Field evaluation of the protective efficacy of Mycobacterium bovis BCG vaccine against bovine tuberculosis

The protective efficacy of Mycobacterium bovis BCG (1 × 10⁶ single dose) was evaluated under field conditions. A total of 140 male Holstein Friesian calves, one to two week-old were selected. Two groups of 70 each were formed, one group was vaccinated and the other was injected with a placebo during their second week of age and followed until 12 months of age. The study considered a positive case of tuberculosis to be an animal that had a positive reaction to the three following tests in a row: tuberculin, IFNγ PPD-B and IFNγ ESAT6-CFP10 during the 12 months of exposure. The results showed a 59.4% efficacy (IC95%: 47.64-71.16). The non-vaccinated calves were 2.4 times more at risk of becoming infected (IC95%: 1.07-5.68) compared to vaccinated animals. As a complementary test a PCR test was performed using nasal exudates in some animals from both groups using a Mycobacterium complex detection kit. All the positive PCR reactions (5/44) were found in the non-vaccinated animals. These findings suggest that the use of the BCG vaccine, even though it is not capable of protecting 100%, does prevent TB vaccinated animals from excreting bacilli in their nasal secretions at their first year of age.     

Assessment of antibody avidity in aborting cattle by a somatic Neospora caninum tachyzoite antigen IgG avidity ELISA

A Neospora caninum IgG avidity ELISA was carried out on the basis of a somatic N. caninum tachyzoite antigen. The test was validated using experimentally infected calves, where a clear maturation of the IgG avidity over time could be demonstrated. At a maximum of 82 days after infection (d.p.i.), all animals showed antibody avidities ranging above 35%, and respective sera were thus defined as highly avid.Sera of 103 naturally infected seropositive cows with abortion (N. caninum association was provided by a N. caninum PCR-positivity of the fetus in 40 cases) and 139 seropositive animals without abortion history were concurrently examined. Significantly lower avidities were observed in aborting cows when compared to animals without abortion problems (P<0.01). While the avidity of sera collected before abortion remained practically constant until abortion, a significant increase of avidity could be observed in samples collected weeks to months after abortion (P<0.01).The avidities of non-aborting animals from farms with or without abortion problems did not differ significantly with time and were mainly located in the high avidity area. These data indicate that low avidities are not necessarily linked to recent N. caninum infection but can also be an indicator for increased abortion risk in cattle.     

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-gamma production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450+/-252), peaked at week 23 (mean 16,000+/-6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467+/-2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 (P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-gamma in similar concentrations to those of infected animals (P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers > or = 200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.     

Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.     

First report of the use of mucosal swabs of the palatine tonsillar crypt for detection of Mycoplasma bovis in naturally infected calves

ABSTRACT

Aims: To compare detection by real-time PCR of DNA from Mycoplasma bovis on mucosal swabs taken from the palatine tonsillar crypt and the mainstem bronchi of clinically asymptomatic calves after slaughter.

Methods: We compared the sensitivity of mucosal swabs taken from two sites: the palatine tonsillar crypt and the mainstem bronchi. Paired samples were taken post-mortem at slaughter from 55 clinically well calves from an infected herd and were tested by real-time PCR for the presence of M. bovis-specific DNA.

Results: Mycoplasma bovis DNA was detected in 51 palatine tonsillar crypt swabs (92.7 (95% CI?=?82.4–98.0)%) and seven mainstem bronchial swabs (12.7 (95% CI?=?5.3–24.5)%). All seven calves with positive mainstem bronchial swabs also had positive palatine tonsillar crypt swabs.

Conclusions: When compared to mucosal swabs of the mainstem bronchi, mucosal swabs of the palatine tonsillar crypt were seven times more sensitive for the post-mortem detection of M. bovis DNA. The viability of detected M. bovis was not assessed, because any cattle carrying viable or non-viable M. bovis DNA were determined to be a potential risk to eradication. Palatine tonsillar crypt mucosa may be a useful anatomical site for real-time PCR detection of M. bovis DNA in naturally infected calves. More work is needed to define the persistence and viability of M. bovis at this anatomical site.

Clinical relevance: The results of this study helped form the basis of surveillance tools used in M. bovis control and eradication efforts. Familiarity with these results may help veterinarians better communicate with their clients about the science behind the eradication efforts.     

Histopathological findings,phenotyping of inflammatory cells,and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067

Background

The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.

Results

The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.

Conclusions

The results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.     

Response of Bali cattle (Bos javanicus) to vaccination with Brucella abortus strain 19 in West Timor

A trial was conducted in two villages (one containing cattle infected with brucellosis and one not containing infected cattle) in Timor, Indonesia to determine the serological response to vaccination with Brucella abortus strain 19 in Bali cattle (Bos javanicus) (n = 599). Mature female cattle were immunised with low-dose strain 19 (2x10(8)-6x10(8) colony forming units) and calves (6-12 months) with high-dose strain 19 (4x10(10)-12x10(10) colony forming units). Other mature females and calves were inoculated with sterile vaccine diluent and formed a non-vaccinated in-contact control group. The seroprevalence and mean titres were highest in the vaccinated cattle 3 months after vaccination. These then receded, however, 1% of vaccinated calves and 1.9% of vaccinated cows from the village without infected cattle were still seropositive on the complement-fixation test (CFT) 24 months after vaccination. Non-vaccinated seropositive animals were more likely to have aborted or had a stillbirth and were less likely to have produced a calf than were seronegative cows from the village containing infected animals. We concluded that strain 19 vaccine induced protection in Bali cattle and that this vaccine might play an important role in the control of bovine brucellosis in Timor.     

Changes in plasma fibrinogen levels in experimental Mycoplasma bovis arthritis in calves

Plasma fibrinogen levels were measured as a means of following the course of an intravenous and intraperitoneal challenge of vaccinated and non-vaccinated animals in an experimental Mycoplasma bovis arthritis in calves. Intraperitoneal challenge failed to induce as much elevation of fibrinogen concentration as intravenous challenge in both the vaccinated and non-vaccinated groups.The elevation of fibrinogen levels among the vaccinated calves remained within the normal range of 300–800 mg% throughout, irrespective of the route of challenge. In contrast, the level rose to over 1600 mg% ten days postchallenge in all but one of the non-vaccinated calves that were challenged intravenously. The relatively low plasma fibrinogen levels in non-vaccinated calves that were challenged intraperitoneally correlated with the absence of arthritis in this group. In general, there was an inverse correlation between high fibrinogen levels and protection from M. bovis arthritis.     

Immune response of calves following the inoculation of Mycoplasma dispar and Mycoplasma bovis

A small but significant reduction in the number of Mycoplasma dispar colonising the respiratory tract after intratracheal challenge was observed in gnotobiotic-calves previously inoculated subcutaneously three times with formalin-killed organisms and oil adjuvant. Injection of M. dispar by the intramuscular route, with oil adjuvant, and 2 weeks later by the intratracheal route, without adjuvant, failed to induce immunity to subsequent intratracheal challenge.Following the subcutaneous injection of killed M. dispar, the amount of antibody detected by single radial haemolysis (SRH) increased markedly with increasing age in groups of calves with average ages of 16 to 155 days when first injected. Most calves aged less than 40 days failed to produce an antibody response to a singel injection of M. dispar. With M. bovis a smaller difference was observed between antibody levels generated in calves of different ages; also, calves less than 40 days old produced a detectable SRH antibody response following a single injection of killed M. bovis.IgG1 and IgG2 antibody to M. dispar and M. bovis were measured by ELISA. IgG1 appeared before IgG2 antibody and this was particularly pronounced in younger calves. Also, for both mycoplasmas IgG2 antibody levels were lower in younger than older calves. The IgG1 response to M. dispar was compared in three groups of calves with average ages of 16, 55 and 155 days and was greatest in the oldest and least in the youngest animals. In contrast, the IgG1 response to M. bovis varied little in calves of different ages. It therefore appears that the immune response of young calves to M. dispar is impaired or defective.     

Larval migration inhibition activity in abomasal mucus and serum from calves infected with Ostertagia ostertagi

The present study investigated whether abomasal mucus from calves naturally infected with gastrointestinal nematodes possessed larval migration inhibition (LMI) activity in vitro, and whether LMI activity was greater in mucus from previously immunised animals, compared to primary infected and uninfected calves. LMI activity was also assessed in serum from calves during both natural and artificial Ostertagia ostertagi infections, in an attempt to monitor the development of acquired immunity. Both abomasal mucus and serum exhibited larval paralysing activity. Although the LMI capacity of the abomasal mucus was very variable, the highest paralysing activity was consistently observed in mucus from previously immunised calves. LMI activity in serum increased significantly during both artificial and natural Ostertagia infections. After a challenge infection, sera from immunised animals showed a significantly higher LMI capacity, compared to previously uninfected calves. Moreover, serum LMI activity was significantly negatively correlated with Ostertagia worm counts after the challenge infection. The present results suggest that LMI activity in serum and/or abomasal mucus reflects a protective immune response against O. ostertagi in the abomasal mucosa.     

Evaluation of single and comparative intradermal tuberculin tests for tuberculosis eradication in caprine flocks in Castilla y León (Spain)

Goats can act as reservoirs for tuberculosis (TB) infection. The main etiological agents of TB in goats are Mycobacterium caprae and Mycobacterium bovis and they infect also a wide range of domestic and wild animals and humans. Control programmes based mainly on the application of single and comparative intradermal tuberculin (SIT and SCIT respectively) tests are being implemented in certain regions of Spain with a high density of caprine flocks as Castilla y León, including goats with epidemiological relationship with cattle. The aim of this study was to evaluate the performance of the intradermal tests in naturally TB-infected caprine flocks from this region. The study was performed using data from 17,450 goats in 54 different flocks that were classified as TB-infected in the control programmes executed in 2010 and 2011. Data from 1237 goats from 7 dairy flocks depopulated after the first intradermal testing were used to estimate the sensitivity (Se) using bacteriology as the gold-standard. Overall Se of the SIT test using the severe interpretation was 43.9% (CI 95%, 40.4–47.4) and decreased to 38.8% (CI 95%, 35.5–42.3) using the standard interpretation. Overall Se of the SCIT test ranged between 21.3% (CI 95%, 17.6–25.4) and 7% (CI 95%, 4.9–9.8) depending of the interpretation criteria. A significant weak positive correlation was found between age and skin fold thickness (Spearman’s test p < 0.05). Results from this study yielded, in general, low Se values probably due the systematic detection and slaughter of reactors as a consequence of the eradication programme in previous years or the presence of factors that may interfere in the diagnosis. Therefore, these results suggest the necessity of including ancillary diagnostic tools and/or strict interpretation criteria to maximize detection of positive animals in infected settings.     

Use of avidity enzyme-linked immunosorbent assay and avidity Western blot to discriminate between acute and chronic Neospora caninum infection in cattle.

Avidity serological tests (avidity enzyme-linked immunosorbent assay [ELISA] and avidity Western blot) were developed and used to differentiate between acute (primary infection, reinfection, and recrudescence) and chronic Neospora caninum infection in cattle. In addition, the pattern of immunoglobulin G (IgG) avidity maturation against different specific antigens of N. caninum tachyzoites was studied. Sequential serum samples were collected from cattle naturally and experimentally infected with N. caninum. Four groups of experimentally infected cattle were included in the study and were representative of primary infection, reinfection, chronic infection, and noninfection. Serum samples were also collected from naturally infected cattle classified into nonaborting and aborting cows on the basis of clinical findings and serological profiles, and a third group composed of seronegative cows that seroconverted during the course of the experiment. All samples were tested by avidity ELISA and avidity Western blot. The IgG avidity ELISA allowed the discrimination between primary and chronic infection because all experimentally primary-infection cows showed low avidity indexes at week 4 postinfection (p.i.) compared with the high avidity values found at week 20 postinfection. However, this test did not allow the discrimination of reinfection or recrudescence from chronic infection. Regarding IgG avidity Western blot results, no antigenic markers correlating with acute (primary infection, recrudescence, and reinfection) or chronic infection were recognized. However, the 17-kD immunodominant antigen was mostly responsible for high avidity values obtained by avidity ELISA because it was intensively recognized by high-avidity antibodies in all chronically infected animals after urea treatment.     

Effect of vaccination of cattle with the low virulence Nc-Spain 1H isolate of Neospora caninum against a heterologous challenge in early and mid-gestation

Live vaccines have emerged as one of the most potentially cost-effective measures for the control of bovine neosporosis. Previous studies have shown that Nc-Spain 1H is a naturally attenuated isolate of Neospora caninum and that immunisation with live Nc-Spain 1H tachyzoites generated a protective immune response in mice. The aim of this study was to evaluate the safety and efficacy of immunisation in cattle. N. caninum-seronegative heifers were immunised subcutaneously twice with 10⁷ live Nc-Spain 1H tachyzoites prior to artificial insemination. No adverse reactions or negative effects on reproductive parameters were recorded following immunisation. In immunised and non-challenged heifers, no foetal deaths were observed, and none of the calves was congenitally infected. The efficacy against N. caninum-associated foetal death and vertical transmission was determined after challenge with high doses of the Nc-1 isolate at 70 and 135 days of gestation, respectively. After the challenge in early gestation, the immunisation induced a protection of 50% against foetal death. In addition, the microsatellite analysis performed in PCR-positive tissue samples from foetuses that died after challenge infection showed that the profiles corresponded to the challenge isolate Nc-1. A degree of protection against vertical transmission was observed after challenge at mid-gestation; calves from immunised heifers showed significantly lower pre-colostral Neospora-specific antibody titres than calves from the non-immunised/challenge group (P < 0.05). Strong antibody and interferon gamma responses were induced in the immunised heifers. This study indicates that the immunisation before pregnancy with the Nc-Spain 1H vaccine isolate appeared to be safe and reduced the occurrence of N. caninum-associated abortion and vertical transmission in experimentally infected cattle. In light of these encouraging results, the next step for testing this live attenuated candidate should be the assessment of its efficacy and safety in naturally infected cattle.     

Evaluation of diagnostic tests for Johne's disease in young cattle

OBJECTIVE: To investigate the development of immune responses in calves experimentally and naturally infected with Mycobacterium paratuberculosis and to evaluate the potential for diagnostic tests to detect infected calves. DESIGN: Sequential testing of four treatment groups of calves over a 2 year period. PROCEDURE: Twenty-nine calves were allocated to four groups. Group D calves were orally dosed with M paratuberculosis, group N calves naturally exposed to M paratuberculosis, group V calves vaccinated for M paratuberculosis, and group C were control calves (not infected or vaccinated). Blood and faecal specimens were collected from each calf at monthly intervals to 18 months of age and then every 2 months until they were slaughtered between the ages of 21 and 29 months. Specimens were tested using absorbed EIA, IFN-gamma EIA and faecal culture. The infection status of the calves was confirmed by extensive histopathological examination and tissue culture. RESULTS: M paratuberculosis infection was confirmed in 10 calves, comprising six of eight orally dosed calves, three of five naturally exposed calves and one of nine vaccinated calves. The six artificially infected calves and one naturally infected calf were detected shedding M paratuberculosis in their faeces. Results with positive absorbed EIA were obtained from one artificially infected calf, one naturally infected calf and three vaccinated calves. All calves including controls had positive results on at least one occasion using the IFN-gamma EIA. In addition, seven calves had positive bovine tuberculosis results using the IFN-gamma EIA, even though bovine tuberculosis has been eradicated from Australia. CONCLUSION: Detection of M paratuberculosis infection in young cattle continues to be difficult using current tests.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

Immunoprophylaxis of experimental Mycoplasma bovis arthritis in calves. Protective efficacy of live organisms and formalinized vaccines

Live Mycoplasma bovis (M. bovis) organisms given subcutaneously or intraperitoneally protected nine of ten calves and eight of nine calves, respectively, from clinical arthritis, while the formalinized vaccine given subcutaneously protected eight of ten calves. In contrast, clinical arthritis was induced in all non-vaccinated calves that were challenged intravenously. The arthritic lesion was more severe in non-vaccinated calves than in the few vaccinated calves that developed clinical arthritis. Unlike formalinized vaccine, live M. bovis culture given subcutaneously provoked a local reaction at the site of injection in most calves in the form of oedematous plaques of about 7–8 cm in diameter. Results suggest that the formalinized vaccine may offer a practical approach to the control of Mycoplasma bovis arthritis in calves.     

T lymphocyte responses during early enteric Mycobacterium avium subspecies paratuberculosis infection in cattle

Johne's disease (JD) is a costly intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map), which is transmitted to perinatal calves by the fecal-oral route. Disease control efforts focus on identification and culling of infected cattle from herds; therefore failure to identify animals early is a major obstacle to reducing transmission. Development of host immunity during early JD remain incompletely characterized so detecting subclinical JD using immunologic techniques is a substantial challenge in the field. Development of a test with high sensitivity and specificity is a major research goal with significant implications for the cattle industry. The objectives of this study were to compare early Map-specific T lymphocyte responses in naive, experimentally Map infected and Map vaccinated calves using a subcutaneous matrigel biopolymer-based assay. We examined the phenotype of recruited lymphocytes and local interferon gamma (IFNγ) production within subcutaneously placed matrigel containing Map antigen 30 days after experimentally induced intestinal Map infection or Map vaccination. We show that IFNγ-secreting CD4+ T cells are recruited to matrigel sites in vaccinated but not infected or naïve calves. γδ T cells recruited to matrigel sites of Map-infected calves were mostly WC1-, while γδ T cells recruited to matrigel sites of Map-vaccinated calves were predominantly WC1+. IFNγ at matrigel sites was a discriminating factor between infected calves, naïve calves and vaccinated calves. These data contribute to our understanding of early anti-Map immunity, and may be useful for detecting early intestinal Map infections in calves or for enhancing our ability to discriminate between Map-infected and Map-vaccinated calves.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Evaluation of three serum i-ELISAs using monoclonal antibodies and protein G as peroxidase conjugate for the diagnosis of bovine brucellosis

Three i-ELISAs using LPS, the immunodominant component of Brucella abortus, were developed with three different conjugates: monoclonal antibodies 1C8 (anti-bovine IgG(1)) and 3H3 (mainly specific for bovine IgG(2) but also reacting with IgG(1)) and protein G (reacts with both bovine IgG subclasses). Using a cut-off value of 2.5U/ml, the i-ELISA with 3H3 as conjugate had a specificity (95% CI: 98.32-99.63%) that was significantly higher than the same assay with 1C8 (95% CI: 96.08-98.26%) or PG (95% CI: 95.83-98.09%). In areas where false positive serological reactions (FPSR) were common, the specificity of the i-ELISAs decreased significantly. The specificity of the i-ELISAs increased with the age of the animals tested, irrespective of the conjugate. The specificity of the i-ELISAs and traditional tests was also examined using sera from animals infected per os with bacteria bearing LPS similar to the Brucella LPS. It appeared that Yersinia enterocolitica O:9, Xanthomonas maltophilia and Salmonella urbana infections induced FPSR both in the i-ELISAs and in the traditional tests, but the 3H3 assay was significantly less prone to produce false positive reactions than the 1C8 and PG assays. The i-ELISAs were more sensitive, allowed earlier detection, and were more persistent than the traditional serological tests both in experimentally and naturally Brucella-infected animals. Weekly i-ELISA monitoring of experimentally infected pregnant heifers (previously vaccinated or not) allowed a prediction of abortion. Furthermore, the 1C8 assay showed significantly higher titres irrespective of day post-infection and vaccination status. The accuracy of the assay could be improved by making use of additional information (e.g. animal age or conjugate) and by selecting appropriate cut-off points on the basis of the prevailing epidemiological situation. The i-ELISAs appear an appropriate choice in order to maintain an official brucellosis-free status because of their sensitivity, early detection and long persistence and, for the same reasons, seem especially valuable for the detection of latent carriers (i.e. animals classified negative by classical serological tests) among imported animals.