 2) than those fed Diet A. HI titre values of embryonic yolk sac from the vaccinated birds fed Diet B were significantly higher (p < .05) than that of the control groups. In addition, HI titre values were significantly higher (p < .05) in the day‐old poults of the birds fed Diet B than that of those fed Diet A. There was significantly (p < .01) positive correlation between serum IgG and IgM of the breeder birds and day‐old chicks. Similarly, there was significantly (p < .05) positive correlation between yolk IgG and IgM after 1‐month experimental feeding and yolk sac IgG and IgM. Positive correlation (p < .05) also existed between yolk sac IgM and day‐old chick serum IgM. Furthermore, the HI titres of breeder birds' serum at 14 days post‐vaccination were positively correlated with their egg yolk after 10 and 15 days post‐vaccination, yolk sac and day‐old chicks. Thus, the study envisaged that a higher immunity in neonate poults from turkey breeders maintained on a higher plane of nutrition may be elicited as there was maternal transfer of antibodies from the serum of breeder birds to their offsprings through their yolk sac.     

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.     

Kinetics of IgM and IgG responses to experimental and naturally acquired Rickettsia rickettsii infection in dogs

The kinetics of specific IgM and IgG antibody response was characterized in four 9-month-old Beagles after inoculation of 2 x 10(2) plaque-forming units (PFU) of Sheila Smith strain of Rickettsia rickettsii. Immunoglobulin M antibodies were first detected by indirect immunofluorescence on postinoculation (PI) day 9, peaked by PI day 20, and were no longer detectable by PI day 80. Immunoglobulin G antibodies became detectable between PI days 22 and 28, peaked by PI day 42, and decreased gradually through PI day 130. Subsequent challenges with R rickettsii on PI days 216 (2 x 10(2) PFU/dog) and 1,029 (5 x 10(4) tissue culture infective dose [TCID50]/dog) resulted in slightly different serologic responses. The initial challenge exposure failed to increase the concentration of IgG antibodies and induced only low concentrations of IgM antibodies. After the second challenge inoculation, IgM antibodies were not detectable and the concentration IgG antibodies increased slightly. Clinical abnormalities and seroconversion were documented in control dogs following each challenge exposure. Examination of acute and convalescent serum samples from 55 dogs in which Rocky Mountain spotted fever was suspected clinically suggested that sole evaluation of IgM antibodies in acute-phase serum would result in inaccurate diagnoses because of false-positive and -negative results. Use of a composite conjugate that detects IgM and IgG antibodies to R rickettsii appears to be satisfactory for diagnostic purposes; however, concurrent quantitation of IgM antibodies may facilitate serodiagnosis in a select group of dogs in which a four-fold increase in convalescent antibody titer is not detected by use of the composite conjugate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental intramammary inoculation with Mycoplasma bovis in vaccinated and unvaccinated cows: effect on local and systemic antibody response.

Four cows were vaccinated with Mycoplasma bovis five times at two week intervals: three times subcutaneously in Freund's complete adjuvant, and two times with M. bovis alone in two of four quarters by intramammary infusion. The effect of vaccination on the immune response was evaluated in the serum and whey of the four vaccinated and control (placebo) cows experimentally challenged in two of four quarters with live M. bovis. Vaccination resulted in markedly increased M. bovis-specific, serum IgM, IgG and IgG2, but not IgA, reactivity. Challenge exposure with live M. bovis by intramammary infusion resulted in high specific serum IgM, IgG1 and IgG2 reactivity and a noticeable IgA response in both vaccinated and control cows. Whey from quarters on vaccinated cows had elevated, specific IgG1 reactivity at the time of challenge but no other differences were observed. Challenge exposure with live M. Bovis resulted in high antibody levels of all isotypes in quarters which were challenged, but highly elevated reactivities in unchallenged quarters occurred only with IgG1 and IgG2. These results indicate that vaccination elevated M. bovis-specific IgG1 but not other immunoglobulin reactivity in quarters on vaccinated cows, and that live organisms are necessary to elicit a local, specific IgA response.     

Comparison of three monoclonal and three polyclonal antibodies in the immunohistochemical diagnosis of canine autoimmune skin diseases

The aim of this study was to evaluate the utility of three monoclonal antibodies (mAbs), and two anticanine IgG and one anticanine IgM polyclonal antibodies (pAbs) for the immunohistochemical diagnosis of canine autoimmune skin diseases. Skin biopsies from 11 cases of pemphigus (7 foliaceus, 3 vulgaris and 1 erythematosus), 12 cases of discoid lupus erythematosus (DLE) and 12 cases of chronic hyperplasic dermatitis were used. The CA4E7 mAb (IgG1 + IgG2) showed similar sensitivity, but higher specificity and lower background than the two anti-IgG pAbs for the immunohistochemical diagnosis of pemphigus and DLE. The CA4F1 mAb (IgG2) and CA3H1 mAb (IgG2) showed moderate and low interepithelial reactivity, respectively, in autoimmune skin diseases, but strong staining of the cytoplasm of plasma cells of the inflammatory infiltrates. These results suggest that the CA4E7 mAb may be valuable in the immunohistochemical diagnosis of such disorders.     

Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice

Traditionally, adjuvants have been administered with antigens to enhance immunity. We studied the effect of several adjuvants such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS), homopolymers of polyinosinic-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U), lithium chloride (LiCl), saponin Quil A and calcium phosphate gel (CaHPO(4)) on the immune response of mice to formalin-inactivated Mycoplasma agalactiae. The specific antibody or cytokine producing splenocytes were detected by ELISAspot and immunocytochemistry, respectively. Depending on the adjuvant given, the number of M. agalactiae-specific antibody producing cells was increased 2.5-6-fold. IgG was the major class of M. agalactiae-specific antibodies followed by IgM, IgA and IgE. Among IgG isotypes, FCA, FIA, Quil A and CaHPO(4) induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes while poly I:C shifted the response toward an IgG2a/IgG3 production. Finally, poly A:U induced an IgG2b response while LPS and LiCl augmented the IgG3/IgG1/IgG2a secretion. FCA augmented IL-4, IL-5 and IL-10 production suggesting a strong Th2 response, while IFN-gamma and IL-12 remained low; poly I:C enhanced IFN-gamma, IL-12 and TNF-alpha eliciting a Th1 response; poly A:U resulted in a IL-10, IL-5, IL-6 and IL-12 secretion; and LPS enhanced the IL-10, IL-6 and TNF-alpha production. Our data show that adjuvants augment M. agalactiae-specific antibody production and lead to B cell isotype-switching via the appropriate cytokine milieu. Certain adjuvants, such as poly I:C, therefore, appear as promising immune enhancers for vaccination against M. agalactiae infections.     

Serological responses of specific pathogen-free foals to equine herpesvirus-1: primary and secondary infection, and reactivation.

Serum antibody (virus neutralisation, complement fixation, IgM and IgG) responses to equine herpesvirus-1 (EHV-1) infection were measured in six foals which were initially free from EHV-1 and EHV-4 infection and maternally-derived antibodies. Following primary infection, high titres of virus neutralisation and complement fixation antibodies were detectable against EHV-1, however, corresponding antibody levels against EHV-4 were low or inapparent, although the two viruses share a number of cross-reactive epitopes. In addition, following the primary infection with EHV-1, IgM levels increased before those of IgG, virus neutralisation and complement fixation antibodies, peaked sooner and thereafter declined. Stimulation of IgM levels was observed on secondary infection with EHV-1 given 61 days later. In contrast, IgG, virus neutralisation and complement fixation antibodies following primary infection were more sustained and no increase in their levels was observed on secondary infection. No consistent changes in IgM or IgG levels were seen after administration of dexamethasone to reactivate latent virus.