Antigen-specific IFN-gamma and IL-4 production in caprine herpesvirus infected goats

The analysis of cytokines secreted by antigen-specific lymphocytes is hampered in goats by the paucity of species-specific reagents yet it is crucial to study immune responses to infections. To overcome this limit, two commercial kits designed to measure soluble bovine IL-4 (by ELISA) and frequencies of bovine IFN-gamma secreting cells (by ELISPOT) were tested for cross-reactivity in goats. In addition, an ELISA specific to bovine/ovine IL-4 and employing two monoclonal antibodies, clones CC313 and CC314, was tested as well. Concanavalin A-stimulated caprine peripheral blood mononuclear cells (PBMCs) cultures were studied and they exhibited high levels of soluble IL-4 and high frequencies of IFN-gamma secreting cells. In addition, the two IL-4 ELISAs detected similar amounts of cytokine. To start defining the cytokine response triggered by caprine herpesvirus 1 (CpHV-1) infection, PBMC cultures were setup from goats naturally or experimentally infected with CpHV-1. High frequencies of IFN-gamma producing cells and low, when detectable, levels of soluble IL-4 were observed in CpHV-1-specific PBMC cultures from both groups of infected goats. Thus, the availability of cross-reactive research tools can expand cytokine studies in goats and can implement the research on immunity to other caprine infections.     

Dominance of Chlamydia psittaci-specific IgG2 subclass in the humoral immune responses of naturally and experimentally infected cattle

Indirect enzyme-linked immunosorbent assays were applied to differentiate Chlamydia (C.) psittaci-specific IgG1 and IgG2 levels in 143 individual serum samples from cattle with naturally occurring chlamydial infections and in 190 sequential serum samples from 26 experimentally infected pregnant cows, calves, and a bull. The mean IgG1:IgG2 ratio of naturally infected cattle was 1:4 indicating a significant (p less than 0.001) IgG2 dominance. Similar ratios were detected in the experimentally infected cattle. The dominance of IgG2 was independent of breed, sex, and age. Twenty-nine cattle had significant immunoglobulin levels to both C. psittaci and Coxiella (C.) burnetii simultaneously. The predominance of C. psittaci-specific IgG2, in contrast to the predominance of C. burnetti-specific IgG1 detected in these same individual serum samples under identical conditions, indicates that the ability to preferentially produce either IgG1 or IgG2 was not limited in these individual cattle. A transient yet significant IgG1 response was also developed in cows following chlamydia-induced abortions (immunotype 1) or in cattle infected with the polyarthritis-serositis-encephalomyelitis agents (immunotype 2). IgG1 levels decreased faster than IgG2 levels. These findings have diagnostic implications and identify the need for determining the immunoglobulin classes and subclasses of the humoral immune responses of animals and man to chlamydial infections.     

Immunoglobulin G subclass distribution in canine leishmaniosis: a review and analysis of pitfalls in interpretation

Infection with Leishmania may have different outcomes in genetically distinct individuals and the course of infection is determined by the nature of the host innate and adaptive immune response. Thus in experimentally infected mice, and in naturally infected dogs or humans, the protective (self-healing or asymptomatic) phenotype is associated with the induction of Th1-regulated cell-mediated immunity. By contrast, a Th2-regulated humoral immune response is associated with severe symptomatic disease. In the murine model system there is strong correlation between clinicopathological phenotype and the nature of the antigen-specific humoral immune response. Symptomatic infection and Th2-regulation is associated with elevation in antigen-specific IgG1 and IgE, whereas asymptomatic infection with Th1-regulation is linked with IgG2a production. IgG subclass restriction is less clear in human disease with only some clinical forms being correlated to a specific serological profile. Although numerous studies have questioned whether infected dogs develop skewed IgG subclass usage, the results of these have been conflicting-suggesting bias towards IgG1 or IgG2 or neither subclass in different investigations. This confusion could relate to the specificity of the commercially available polyclonal antisera used to detect the canine IgG1 and IgG2 subclasses. More meaningful results might be obtained by the use of the panel of monoclonal antibodies with well-validated specificity for all four canine IgG subclasses.     

IgG subclass responses in a longitudinal study of canine visceral leishmaniasis

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an important disease of domestic dogs. Here, we present data on the IgG subclass antibody response to crude L. infantum antigen in a cohort of naturally infected Brazilian dogs. Specific IgG1-IgG4 responses could be detected in 98, 58, 70 and 82%, respectively of 57 dogs that were seropositive for specific IgG. Levels of all IgG subclasses were strongly inter-correlated. Levels of all IgG subclasses increased at the time of seroconversion, before reaching a plateau after several months. Levels of all IgG subclasses were higher in sick dogs than healthy dogs, and levels of all except IgG2 were higher in parasite-positive (by PCR) than parasite-negative dogs. However, levels of IgG2 relative to IgG1 were lower in sick or parasite-positive dogs compared to healthy or parasite-negative infected dogs. In contrast to previous studies, the results suggest that canine VL is associated with upregulation of specific antibody of all IgG subclasses, particularly IgG1, IgG3 and IgG4.     

Serological comparisons of antigenically related herpesviruses in cattle,red deer and goats

Serological comparisons were made using related herpesviruses from cattle (bovid herpesvirus 1), red deer (herpesvirus of cervidae 1) and goats (bovid herpesvirus 6) by virus neutralization and enzyme-linked immunosorbent assays. The test samples comprised field sera from British cattle, red deer and goats and sera from experimentally infected or immunized animals. Both the cervine and caprine viruses appeared to be more closely related to bovid herpesvirus 1 than they were to each other. Cattle sera reacted most strongly with the bovine virus and deer sera with the cervine virus. Antibodies to the caprine virus were not detected in the samples from British goats.     

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.     

Induction of a Th-1-biased IgG subclass response against equine herpesvirus type 1 in horses previously infected with type 4 virus

An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were na?ve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses na?ve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.     

Analysis of caprine IgG1 and IgG2 subclass responses to Chlamydia psittaci infection and vaccination

Enzyme-linked immunosorbent assays (ELISAs) specific for caprine IgG(H+L), IgG1 and IgG2 were developed and evaluated for serodiagnosis of Chlamydia psittaci infections in a Tunisian goat flock with currently occurring chlamydial abortions and a clinically inapparent goat flock of an animal research facility. Additionally, ELISAs were applied to record the IgG1 and IgG2 dynamics of four goats vaccinated with inactivated Chlamydia psittaci and Coxiella burnetii. For screening purposes, the IgG(H+L) ELISA proved to be superior to the complement fixation test because it detected a larger number of chlamydial abortions and was easier to perform and to interpret. Analysis of Chlamydia psittaci-specific IgG1 and IgG2 responses to naturally occurring infections by ELISA revealed high IgG1 levels associated with IgG2 in goats with current abortions, whereas clinically inapparent, but seropositive goats were characterized by significantly lower IgG1 levels only (P less than 0.001). Similarly, the four vaccinated goats responded initially with Chlamydia psittaci-specific IgG1, whereas second and third vaccinations induced (as in goats with chlamydial abortions) predominantly IgG1, but also IgG2. The results indicated that clinically inapparent chlamydial infection may be distinguished from overt disease by analysing specific IgG1 and IgG2 responses. Applying Coxiella burnetii- specific ELISAs on field samples, IgG1 alone could be detected in eight, IgG2 alone in one and IgG1 combined with IgG2 in nine goats. The coxiella-specific antibody response of the four vaccinated goats was--in contrast to the chlamydia-specific response--characterized by IgG2 dominance.     

Serological evidence of caprine herpesvirus 1 infection in Mediterranean France

Caprine herpesvirus 1 (CpHV-1) is responsible of a systemic disease in kids and genital diseases inducing abortions in adult goats. In Europe, CpHV-1 is widespread in Mediterranean countries such as Greece, Italy and Spain. As France is geographically close to these countries, a survey was conducted to investigate the presence of CpHV-1 in goats in a Mediterranean department (Corse-du-Sud) and in continental departments (Dordogne and Vendée) of this country. Taking into account the close antigenic and genetic relationships between bovine herpesvirus 1 (BoHV-1) and CpHV-1, the serological detection was performed by using BoHV-1 glycoproteins B (gB) and E (gE) blocking ELISAs. The analysis of 2548 serum samples in a BoHV-1 gB blocking ELISA revealed that a ruminant alphaherpesvirus infection related to BoHV-1 was widespread in Corse-du-Sud whereas no positive animals was detected in Dordogne and Vendée. Furthermore, the specificity and the sensitivity of the BoHV-1 gB blocking ELISA to detect a BoHV-1 related infection in goats were evaluated. A subsequent analysis by a BoHV-1 gE blocking ELISA demonstrated that 22.6% of gB-positive serum samples were also gE-positive. Cross-seroneutralisation assays afforded the unambiguous identification of antibodies against CpHV-1 in gB-positive goats. The likely presence of CpHV-1 in Corse-du-Sud supported by a high seroprevalence (61.9%) in all investigated flocks extends the number of countries infected with CpHV-1. Moreover, the difference observed between Corse-du-Sud and Dordogne and Vendée suggests that CpHV-1 is more prevalent in Mediterranean countries or regions than in central and northern Europe.     

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses

OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.     

Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus

A panel of 40 monoclonal antibodies (MAb) specific for bovine viral diarrhea virus (BVDV) was produced, and each MAb was characterized and grouped according to its viral protein specificity, immunoglobulin subclass, virus-neutralizing activity, and immunoreactivity with a large collection of BVDV isolates. The MAb were found to be specific for 1 of 3 sets of related viral-induced proteins found in cells infected with the Singer strain of BVDV. Group-1 MAb were specific for the 80- and 118-kilodalton (kD) proteins of BVDV. Group-2 MAb recognized 3 proteins with molecular sizes of 54, 56, and 58 kD. Group-3 MAb recognized a 43- and a 65-kD protein. The MAb belonged to either the IgG1, IgG2a, IgG2b, IgG3 subclasses or the IgE class of mouse immunoglobulin. All MAb in group 2 were able to neutralize BVDV and had neutralization titers that ranged from 24 to 1,600,000. The reactivity of the MAb with numerous field isolates of BVDV was highly variable. Both cytopathic and noncytopathic biotypes of BVDV were examined and had the same degree of antigenic variation. The greatest degree of variation was detected with group-2 MAb. The data demonstrate that BVDV isolates have a high degree of antigenic variation that is largely confined to the envelope glycoproteins associated with virus neutralization. The results also suggest that antigenic variability of this virus is important in the development and severity of the disease it causes.     

Canine visceral leishmaniasis: relationships between clinical status, humoral immune response, haematology and Lutzomyia (Lutzomyia) longipalpis infectivity

The main source of Leishmania infantum infection in humans is a naturally infected dog. This study reports on the infectivity to phlebotomine sandflies (Lutzomyia longipalpis) of serologically positive mongrel dogs that differed in clinical status, haematology and humoral responses to immunoglobulin (Ig) G(T) (total anti-Leishmania IgG), IgG(1) and IgG(2) subclasses of antibody to crude antigen of L. infantum. Forty-five female L. longipalpis were allowed to feed directly on the ears of dogs classified as asymptomatic, oligosymptomatic or symptomatic before being dissected five days later. Promastigotes were detected in 88% of the dissected sandflies. The highest rate of infectivity to sandflies was found in symptomatic dogs, followed by oligosymptomatic and asymptomatic animals. The results suggest that dogs naturally infected with L. infantum with higher total IgG and IgG(2) concentrations and lower haematocrit levels were able to infect the highest proportion of L. longipalpis. No correlation was observed between anaemia and the intensity of clinical signs. Symptomatic dogs presented the highest infection rate and intensity of infection.     

Neospora caninum IgG avidity tests: an interlaboratory comparison

Avidity tests can be used to discriminate between cattle that are acutely and chronically infected with the intracellular parasite Neospora caninum. The aim of this study was to compare the IgG avidity ELISA tests being used in four European laboratories. A coded panel of 200 bovine sera from well documented naturally and experimentally N. caninum infected animals were analysed at the participating laboratories by their respective assay systems and laboratory protocols. Comparing the numeric test results, the concordance correlation coefficients were between 0.479 and 0.776. The laboratories categorize the avidity results into the classes “low” and “high” which are considered indicative of recent and chronic infection, respectively. Three laboratories also use an “intermediate” class. When the categorized data were analysed by Kappa statistics there was moderate to substantial agreements between the laboratories. There was an overall better agreement for dichotomized results than when an intermediate class was also used. Taken together, this first ring test for N. caninum IgG avidity assays showed a moderate agreement between the assays used by the different laboratories to estimate the IgG avidity. Our experience suggests that avidity tests are sometimes less robust than conventional ELISAs. Therefore, it is essential that they are carefully standardised and their performance continuously evaluated.     

IgG antibody subclass response against equine herpesvirus type 4 in horses

In this study, IgG subclass responses against equine herpesvirus type 4 (EHV-4) were examined by enzyme-linked immunosorbent assay (ELISA) using a type-specific region of EHV-4 glycoprotein G (gG). ELISA using sera collected from horses experimentally infected with EHV-4 revealed that IgGa and IgGb antibodies were detected at high level, but IgGc and IgG(T) antibody responses were detected at low level or were undetectable. The IgGa antibody response reached its peak on day 10 post-infection, and then dropped. The IgGb antibody response reached its maximum level on day 12 post-infection, and then the level was sustained during at least 28 days after infection. Forty healthy racehorses that had already been infected with EHV-4 possessed antibody against EHV-4. Although IgGa antibodies specific for EHV-4 were not detected in any horses, IgGb antibodies were detected and the levels correlated with total IgG antibodies against EHV-4 gG. The results suggest that EHV-4-specific IgGa and IgGb antibodies are induced in EHV-4-infected horses, and that IgGb antibody, but not IgGa, is long lasting.     

Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae)

Antigen-specific antibody responses, T cell subsets and cytokine profiles were studied in 7 heifers naturally infested by Hypoderma sp. in Northwestern Spain. Immunoglobulin G (IgG) levels increased significantly at the end of the endogenous life cycle of the parasite (Mr). Similarly, IgG1 subclass increased considerably when first instars (L1) started their migration towards the back (Nv-Ja), whereas IgG2 increased earlier, coinciding with the arrival of L1 to the resting sites (Jn-Jl). Both subclasses decreased significantly when L3 began to leave the host. IgM levels and CD4 and CD8 profiles hardly oscillated throughout the life cycle of the parasite into the host. The CD4/CD8 ratio showed helper T cell predominance. Serum interferon-γ (IFN-γ) concentrations decreased from October to the end of the study. Interleukin 4 (IL-4) concentrations decreased in January and increased in February and May. There were a significant positive relationship between IL-4 and IgG2 subclass and a negative correlation between IFN-γ, IgG and IgG1 and also between IgM and CD2 and CD8 counts. These results suggest that in the early phases of natural primoinfestations by Hypoderma there is a slight predominance of a Th1 response, characterized by high IgG2 and IFN-γ levels, which is followed by a Th2 response with a clear predominance of IgG1 and IL-4.     

Transmission of caprine herpesvirus 2 in domestic goats

Caprine herpesvirus 2 (CpHV-2) is a recently recognized gammaherpesvirus that is endemic in domestic goats and has been observed to cause clinical malignant catarrhal fever (MCF) in certain species of deer. In this study, transmission of CpHV-2 in goats was examined. A total of 30 kids born to a CpHV-2 positive goat herd were selected and divided into two groups: group 1 (n=16) remained in the positive herd; group 2 (n=14) was separated from the herd at 1 week of age after obtaining colostrum. Peripheral blood samples from each kid were examined regularly by competitive ELISA for MCF viral antibody and by PCR for CpHV-2 DNA. Fifteen out of 16 goats (94%) that remained with the positive herd seroconverted and became PCR-positive for CpHV-2 by 10 months of age. In contrast, all kids (100%) that were separated from the positive herd at 1 week of age remained negative until termination of the experiment at 1 year of age. Additional transmission experiments revealed that all CpHV-2-free adult goats were susceptible to CpHV-2 or ovine herpesvirus 2 (OvHV-2) infection. The data indicate that the transmission pattern of CpHV-2 in goats is similar to the pattern of OvHV-2 in sheep and that CpHV-2-free goats can be established by early separation of kids from positive herds, which has significant implications for MCF control programs.     

A live attenuated glycoprotein E negative bovine herpesvirus 1 vaccine induces a partial cross-protection against caprine herpesvirus 1 infection in goats

Taking into account the close antigenic relationship between bovine herpesvirus 1 (BoHV-1) and caprine herpesvirus 1 (CpHV-1), a live attenuated glycoprotein E (gE) negative BoHV-1 vaccine was assessed in goats with the aim to protect against CpHV-1 infection. Vaccine safety was evaluated by intranasal inoculation of two groups of goats with either a gE-negative BoHV-1 vaccine or a virulent BoHV-1. The length of viral excretion and the peak viral titre were reduced with the gE-negative vaccine. To assess the efficacy, two goats were inoculated intranasally twice 2 weeks apart with a gE-negative BoHV-1 vaccine. Four weeks later, immunised and control goats were challenged with CpHV-1. A 2 log(10) reduction in the peak viral titre was observed and the challenge virus excretion lasted 2 days more in immunised than in control goats. These data indicate the safety and the partial efficacy of a live attenuated gE-negative BoHV-1 vaccine intranasally administrated in goats.     

Production and characterization of monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2

Monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2 were produced using conventional cell fusion techniques. Monoclonal antibodies detected by preliminary screening assays were further characterized and their specificity verified by titration of ascites in radioimmunoassay or passive haemagglutination using pure sheep IgG1 or IgG2. Further evidence for the subclass specificity of the antibodies was obtained from immunoelectrophoretograms of sheep serum or purified proteins developed with monoclonal antibodies. Reaction of monoclonal antibodies with various IgG fragments showed that the determinants recognised were located on the pFc' portion of the heavy chain.     

Bovine herpesvirus-1 infection of cattle: kinetics of antibody formation after intranasal exposure and abortion induced by the virus

The kinetics of antibody formation in Holstein heifers after primary and secondary intranasal inoculation of bovine herpesvirus-1 (BHV-1) and after BHV-1-induced abortion was determined. Sera were fractionated by gel filtration and ion-exchange chromatography. The antibody activity within serum immunoglobulin (Ig) isotypes was assessed, using a plaque-reduction neutralization assay. The primary immune response to BHV-1 infection was characterized by the appearance of IgM and IgG antibodies in serum by postinoculation day (PID) 7. Maximal IgG antibody activity occurred at PID 35 in nonpregnant heifers and at PID 14 in pregnant heifers. Thereafter, IgG antibody activity declined slowly in both groups of heifers. Maximal IgM antibody activity occurred at PID 14 in both groups of heifers and declined rapidly thereafter. The IgG antibody activity during primary immune responses was restricted to the IgG1 subclass. Secondary responses were characterized by anamnestic IgG antibody responses. Antibody activity was present within the IgG1 and IgG2 subclasses during secondary immune responses, but the increase in antibody activity during this period was primarily in the IgG2 subclass. Secondary IgM antibody formation was elicited by abortion induced by the intra-amniotic inoculation of BHV-1, but not by reexposure by the intranasal route. Abortion occurred in 1 heifer 28 days after intranasal BHV-1 inoculation. Abortion in this heifer was not associated with a secondary antibody response. The nature of BHV-1 antigenic exposure in the bovine determined the relative distribution of anti-BHV-1 antibody activity in serum IgM, IgG1, and IgG2. The formation of IgM antibody, with the exception of secondary intranasal exposure, indicated recent BHV-1 antigenic exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.