Pathogenicity of Rhodococcus equi expressing a virulence-associated 20 kDa protein (VapB) in foals

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.     

A modified live PRRSV vaccine and the pathogenic parent strain induce regulatory T cells in pigs naturally infected with Mycoplasma hyopneumoniae

Rhodococcus equi is an important respiratory pathogen of young foals for which a vaccine has long been sought. Two major impediments to effective vaccination are the functionally immature type I immune responses of neonatal foals and early exposure to the bacterium via the environment. Despite these obstacles, it appears that under specific circumstances foals can develop a protective immune response. In this study we investigated the protective mechanisms behind oral inoculation of foals with virulent R. equi bacteria. Two foals receiving an oral inoculum demonstrated accelerated development of R. equi specific cytotoxic T lymphocytes (CTL) as evidenced by significant lysis of R. equi infected, ELA-A mismatched cells at 3 weeks of age. As in a previous study, CTL were not detected until 5-6 weeks of age in two control foals. At each time point the ability of foal peripheral blood mononuclear cells (PBMC) to produce IFN-γ following stimulation with live R. equi or extracted cell wall lipids was similar to that of an adult horse control and between foals, regardless of treatment. These results provide a potential mechanism of protection which has previously been shown to occur following oral inoculation, and suggest that the early detection of CTL may be a useful marker for induction of protective immunity.     

Enzyme-linked immunosorbent assay for diagnosis of Corynebacterium (Rhodococcus) equi infection in foals

An enzyme-linked immunosorbent assay (ELISA) was used to diagnose Corynebacterium (Rhodococcus) equi infection in foals. In tests done with different antigen-extraction procedures (sodium dodecyl sulfate, sodium deoxycholate, polyoxy-ethylene [9] p-tert-octylphenol, polyoxy-ethylene [9-10] p-tert-octylphenol, sonification, homogenization, and heat treatment at 121 C), Tween 20 was a satisfactory reactive antigen. Using hyperimmune rabbit sera or infected foal sera, we investigated the specificity and the sensitivity of the ELISA with the Tween 20 antigen of the different serotypes or of the isolates. Corynebacterium equi strain ATCC 6939 antigen had the best activity for detecting antibodies to C equi in foals. Sera from 218 healthy horses, 11 healthy foals, 17 healthy newborn foals, a foal with suspected C equi infection, and 5 infected foals were evaluated for antibodies to C equi, using ELISA. The optical density values of 206 healthy horses, 17 healthy newborn foals, and 9 healthy foals were less than 0.1. Infected foal sera, except from foal 3, and serum from a foal with suspected C equi infection had higher optical density values. Using ELISA, specific antibodies against C equi were detected in a naturally infected 6-week-old foal after the foal had a rapid increase in the number of bacteria in the feces and after the initial development of clinical signs of illness at 5 weeks of age. Therefore, ELISA was useful for the early diagnosis of C equi infection in foals.     

Rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals

The immunoprophylactic capacity of specific immune plasma was evaluated in pony foals infected experimentally with Rhodococcus equi. Immune plasma, produced by repeated parenteral administration of viable R. equi to adult horses, was harvested and frozen. Group I (six control foals) and Group II (six principal foals) received lactated Ringers solution and immune plasma respectively at three and five days of age. R. equi were aerosolised into a caudal lung lobe of all foals at seven days of age. Clinical signs, haematological alterations, immune responses, thoracic radiographs and technetium99m pulmonary perfusion scans were monitored. All foals were destroyed and complete post mortem examinations performed. All foals developed pneumonia as evidenced by clinical, radiographic and perfusion alterations, but the survival rate of principal foals was significantly (P less than 0.01) greater than that of control foals. Five control foals developed terminal disease, whereas all principal foals recovered. There was no significant (P greater than 0.05) difference in temperature response, or peripheral blood leucocyte, neutrophil or fibrinogen concentrations between groups. ELISA values for R. equi antibody were significantly (P less than 0.001) greater in principal foals following treatment, but there was no significant (P greater than 0.05) difference in IgG or IgM concentrations between groups. Results of the haemolysis inhibition assay indicated that equi factor neutralising antibodies were transferred by immune plasma to the principal foals. Post mortem examinations of five control foals destroyed at approximately three weeks post infection because of terminal disease, revealed severe pyogranulomatous pneumonia. One control and all principal foals were either free of lesions or had resolving lesions and/or minimal scar formation at three months post infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equine humoral immune response to Rhodococcus (Corynebacterium) equi

An enzyme-linked immunosorbent assay was developed to test equine serum for the presence of antibodies to Rhodococcus (Corynebacterium) equi. Experimental ponies had no detectable antibody to R equi before exposure to the bacterium. After experimental inoculation, animals in groups that received live R equi subcutaneously or intranasally/intratracheally developed high titers to R equi. Noninoculated controls remained seronegative. Serum was also collected from horses of various ages that were naturally exposed to R equi. There was a wide range of anti-R equi titers in these horses. Because experimentally infected horses seroconverted when some naturally infected foals did not seroconvert, the function of antibody in resistance to R equi infection remains unknown.     

Systemic and local immune responses of gnotobiotic calves to respiratory infection with Mycoplasma bovis

Gnotobiotic calves were inoculated by the intratracheal route with Mycoplasma bovis and the specific antibody response in sera and tracheo-bronchial washings examined by radioimmunoassay. In sera an IgM response which reached a peak two weeks post infection was followed by IgG1 and IgG2 antibody responses. Low levels of IgA antibody were detected in sera three and four weeks after infection. The predominant antibody in tracheo-bronchial washings 2 weeks after infection was IgA. Four weeks after infection IgG1 antibody predominated, but IgG2 and IgA antibodies were also present. Cells containing Ig were present in the cellular accumulations around the necrotic zones produced by M. bovis in the lung parenchyma two and four weeks after infection. IgG1 containing cells predominated in these cellular infiltrates. IgG2 producing cells were the next in frequency. It is concluded that the lung lesion caused by M. bovis is partly due to the host's immune response, presumably contributing to the control of the infection, and that the cells infiltrating the lung are a major source of the local and systemic IgG antibody that is detected after infection. IgA staining cells were observed in the submucosa of tissues from nasal cavity and trachea. These cells are probably the source of IgA in tracheo-bronchial washings and sera since IgA-producing cells were not a predominant component of the lesion in the lung parenchyma.     

The pathogenesis of Rhodococcus equi pneumonia in foals

The pathogenesis of Rhodococcus equi pneumonia in foals is reviewed. The main routes of infection are respiratory and alimentary. The latter is probably the chief route of exposure in all foals and probably leads to development of specific immunity. Susceptible foals, those whose maternal immunity wanes before generation of their own immune response, readily develop disease if exposed aerogenously to sufficient numbers of R. equi. Management and environmental circumstances have a major role to play in determining the magnitude of this challenge and, therefore, in the prevalence of the disease. Infection of a naive foal leads to severe, suppurative bronchopneumonia with suppurative lymphadenitis of regional nodes and, in approximately 50% of animals, to necrotizing enterocolitis. The foal is uniquely susceptible to R. equi pneumonia; comparable experimental infections do not produce progressive destructive pulmonary lesions in other animal species. In the naive foal lung, R. equi behaves as a facultative intracellular pathogen, avoiding destruction within the alveolar macrophage by inhibiting phagolysosome fusion and possibly by causing lysosomal degranulation. The role of putative virulence factors, such as equi factor, remains to be elucidated.     

Recognition of a B-cell epitope of the VapA protein of Rhodococcus equi in newborn and experimentally infected foals

The aim of this study was to evaluate the usefulness of the previously identified B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein of Rhodococcus equi and its association with R. equi pneumonia. A modified peptide designated PN11-14 corresponding to the epitope was recognized by all sera from experimentally infected foals with virulent R. equi ATCC103+ containing the virulence plasmid but not by its plasmid-cured derivative ATCC103- strain. Marked levels of VapA-specific immunoglobulin (Ig)G were detected in all sera from the ATCC103+ infected foals at 2 weeks after the infection. One control animal had high titres as determined by the peptide enzyme-linked immunosorbent assay (ELISA), indicating the ELISA may not absolutely differentiate between foals with R. equi pneumonia and healthy exposed foals in farms where the prevalence of disease is high. However, numbers of animals used were small. Further evaluation of the peptide ELISA with field samples is necessary to determine whether the assay is diagnostically useful. This study showed that levels of passive transfer of maternal IgG antibodies to the epitope in newborn foals could be measured. Interestingly, the maternally derived antibodies were found to significantly (P<0.05 by Student's t-test) decline 2 weeks after birth. Seroconversion against naturally occurring VapA expressing R. equi could be detected in some foals at 4 weeks of age. Antibodies to the epitope peaked and were significantly (P<0.05) greater in foals aged between 6 and 8 weeks. These results indicated that the peptide ELISA could be used to monitor anti-VapA antibodies in foals, particularly those at the age of 4-6 weeks. It is possible that the ELISA may be of some use as a diagnostic test on farms where R. equi is non-endemic. Further studies using large number of field samples are needed to verify this assumption.     

Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody

The intestinal and systemic antibody response of calves vaccinated and/or challenged with rotavirus was studied employing isotype-specific ELISAs for the detection of IgG1, IgG2, IgM and IgA antibodies to rotavirus. Monoclonal antibodies to bovine immunoglobulin isotypes of proven specificity were used as conjugated or catching antibody. Five days after oral inoculation (dpi) of a 5-day-old gnotobiotic calf with rotavirus, IgM rotavirus antibodies were excreted in faeces, followed 5 days later by IgA rotavirus antibodies. The increase in IgM rotavirus antibody titre coincided with the inability to detect further rotavirus excretion. Faeces IgM and IgA rotavirus antibody titres fell to low levels within 3 weeks post infection. IgG1 and IgG2 rotavirus antibodies were not detected in faecal samples. In serum, antibodies to rotavirus of all four isotypes were detected, starting with IgM at 5 dpi. Two SPF-calves, which were fed colostrum free of rotavirus antibodies, were vaccinated with a modified live rotavirus vaccine and challenged with virulent rotavirus 6 days later. Upon vaccination, the calves showed an antibody response similar to the response of the infected gnotobiotic calf. Intestinal IgM rotavirus antibodies were excreted before or on the day of challenge and appeared to be associated with protection against challenge infection with virulent virus and rotavirus-induced diarrhoea. In 3 control calves, which were challenged only, the antibody patterns also resembled that of the gnotobiotic calf and again the appearance of IgM rotavirus antibodies coincided with the end of the rotavirus detection period. Two other groups of 3 SPF-calves were treated similarly, but the calves were fed colostrum with rotavirus antibodies during the first 48 h of life. These calves excreted passively acquired IgG1 and IgG2 rotavirus antibodies in their faeces from 2 to 6 days after birth. After vaccination, no IgM or IgA antibody activity in serum or faeces was detectable. Upon challenge, all calves developed diarrhoea and excreted rotavirus. Seven to 10 days after challenge low levels of IgM rotavirus antibody were detected for a short period. These data indicate that the intestinal antibody response of young calves to an enteric viral infection is associated with the excretion of IgM antibodies, immediately followed by IgA antibodies. This response is absent or diminished in calves with passively acquired specific antibodies which may explain the failure to induce a protective intestinal immune response by oral vaccination with modified live rotavirus of calves fed colostrum containing rotavirus antibodies.     

Colostral and serum IgG, IgA, and IgM concentrations in Standardbred mares and their foals at parturition

Immunoglobulin G, IgM, and IgA concentrations were measured in serum collected from 36 Standardbred mares within 12 hours of foaling, in colostrum collected within 6 hours of foaling, and in serum collected from foals 24 to 48 hours after birth. In serum collected from mares after parturition, mean concentrations of IgG, IgM, and IgA were 2,463.9 +/- 1,337.3 mg/dl, 136.4 +/- 218 mg/dl, and 305.2 +/- 237.5 mg/dl, respectively. In serum from foals, mean concentrations of IgG, IgM, and IgA were 1,953.3 +/- 1,635 mg/dl, 33.8 +/- 30.4 mg/dl, and 58.4 +/- 42.2 mg/dl, respectively. In colostrum, mean concentrations of IgG, IgM, and IgA were 8,911.9 +/- 6,282.2 mg/dl, 957 +/- 1088.1 mg/dl, and 122.9 +/- 77.3 mg/dl, respectively. The IgG concentrations in foal serum were poorly correlated with IgG concentrations in colostrum (r = 0.462, P less than 0.01). Correlations of IgM or IgA concentrations in serum from foals with IgM or IgA concentrations in colostrum and correlations of IgG concentrations in serum from mares with those in colostrum were not significant (P less than 0.01). Of 36 foals, 1 (2.8%) had a serum IgG concentration less than 400 mg/dl. Of 36 foals monitored for 4 months, 6 developed infectious respiratory tract disease requiring antimicrobial therapy at ages varying from 55 to 113 days; these infections were probably not related to failure or partial failure of passive transfer of antibody.     

Humoral and cellular immune responses of pigs inoculated with Mycoplasma hyopneumoniae

Cellular and humoral immune responses of pigs inoculated with Mycoplasma hyopneumoniae were investigated at postinoculation weeks (PIW) 2, 4, and 6. The response of blood lymphocytes (BL) and bronchial lymph node lymphocytes (LNL) to stimulation by M hyopneumoniae antigens was evaluated by a lymphocyte-stimulation test. Specific antibodies in serum and lung washing samples were assayed by ELISA. Immunoglobulin-positive cells in lungs and bronchial lymph nodes were identified by indirect fluorescent antibody test, using isotype-specific monoclonal antibodies. At PIW 0 to 6, BL from control and M hyopneumoniae-inoculated pigs were stimulated by M hyopneumoniae cells; however, BL from inoculated pigs generally had higher stimulation indices, especially at PIW 6. The response of LNL was influenced by previous exposure to M hyopneumoniae, as indicated by higher stimulation indices (P less than 0.01) of LNL from inoculated pigs killed at PIW 2 and 6. Specific ELISA antibodies to M hyopneumoniae in lung washings from inoculated pigs consisted mainly of IgG and IgA isotypes. Examination of lung sections by indirect immunofluorescence revealed that cells producing IgM and IgA were in controls as well as M hyopneumoniae-inoculated pigs, but IgG-positive cells were only in lungs of inoculated pigs. Resolution of pneumonia appeared to correlate with development of increased sensitization of BL, as well as development of marked increases in immunoglobulins, particularly IgG in lung washings at PIW 6.     

Rapid immunohistochemical detection of Rhodococcus equi in impression smears from affected foals on postmortem examination

The first objective of this study was to develop an immunohistochemical procedure for rapid detection of Rhodococcus equi in impression smears from affected organs of foals on postmortem examination. The second aim was to demonstrate whether R. equi can be detected in smears of tracheal exudates collected from the same foals using an immunohistochemical method. Impression smears and cryostat and paraffin-embedded sections were made from the lungs and mediastinal lymph nodes of three foals (A, B and C) that had died of respiratory disease caused by R. equi, and also from the caudal mesenteric lymph node of foal A. Impression smears were made from the tracheal exudates of all foals. An affinity purified rabbit IgG was used for the immunohistochemical demonstration of R. equi. This antibody reacted with serotype 1 of R. equi in Ouchterlony's immunodiffusion and in the passive haemagglutination test, but not with other serotypes or with Streptococcus equi ssp. equi or Staphylococcus aureus, and failed to give an immunohistochemical reaction with Mycobacterium bovis or M. paratuberculosis. The immunohistochemical method proved to be of identical sensitivity to bacterial culture; moreover, from the lungs and mediastinal lymph nodes of one foal, R. equi could only be detected by this method. R. equi was demonstrated in smears of the tracheal exudates of all three foals. The results of this study indicate that the immunohistochemical method may be used for the rapid detection of R. equi in impression smears from the affected organs, especially abscesses, obtained postmortem, and possibly as a tool for diagnosing R. equi pneumonia in live foals by examining smears of tracheal aspirates.     

Immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses

OBJECTIVE: To determine concentrations of IgA and IgG subclasses in serum, colostrum, milk, and nasal wash samples of adult horses and foals. ANIMALS: Seven 2-year-old Welsh ponies, 27 adult mixed-breed horses, and 5 Quarter Horse mares and their foals. PROCEDURE: Serum was obtained from ponies and adult horses. Colostrum and milk were obtained from mares and serum and nasal wash samples from their foals immediately after parturition and on days 1, 7, 14, 28, 42, and 63. Nasal wash samples were also obtained from 23 adult horses. Concentrations of immunoglobulins were determined by use of inhibition ELISA. To determine transfer of maternal isotypes to foals, concentrations in colostrum and milk were compared with those in foal serum. Serum half-lives of isotypes in foals were also determined. RESULTS: IgGb was the most abundant isotype in serum and colostrum from adult horses, whereas IgA was the predominant isotype in milk. The major isotype in nasal secretions of adult horses and foals > or = 28 days old was IgA, but IgGa and IgGb were the major isotypes in nasal secretions of foals < or = 14 days old. Serum half lives of IgGa, IgGb, IgG(T), and IgA in foals were 176, 32, 21, and 3.4 days, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The early immunoglobulin repertoire of neonatal foals comprised IgGa, IgG(T), and IgA; endogenous synthesis of IgGb could not be detected until 63 days after birth. The restricted repertoire of immunoglobulins in foals may influence humoral immune responses to vaccination.     

Unusual Patterns of Serum Antibodies to Streptococcus Equi in Two Horses with Purpura Hemorrhagica

An enzyme-linked immunosorbent assay (ELISA) was developed for use in horses to determine serum titers of antibodies of the immunoglobulin classes IgA, IgG, and IgM to Streptococcus equi M-like protein and culture supernatant protein antigens. Serum antibodies were determined in 28 adult horses, including 9 horses with recent S. equi infections, 17 horses without known exposure to S. equi, but without a history of respiratory disease in the preceding 4 months, and 2 horses with clinical purpura hemorrhagica. Serum IgA titers to culture supernatant protein antigen were highest in recently infected horses (P less than 0.001). Serial determinations of antibody titers in the horses with purpura showed that IgG antibodies to both S. equi M-like protein and culture supernatant protein antigens were undetectable initially, but later rose coincidental with clinical recovery from the disease. Possible mechanisms for these findings are discussed.     

Peripheral Blood Lymphocyte Subpopulations and Immunoglobulin Concentrations in Healthy Foals and Foals with Rhodococcus equi Pneumonia

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.     

Electron microscopic investigation of intracellular events after ingestion of Rhodococcus equi by foal alveolar macrophages

It has been suggested that R. equi causes pulmonary disease in foals by persisting within the lung as a facultative intracellular parasite of alveolar macrophages. This paper describes an ultrastructural study of the intracellular events after ingestion of R. equi by foal alveolar macrophages, in an attempt to determine the mechanism of intracellular survival of R. equi. Secondary lysosomes of alveolar macrophages recovered from foals by bronchoalveolar lavage were labelled with electron-dense ferritin, and the cells were challenged with either viable or formalin-killed R. equi. After 0-, 3-, 8- or 24-h incubation, the cells were fixed and processed for electron microscopy. There was no evidence of phagosome-lysosome fusion after ingestion of either viable or non-viable R. equi by foal alveolar macrophages. Rhodococcus equi persisted and multiplied within dilated phagosomes, which were often lined by elongate microvillous structures. After 24-h incubation, 75% of the ingested bacteria were still structurally intact. Macrophages with ingested viable R. equi were irreversibly damaged and released intracellular bacteria into the surrounding medium. These data confirm that R. equi is a facultative intracellular parasite of foal alveolar macrophages and is able to persist and multiply within the phagosome, apparently inhibiting phagosome-lysosome fusion by some as yet unknown mechanism.     

Diagnosis of Rhodococcus equi infection in foals by the agar gel diffusion test with protein antigen

A protein antigen that reacted in the agar gel diffusion (AGD) test and which had equi factor(s) activity, was partially purified from the culture supernatant of Rhodococcus equi by successive column chromatography on diethylaminoethyl cellulose and Sepharose 4B. Employing a standard foal serum, the concentration of this antigen was adjusted for the AGD test. Optimal dilutions of the antigen reacted in the AGD test with sera from foals naturally infected with serologically different R. equi. The antigen prepared was considered suitable for use in field surveys of R. equi infection. Accordingly, four groups of sera were tested: those from 18 foals diagnosed as being infected with R. equi, those from 54 control foals with culture-negative R. equi pneumonia, arthritis or cellulitis, those from 46 diseased foals suspected of having R. equi infection and those from 51 clinically normal foals. A positive precipitation reaction was observed with sera from 100% of the first group, 69.5% of the third group and 17.7% of the fourth group. A negative reaction was obtained with sera from 100% of the second group.     

Change of antibody levels to ferritin in the sera of foals after birth: Possible passive transfer of maternal anti‐ferritin autoantibody via colostrum and age‐related anti‐ferritin autoantibody production

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi‐quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co‐immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co‐immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin‐binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.     

Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs

The use of the four-layer enzyme immunoassay (EIA) for the detection of IgG, IgM and IgA antibodies against Aujeszky's disease virus in blood and oropharyngeal swabs of infected and vaccinated pigs is described. Mean antibody titres obtained using the four-layer EIA were 6.1 and 3829 times higher compared with the indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test, respectively. The VN test detected mainly IgG antibodies, while the IgM antibodies did not react. Using the EIA, the first antiviral antibodies in sera were demonstrated on Days 5-7 after infection or vaccination. Up to the 7th day, demonstrable antibodies were almost exclusively of the IgM class. In infected pigs high titres of IgM antibodies were still detected on Day 18, while in vaccinated animals they were absent by this time. Antibodies of the IgG class appeared in infected pigs sooner (Day 7) than in vaccinated pigs (Day 10) and reached higher mean titres. Antibodies of the IgA class were demonstrable from Day 10 only in samples from infected pigs. Similar antibody dynamics and distribution were detected in oropharyngeal swabs, except that the IgG and IgM titres were roughly 100 times lower than in sera. However, titres of IgA antibodies in oropharyngeal swabs were two times higher than in sera. The greatest differences between both groups of animals were recorded on Day 18; in the infected pigs, IgG, IgM and IgA antibodies were present in sera and oropharyngeal swabs at that time, while in vaccinated pigs only IgG antibodies were demonstrable. The effect of infection and vaccination on the pattern of the immune response as well as the importance of the detection of individual immunoglobulin classes for the specificity of the enzyme immunoassay are discussed.