Humoral antibody response of rabbits to experimental infection with Encephalitozoon cuniculi

Six female rabbits (Oryctolagus cuniculus) were experimentally infected intravenously with approximately 1.5 X 10(7) live spores of Encephalitozoon cuniculi. Head tilt was observed as the single clinical sign in only one of the six animals. Antibody response was registered over 68 days postinfection using the indirect immunofluorescence test (IFT) for IgM and IgG, and the carbon immunoassay (CIA). IgG titers reached a level of 160-2560 after a latent phase of 13-28 days, followed by a 2-4 week relatively steep increase. The IgM seroconversion was faster than that of IgG and occurred at the beginning of the antibody response. Thus, simultaneous detection of both IgM and IgG allowed the infection to be identified as recent. Long, short, and episodic antibody responses could be distinguished: the IgG titer continued to increase on Day 68 in one animal (long response) and began to decrease between Days 45 and 63 in three other animals (short response). In two additional animals the seroconversion was very short, occurring between Days 13 and 41, and 28 and 52, respectively (episodic response). The CIA proved to be specific, reliable, and simple to perform; titers were slightly higher than in the IFT. Parasite pseudocysts were detected scattered throughout the brain on Day 68 in four of the six rabbits. The persistence of antigen in the brain did not correlate with antibody response, which in most cases was shorter.     

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.     

Antibody Development in Pigs Inoculated with Live or Killed Cultures of Brucella Abortus

As shown by density gradient ultracentrifugation and column chromatography, pigs formed IgM antibodies during the first week following vaccination with Brucella abortus, strain 19. At this time their sera reacted in both plate and tube agglutination but not in complement-fixation tests. A few days later, when IgG antibodies had developed, agglutination titers were still high and some activity was recorded in hemolytic complement-fixation tests. A similar sequence was observed in pigs repeatedly inoculated with phenol-killed suspensions of B. abortus. As the proportion of IgM to IgG antibodies decreased, agglutinin titers fell in relation to complement-fixing titers. In some animals the conglutinating complement absorption test became positive earlier than the plate agglutination.     

Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2)

Immunological impairment by porcine circovirus type 2 (PCV2) infection is well documented in pigs suffering from postweaning multisystemic wasting syndrome. Nonetheless, little is known about immune status of pigs that remain PCV2 subclinically infected. Thus, seven pigs successfully infected in an experimental inoculation and without developing disease and nine control non-inoculated pigs were examined. Serological, virological and immunological determinations were done throughout ten weeks post-infection (PI). At week 3 PI, inoculated animals presented the peak of viremia and produced higher levels of IL-10 than the controls; correlation between viral load and IL-10 amounts was observed (p<0.05). Also, the ratio IgM/IgG suffered a shift skewing IgM production towards an IgG response. By 10 weeks PI, levels of IL-10 disappeared and the viremia decreased. In summary, subclinically PCV2-infected pigs developed a transient PCV2-specific IL-10 response during the viremic phase of infection which coincided with the inversion of the IgM/IgG ratio.     

Immunocompetence of fattening pigs fed organic versus conventional diets in organic versus conventional housing

The effect of organic or conventional feeding on the immune response of pigs was determined using organic or conventional housing in a pig fattening unit. The experimental design involved four pens of four animals per housing and diet combination (organic housing and organic nutrition; organic housing and conventional nutrition; conventional housing and organic nutrition and conventional housing and conventional nutrition). The IgM, IgA and IgG responses against intramuscularly injected bovine thyroglobulin were determined as indicators of the antigen-specific immune responsiveness. Some general health and welfare related parameters were evaluated by measuring haptoglobin concentrations at selected times; blood lactate concentration was measured at slaughter. Conventional housing led to a higher IgG response three weeks after the first immunisation. Organic housing led to lower haptoglobin and lactate concentrations at slaughter, indicating a higher stress resistance in these pigs. No major differences between the two feeding types were found. We conclude that the immune responses following either a conventional or an organic diet are comparable, whereas organic housing can increase stress resistance at slaughter compared to conventional housing.     

Antibody response in calves experimentally or naturally exposed to Mycoplasma bovoculi

An antiglobulin-ELISA has been developed to detect antibody activity to Mycoplasma bovoculi in sera, nasal fluids and lacrimal fluids of field and experimentally exposed calves. Low IgG activity with no IgM or IgA was detected in sera of experimental calves. In nasal and lacrimal fluids, IgA appeared as early as the first week following exposure to M. bovoculi and predominated in both of these fluids throughout the 9 wk observation period. Sera from field-exposed animals showed high IgG and IgM activities. The metabolic-inhibition (MI) test was applied to detect growth inhibition of M. bovoculi in those fluids. This property was found only in sera of exposed animals and thus could be used to test for M. bovoculi infection. The ELISA test and the MI test were considered reliable tests for the detection of antibodies to M. bovoculi infection. The implications of finding no growth-inhibiting activity in nasal and lacrimal fluids concurrent with a high IgA activity are discussed.     

Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm

The objective of the present study was to determine the pattern of Hepatitis E virus (HEV) infection in a naturally infected, farrow-to-finish herd. For that purpose, a prospective study was conducted in randomly selected 19 sows and 45 piglets. Blood samples were collected from sows at 1 week post-farrowing and from piglets at 1, 3, 6, 9, 12, 15, 18 and 22 weeks of age. Furthermore 3 or 5 animals were necropsied at each bleeding day (but at 1 week of age), and serum, bile, liver, mesenteric lymph nodes and faeces taken. HEV IgG, IgM and IgA antibodies were determined in serum and viral RNA was analysed in all collected samples by semi-nested RT-PCR. Histopathological examination of mesenteric lymph nodes and liver was also conducted. From 13 analysed sows, 10 (76.9%) were positive to IgG, one to IgA (7.7%) and two to IgM (15.4%) antibodies specific to HEV. In piglets, IgG and IgA maternal antibodies lasted until 9 and 3 weeks of age, respectively. IgG seroconversion occurred by 15 weeks of age while IgM and IgA at 12. On individual basis, IgG was detectable until the end of the study while IgM and IgA antibody duration was of 4-7 weeks. HEV RNA was detected in serum at all analysed ages with the highest prevalence at 15 weeks of age. HEV was detected in faeces and lymph nodes for the first time at 9 weeks of age and peaked at 12 and 15 weeks of age. This peak coincided with the occurrence of hepatitis as well as with HEV detection in bile, liver, mesenteric lymph nodes and faeces, and also with highest IgG and IgM OD values at 15 weeks. Finally, different HEV sequences from this farm were obtained, which they clustered within 3 different groups, together with other Spanish sequences, all of them of genotype 3. Moreover, the present study also indicates that the same pig can be infected with at least two different strains of HEV during its productive life. This is the first study characterizing HEV infection in naturally infected pigs with chronological virus detection and its relationship with tissue lesions throughout the productive life of the animals.     

Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses.

Recently a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting antibody against H1N1 swine influenza virus (SIV) has been made available to diagnosticians and veterinary practitioners. Because the hemagglutination inhibition (HI) test has been considered the standard test for SIV serology, diagnostic performance of the new ELISA was evaluated using positive (n = 60) and negative (n = 188) serum samples from young pigs with known status of SIV infection and compared with that of the HI test. Both ELISA and HI test identified all negative animals correctly. None of the serum samples (n = 64) from pigs inoculated with H3N2 SIV was positive by ELISA for SIV antibody. The H1N1 SIV antibody detectable by ELISA appears to develop more slowly in comparison with antibody detectable by HI test. Although antibody was detected by HI test in all inoculated animals (n = 20) by day 7 postinoculation (PI), antibody was detected by ELISA in 0%, 75%, and 100% of the inoculated animals on days 7, 14, and 28 PI, respectively. Discrepancy in test results between the 2 serologic tests appeared to be because of differences in antibody isotypes detected by each test. Enzyme-linked immunosorbent assay mainly detected IgG antibody, whereas the HI test detects IgM antibody very efficiently as well as IgG antibody. Collectively, the commercial ELISA is highly specific for antibody to H1N1 SIV but may not identify positive animals at the early stage of infection as effectively as the HI test, particularly when SIV is introduced to a na?ve swine population.     

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.     

Evaluation of the ELISA for the serological diagnosis of trichinosis in Canadian swine

An ELISA using a Trichinella spiralis spiralis excretory-secretory antigen was evaluated as a procedure for the diagnosis of trichinosis in swine in Canada. Field and experimental trials were carried out using both indirect serological (ELISA) and direct parasitological (pepsin-digestion) methods concurrently on serum and musculature, respectively, from each animal. The ELISA is a sensitive and specific test for the detection of Trichinella antibodies in porcine sera when present. The development of Trichinella antibodies appears to be dependent on the magnitude of the infection established, age of the infection when the animal is tested and the immunocompetence or response to infection of individual animals. False negative reactions were recorded in both field and experimental trials. In the field study, five of the 1009 swine examined were parasitologically positive with light infections ranging from 0.01 to 0.046 larvae per gram (la/g) of musculature yet all were serologically negative. Experimentally it was shown that Trichinella antibodies develop slowly, at least two to three months postinfection, in pigs with very light infections. Even in pigs which developed infections of 33 to 55 la/g of musculature, seroconversion occurred greater than 23 and less than 30 days postinfection. The immunocompetence or response to infection of individual pigs was variable as illustrated by one pig inoculated with 3000 infective larvae which had consistently lower titers compared to others in the same group despite the establishment of a muscle infection of 8.5 la/g of musculature. One false positive reaction was recorded in the experimental trial in an animal which had received 100 larvae and seroconverted at about three months postinfection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of specific anti-leptospiral immunoglobulins M and G in sera of experimentally infected dogs by solid-phase enzyme-linked immunosorbent assay

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) to detect specific anti-leptospiral IgM and IgG in sera of dogs experimentally infected with Leptospira interrogans serotype canicola are reported. In all dogs specific anti-leptospiral IgM was detected from the second half of the first week after infection, the maximum being attained during the second week. Subsequently the IgM titre gradually decreased. Specific anti-leptospiral IgG was detected later and increased gradually to reach almost the same level as the IgM titre after two to three months. During the initial stage of the infection, when the microscopic agglutination titre was still negative or very low, a high IgM titre was accompanied by a negative or very low IgG titre in every case. After the initial stage a substantial IgG titre was also detectable. It is suggested that the test is suitable for serodiagnostic purposes, particularly for the diagnosis of a current infection in an individual.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Circulating lymphocytes, neutrophils and immune complexes in pigs after feeding

There are reports in the literature claiming that pigs experience a lymphopenia after feeding. This is not observed in other species. We set out to investigate this phenomenon because of the unusual route of lymphocyte recirculation in pigs. for it suggested that this might be a specific response to dietary antigens. We were unable to confirm this phenomenon. A lymphopenia was observed in both test and control groups as a non-specific response to restraint and blood sampling. In contrast, feeding was associated with a significant neutrophilia (mean 58%, P less than 0.002). There were no increases in circulating IgA, IgG or IgM immune complexes after feeding but IgG immune complexes remained at a higher level than in the control group.     

囊虫病猪血清CA效价与虫负荷的相关性及CA与Ab的动态观察

应用ELISA双抗体夹心法检测囊虫病猪血清中的循环抗原(CA),对CA效价与虫负荷的相关性作了初步观察,发现两者密切相关.轻度感染猪血清CA效价≤000,中度为1000～10000,重度为10000～100000.人工感染病猪血清一般在感染后1周检出CA,于感染后4～5周达高峰;抗体一般于感染后2周检出.这种方法可检出的最小虫负荷约为20个/猪     

Preparation of heavy chain specific antisera to bovine IgA, IgM, IgG1 and IgG2

Goats, guinea pigs and rabbits were immunized with bovine IgM or with intact molecules, heavy chains, Fc portions or light chains of bovine IgG1 and IgG2. Rabbits and guinea pigs were immunized with bovine secretory IgA. Goats and guinea pigs produced heavy chain specific antisera to intact IgM whereas rabbits produced anti-light chain antibody and in one instance anti-alpha 2-macroglobulin antibody in addition to the anti-mu response. Goats and guinea pigs produced antisera to bovine IgG1 and IgG2 and their Fc portions that needed little absorption to render them monospecific for the heavy chain. In addition to antibody to the heavy chains, rabbits produced anti-light chain antibody when immunized with intact IgG1 or IgG2 molecules. These latter sera, and those produced by rabbits immunized with Fc portions of IgG1 or IgG2 required extensive absorption before they were monospecific for their respective heavy chains. Heavy chains were poor immunogens in all three species. Rabbits immunized with IgA produced both anti-alpha and anti-light chain antibodies while guinea pigs produced sera with antibody activity to the alpha chain only.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Cytokine and antibody subclass responses in the intestinal lymph of sheep during repeated experimental infections with the nematode parasite Trichostrongylus colubriformis

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-alpha and IFN-gamma genes, and parasite-specific IgM, IgG1, IgG2, IgA and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-gamma did not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and na?ve S sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge. Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous exposure to nematodes. Genes for TNF-alpha and IFN-gamma were also transiently up-regulated, but to a lesser extent and more typically after primary challenge. Na?ve sheep of both genotypes produced relatively little antibody response after primary challenge. A second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for adult antigen-specific IgA and larval antigen-specific IgG1. In na?ve S line sheep, the larval-specific IgA and IgG2 response appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-gamma gene expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total IgE in lymph plasma.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for detection of Taenia saginata cysticercosis in cattle.

Serum IgG response of cattle with cysticercosis caused by Taenia saginata was studied in an enzyme-linked immunosorbent assay (ELISA) where a T. saginata metacestode surface extract was used as antigen. In experimentally infected calves, a sharp rise in specific antibody levels was found 3-4 weeks after the infection followed by a logical level of detection corresponded to about 25 cysts. The ELISA was employed in cattle herds where cysticercosis outbreaks had occurred and also in supposedly uninfected herds. Significantly increased antibody levels were found in the herds with massive cysticercosis cases. The test was not adapted for individual diagnosis as some animals of the uninfected herds, especially within the older age groups, had elevated antibody values. The ELISA was, however, useful in the investigation of outbreaks to determine the extent and pattern of the infection in the herd. The rate of decline in antibody levels in these herds was studied by follow up sampling. The increased antibody levels in the infected herds were also reflected in colostrum-fed calves. This observation was employed to estimate the time of infection.     

Isotype- and subclass-specific responses to infection and reinfection with parainfluenza-3 virus: comparison of the diagnostic potential of ELISAs detecting seroconversion and specific IgM and IgA.

Isotype- and subclass-specific indirect enzyme-linked immunosorbent assays were developed to detect parainfluenza-3 virus-specific IgG1, IgG2, IgM, and IgA responses. Sera were treated with protein G-agarose prior to testing for specific IgM and IgA to eliminate the possibility of false-positive results due to IgM-rheumatoid factor and to remove interisotypic competition due to specific IgG. IgM and IgA absorbance values were expressed as a percentage of the absorbance values of positive reference sera included on each plate (S/P%), and respective positive/negative threshold values of 15.0% and 28.0% were determined. The mean interval between experimental infection of 3 calves and initial detection of specific IgG1 and IgG2 responses was 8.0 and 9.3 days respectively, rising rapidly to an initial plateau 13.7 and 11.0 days postinfection (dpi). Reinfection of these calves at 30 dpi resulted in further rapid increases, with higher plateau values reached 13.0 (IgG1) and 13.7 (IgG2) days later. The mean interval between infection and the first positive IgM and IgA responses was 6.7 and 12.3 days, respectively. IgM S/P% values peaked at 13.0 dpi, with all 3 calves showing a secondary anamnestic response to reinfection, peaking 4.7 days later. The IgA response to initial infection was weak, with only 2 calves showing an obvious peak response at 15.0 dpi. A strong anamnestic IgA response to reinfection occurred in 2 calves, with a peak response 9.5 days later. Apparent biphasic and triphasic IgM and IgA responses were evident in some calves. Acute and convalescent serum samples from 80 calves involved in 17 outbreaks of respiratory disease were tested for specific IgM and IgA. Positive IgM results were detected in 15 outbreaks, with 71 sera from 44 calves testing positive. Although IgA-positive results were detected in the same 15 outbreaks, only 42 sera from 31 calves were positive. In a previous study, seroconversion was detected in 21 of these calves from 10 outbreaks. Thus the diagnostic potential of the assays was in the order IgM > IgA > seroconversion. The correlations between IgM and IgA, IgM and seroconversion, and IgA and seroconversion results for each calf were 73.8%, 58.8% and 62.5%, respectively.