Crossed immunoelectrophoretic analysis of the antibody response in heifers and dry cows to Peptococcus indolicus in experimental and natural heifer mastitis and dry cow mastitis

Crossed immunoelectrophoresis with intermediate gel was used to investigate the antibody response of experimentally infected heifers and of naturally infected heifers and dry cows at grass with mastitis against soluble extracellular antigens of Peptococcus indolicus. Using both precipitin titres and precipitin scores (the sum of precipitin titres against individual antigens) rising titres of antibodies against antigend of P. indolicus were demonstrated in sera from experimentally infected pregnant and non-pregnant heifers. Statistically significant differences in precipitin scores against extracellular antigens were also shown between pregnant heifers with mastitis and healthy pregnant heifers and between dry cows with mastitis and health dry cows. The biochemical nature of the principal reactive antigens was not elucidated. These serological investigations lend strong support to the pathogenic role and significance of P. indolicus in mixed infections with Corynebacterium pyogenes in the aetiology of heifer and dry cow mastitis.     

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.     

IgG response in natural and experimental infections of cattle with Fasciola gigantica

A specific IgG response was detectable in serum and bile of cattle infected with Fasciola gigantica. Although there was an increase in both specific and total IgG concentration in the sera of fluke-infected cattle, there was no significant correlation between IgG1 concentration and the worm burden (r = 0.17; P greater than 0.05) in chronic fascioliasis. However, there was a highly significant correlation between the levels of IgG1 in the serum and bile of infected cattle (r = 0.87; P less than 0.01), indicating that the presence of IgG1 in the bile may be due to a leakage of serum antibody into the bile duct.     

Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections

Sera from three groups of cats under different experimental conditions were studied by ELISA to assess the host's immune response against synthetic peptides derived from Dirofilaria immitis (Dipp) and against the surface protein of its endosymbiont, Wolbachia (WSPr). In experimentally infected cats (Group 1), an increase of IgG antibody against both Dipp and WSPr was observed from 2 months post-infection until the end of the study, 6 months post-infection. In experimentally infected cats, treated against infective larvae (Group 2), anti-Dipp IgG decreased dramatically from 4 months post-infection (3 months post treatment), showing very low values till the end of the study (6.5 months from infection, 5.5 months from treatment), while anti-WSP IgG increased constantly till the end of the study. Of 49 outdoor, asymptomatic cats exposed to a high risk of natural infection (Group 3), 9 were positive for anti-Dipp IgG and for a validated, in-clinic commercial antibody diagnostic kit for cats. Two cats were also found positive for circulating antigens of adult female worm. Anti-WSPr IgG were found in five of nine anti-Dipp IgG-positive sera and from eight ELISADipp-negative sera. Our results confirm the strong IgG response in heartworm infected cats and demonstrate the involvement of the Wolbachia endosymbiont in the immune reaction to the parasite both in experimentally infected cats and in cats exposed to a high risk of natural infection.     

Circulating antibody response to Eimeria tenella oral and subcutaneous infections in chickens

Immune responses in chickens to Eimeria tenella using oral and subcutaneous routes of infection were investigated. The results obtained indicated that sporulated oocysts inoculated subcutaneously in doses up to 50 000 oocysts per bird were not fatal to 21-day-old chicks. Subcutaneous inoculation of oocysts was found to be less immunogenic than oral administration. The dynamics of the antibody responses were different for the two routes of infection. Orally administered oocysts stimulated a dramatic primary increase in the serum antibody titre with a tendency towards a decrease in the titre 14 days post infection irrespective of second infections at that time. However, a third oral dose of oocysts stimulated a slight increase in antibody titre. Two doses of oocysts injected subcutaneously induced only a slight increase in serum antibody titre. Such a low titre was dramatically increased following a subsequent oral dose of oocysts. Antibodies specific to E. tenella are IgM and IgG immunoglobulins. IgA immunoglobulin was not investigated.     

Immunoblot analysis of IgG antibody response to Sarcoptes scabiei in swine

This study was performed to determine the frequencies and specificities of IgG antibodies binding to component of Sarcoptes scabiei extracts in swine with hypersensitive and chronic mange. The hypersensitive form is characterised by pruritus and the presence of small red papules over the flanks and belly. The chronic form is characterised by crusts, which contain large numbers of mites and are attached to the skin; the lesions are most commonly found on the internal pinna extending into the auditory canal. S. scabiei mite extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with subsequent immunoblotting. IgG-binding proteins were detected with individual sera from 30 hypersensitive and 21 chronically infected pigs; eight "Specific Pathogen Free" pigs were used as negative controls. Seven protein bands with molecular weights ranging from >220 to 30 Kilodalton (KDD) (>220, 218, 110, 80, 66, 52, 36 KDD) strongly bound with IgG antibodies; five out of these seven components (218, 110, 80, 66, 52 KDD) bound also with sera from negative pigs. There is a statistically significant difference in the antigenic recognition spectra between hypersensitive and chronically infected pigs; component of >220 KDD is more frequently recognized by chronically infected pigs (P=0.0006, chi(2)=11.74), in contrast component of 36 KDD is more frequently recognized by hypersensitive pigs (P=0.001, chi(2)=10). Our results clearly indicate there is a difference in the reactivity to antigenic peptides/proteins of S. scabiei mite between hypersensitive and chronically infected pigs, and revealed that only two antigens may be considered S. scabiei-specific and used for diagnostic purposes in swine.     

Serum IgG and IgM antibody response in cattle to antigens of Pasteurella haemolytica

The serum IgG and IgM antibody responses of 48 cattle vaccinated with live Pasteurella haemolytica (LIVE), formalin-killed P. haemolytica in Freund's incomplete adjuvant (FIA), or formalin-killed P. haemolytica in aluminum hydroxide adjuvant (ALH) to a variety of P. haemolytica antigens were evaluated. Enzyme-linked immunosorbent assays (ELISAs) were used to determine the sequential and day 21 IgG and IgM antibody responses to whole P. haemolytica (WB), a capsular carbohydrate-protein subunit (CPS) extracted from the organism, P. haemolytica capsular carbohydrate (CC), and P. haemolytica leukotoxin (LT). LIVE and FIA vaccinates developed generally higher IgG and IgM responses to all antigens compared to ALH vaccinates. LIVE vaccinates developed IgG responses to LT which were significantly higher (P less than 0.05) than all other vaccinates. In contrast, FIA vaccinates developed significantly higher IgG responses to CPS than all other vaccinates. On the basis of the ELISA results, similar or cross reacting antigenic sites were present in preparations containing surface antigens (WB, CPS and CC), but not LT. Disease resistance, as determined by experimental lesions induced in the 48 calves by transthoracic challenge with P. haemolytica, was significantly greater in the LIVE and FIA vaccinates compared with ALH vaccinates. No significant difference in resistance was detected between LIVE and FIA vaccinates. Lesions in ALH vaccinates were not significantly different than those in phosphate-buffered saline (PBS) controls. Increased IgG responses to all antigens were significantly associated with resistance to experimental disease; however, IgG responses to CPS were most highly correlated with resistance. The only IgM response which was significantly correlated with resistance was the response to CPS. These studies indicate that serum IgG antibody responses to various surface antigens of P. haemolytica, as well as LT, can enhance resistance to experimental pneumonic pasteurellosis. Serum IgM responses, however, do not appear to play a major role in resistance to experimental disease.     

Effect of damage to the teat end on the experimental induction of mastitis in dry cows with Corynebacterium pyogenes

The teat ends of 12 dry cows were contaminated with Corynebacterium pyogenes. To determine whether a pre-existing (an)aerobic bacterial infection of the udder was a predisposing factor for a C pyogenes mastitis they included infected and uninfected quarters. Anaerobic bacteria could not be found and mastitis was not induced. When the teats were contaminated with C pyogenes after the teat ends had been injured 30 of the quarters became infected, and anaerobic bacteria were demonstrated in many quarters.     

Serum antibody response to Mycoplasma hyopneumoniae measured by enzyme-linked immunosorbent assay after experimental and natural infection of pigs

Studies were conducted under experimental and field conditions to determine the effect of infection with M. hyopneumoniae on the immune response in serum as measured by ELISA. Following intratracheal challenge or contact exposure, serologically negative pigs derived from mycoplasma-free piggeries developed an immune response within 10 days. This response continued to rise for a further 50 days. In a field study in a commercial piggery, no animals (0/44) were observed to have M. hyopneumoniae antibodies at day 86 of life. However between day 86 and day 144, 97.7% (42/43) animals sero-converted. These results are discussed in terms of infection spread, particularly in the grower/finisher shed.     

Comparison of adjuvants with respect to serum IgG antibody response in orally immunized chickens

We have previously shown that oral immunization with non-replicating antigens hardly induced serum IgG antibody response in chickens and addition of sodium fluoride (NaF) to the immunogen markedly improved their immunological states. In the present study, taurine, lithium and Quillaja saponin (Q-SAP) were compared with NaF with respect to their enhancement of serum IgG antibody response in chickens after oral immunization. The antibody titer of chickens which received Q-SAP as the mucosal adjuvant tended to be higher than that of chickens which received antigen plus NaF. Simultaneous administration of antigen with lithium or taurine elicited a higher antibody titer in chickens compared to those of chickens orally immunized with antigen alone, but the effect of these two adjuvants was less efficient compared with that of NaF. These results suggested that Q-SAP as well as NaF is useful as an oral adjuvant for chickens.     

The specificity of antibody response in experimental and natural bovine paratuberculosis studied by crossed immunoelectrophoresis with intermediate gel

The sonicate antigen (MPS) of a local strain (IVRI) of Mycobacterium paratuberculosis and a commercial lysate of Strain 18 were analysed using hyperimmune rabbit and calf antisera to MPS in crossed immunoelectrophoresis with intermediate gel (CIE-ig) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The rabbit antiserum was more potent than the calf antiserum and it precipitated 35 and 15 antigens, respectively, among MPS and lysate antigens. SDS-PAGE resolved 50 and 32 peptides among these antigens respectively, of which, 35 and 15 were precipitated by rabbit antiserum. A CIE-ig reference system, with 30 MPS antigens, was standardized and used to analyse antibody specificities among sera derived from animals experimentally and naturally infected with bovine paratuberculosis. Fourteen antigens of MPS were found to be reactive with these sera and among these, Antigens 2 and 5 were found to be serodominant; sonicate antigens of M. bovis BCG and M. avium did not contain these antigens. Both were high molecular weight (greater than 60 kDa) antigens which may be of serodiagnostic value.     

Viremia, virus shedding, and antibody response during natural avian polyomavirus infection in parrots

OBJECTIVE: To determine rapidity of spread and onset and duration of viremia, virus shedding, and antibody production in parrots naturally infected with avian polyomavirus (APV). DESIGN: Case series. ANIMALS: 92 parrots in 2 aviaries. PROCEDURE: Blood samples were obtained from parrots naturally exposed to APV during a 3- to 4-month period for determination of serum virus neutralizing antibody and detection of viral DNA. Nestlings from the next year's hatch were monitored for APV infection. RESULTS: The first indication of inapparent infection was viremia, which developed simultaneously with or was followed within 1 week by cloacal virus shedding and antibody production. Cloacal virus shedding continued after viremia ceased. During viremia, viral DNA was detected continuously in blood samples. Viral DNA was detected in serial cloacal swab specimens in most birds, but it was detected inconsistently in 6 birds and not detected in 3 birds, even though these birds were viremic. Duration of cloacal virus shedding was < or = 4.5 months. In 1 aviary, prevalence of infection was 88% and dissemination of virus through the 3-room building required 4.5 months. In the second aviary, a single-room nursery, prevalence of infection was > or = 90%. For all affected birds, infection could be detected 18 days after the first death. CONCLUSIONS AND CLINICAL RELEVANCE: If a single sampling is used for polymerase chain reaction detection of viral DNA, blood and cloacal swab specimens are required. In nestling nonbudgerigar parrots, cloacal virus shedding may persist for 4.5 months. Management protocols alone are sufficient to prevent introduction of APV into a nursery.     

Specific binding of bovine, ovine, caprine and equine IgG subclasses to defined types of immunoglobulin receptors in gram-positive cocci

One hundred and twenty bacterial strains were tested for non-immune binding of radiolabelled bovine, ovine, caprine and equine immunoglobulins. Bacteria possessing previously defined IgG receptors interacted in a well defined manner with purified IgG subclass immunoglobulins. Human group C and G streptococci carrying IgG receptors type III were capable of binding all IgG subclasses in the four mammalian species studied. Protein A-containing staphylococci demonstrated a restricted specificity with binding of bovine IgG1, ovine IgG1, caprine IgG1 and IgG2 as well as equine IgG(ab). Group A streptococci which can bind human IgG did not show specific reactivity. A new type of binding unrelated to the regular Fc-mediated binding was observed with equine IgG(T).The differences in specificity for IgG subclasses suggest that structures with binding capacity to streptococcal type III Fc receptors are different from staphylococcal protein A reactive sites. Inhibition experiments performed with purified immunoglobulins showed that individual IgG subclasses differed greatly in their inhibiting capacity reflecting differences in avidity.The high avidity and the broad, unrestricted immunoglobulin G reactivity of streptococcal IgG receptor type III indicate that human group C and G streptococci may provide a valuable tool for solid phase absorption of immunoglobulins from several mammalian species.     

Analysis of IgG response to experimental infection with RH Toxoplasma gondii in goats.

The IgG response of goats experimentally infected with RH Toxoplasma gondii has been analysed using an indirect ELISA and Western-blot analysis. Specific IgG antibodies were first detected at 14 days post-inoculation (p.i.), reaching a peak by day 35 p.i. and showing slight fluctuations until the end of the experiment (91 p.i.). Specific IgG showed a reactivity over a whole range of peptides (125-24 kDa approximately), but the highest reactivity was observed against a group of antigens with a molecular weight between 34 and 28 kDa, in particular against a 30 kDa fraction which is considered to represent the major surface protein of T. gondii named p30 or SAG-1.     

Immunologic and pathologic responses of cows to naturally occurring Mycoplasma bovis mastitis

Systemic humoral and cell-mediated immune responses of four Holstein cows with natural Mycoplasma bovis mastitis were evaluated to determine whether a relationship exists between systemic cellular and humoral responses and the pathogenesis and resolution of infection. In vitro lymphocyte activation tests of peripheral blood lymphocytes and in vivo skin tests with M. bovis antigens provided evidence that cell-mediated immune responses against M. bovis may be involved in successful resolution or containment of infection. In several observation it appeared that viable M. bovis and their aqueous extracts are suppressive to cell-mediated responses.Humoral responses were determined by the serum indirect hemagglutination (IHA) test and the growth inhibition test. The IHA titers after approximately 2 weeks of infection were elevated; however, 75–87% of the IHA activity was in the IgM antibody class.The cell-mediated immune responses may be necessary for resolution of mycoplasmal mastitis both directly and via their helper cell function on antibody production. However, it appears that immune injury to mammary tissue results from the immunologic response to infective mycoplasma. Presence of locally secreted antibody and locally active immune cells may provide a better indication of those animals in the process of resolving the infection than was observed using systemic indicators of immune responsiveness such as indirect hemagglutination or growth inhibition tests.     

Antigen-specific IgG(T) responses in natural and experimental cyathostominae infection in horses

Equine clinical larval cyathostominosis is caused by simultaneous mass emergence of previously inhibited larvae from the mucosa of the colon. Clinical signs include diarrhoea, colic, weight loss and malaise, and in up to 50% of cases, the disease results in death. Cyathostominae spend a large part of their life cycle as larval stages in the intestinal mucosa. Definitive diagnosis is difficult due to the lack of diagnostic methods for pre-patent infection. In the present study, the enzyme-linked immunosorbent assay (ELISA) was used to investigate isotype responses to larval cyathostominae somatic antigen. Measurement of anti-larval IgG(T) responses appeared to have the most immunodiagnostic potential. An increase in IgG(T) response was detected to crude larval antigen by 5 weeks post-infection (PI) in individual infected ponies. Subsequently, IgG(T) responses to larval and adult somatic extracts were examined by Western blotting using sera from experimentally-infected horses and helminth-naive animals (n=6). Two antigen complexes, designated A and B, in larval somatic antigen were recognised specifically by the infected animals by 7 weeks PI. Sera taken from 23 endemically-infected animals, whose cyathostominae burdens had been enumerated, were also used to identify putative diagnostic antigens. Eighteen horses had positive mucosal worm burdens (range 723-3,595,725) and all but two of these animals had serum IgG(T) antibody specific to either complex. Moreover, IgG(T) responses specific to antigen complexes A and B were absent in all five parasite negative horses that were tested. Serum IgG(T) responses to either of the two complexes were identified in five clinical cases tested. IgG(T) responses to adult antigen somatic extracts were more heterogeneous, with no clear pattern between experimentally-infected ponies and helminth-free controls. The results indicate that increases in serum IgG(T) to mucosal larvae occur in the pre-patent period and that two antigenic complexes within somatic preparations of these stages have immunodiagnostic potential.     

Diminished antibody response to rinderpest vaccination in cattle undergoing experimental East Coast fever.

The immunocompetence of cattle undergoing East Coast Fever (ECF) reactions of varying degrees of intensity was evaluated using the neutralising antibody response of these cattle to the vaccine response was found in animals undergoing severe ECF reactions. The results suggest that the massive lymphoid cell involvement of such severe ECF reactions diminishes the immune responsiveness of cattle to rinderpest vaccine.     

Onchocerca ochengi acquisition in zebu Gudali cattle exposed to natural transmission: parasite population dynamics and IgG antibody subclass responses to Ov10/Ov11 recombinant antigens

Ngaoundere Gudali zebu cattle naturally exposed to Simulium damnosum s.l. and Culicoides spp. bites were examined during 4 years for O. ochengi adult worm acquisition, Onchocerca ochengi and Onchocerca gutturosa skin microfilaria dynamics, and IgG1 and IgG2 antibody subclass responses. Eleven animals acquired a total of 465 O. ochengi nodules (average of 17 per female and 72 per male). The O. ochengi nodule load was highly variable in individual animals and exacerbated in mature male cattle. Three patterns of acquisition of O. ochengi (resistant to new infestation, early susceptibility and late susceptibility), not associated with Simulium biting intensity (P > 0.05), were distinguished. The minimum prepatent periods for O. ochengi nodules, O. ochengi microfilariae and O. gutturosa microfilariae were 10, 20 and 21 months, respectively. The O. ochengi microfilaria density significantly (P < 0.001) increased with age, was higher in young mature bulls than female animals (P < 0.001) and finally reached highest levels (P < 0.005) during the dry season. Antibody responses to Ov10/Ov11 recombinant O. volvulus antigens were predominantly of the IgG1 subclass. High levels of this subclass (not IgG2) observed in new born calves declined to almost zero levels at the age of 5-8 months but IgG1 levels significantly increased (P < 0.05) with age subsequently during patency. Put together the acquisition and accumulation of O. ochengi parasites in zebu cattle, apart from being season, sex (gender) and host age associated, may also suggest a density-dependent regulation of parasite establishment in a proportion of the exposed population.     

Comparison of serological tests for the detection of antibody to natural and experimental murine cytomegalovirus.

Three serological tests, i.e. complement fixation test, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA) were compared for sensitivity in the detection and titration of murine cytomegalovirus antibody. The three tests were compared using sera from experimentally inoculated and naturally infected mice bled at intervals from 3 to 140 days postinfection. In the acute infection, complement fixation and indirect immunofluorescent assay tests were of comparable sensitivity for early detection of antibody, whereas the ELISA was less sensitive. In persistent infection, higher titers were recorded with ELISA. Since murine cytomegalovirus has been shown to exert significant effects on the immune response of infected mice, this antigen should be included routinely in viral antibody screening programs.