Differential antibody responses to Corynebacterium pseudotuberculosis in sheep with naturally acquired caseous lymphadenitis

Enzyme-linked immunosorbent assays (ELISA) with Corynebacterium pseudotuberculosis cell wall and bacteria-free supernatant with exotoxin preparations as antigens, and hemolysis inhibition tests were used to detect antibodies in the sera of adult range sheep with naturally acquired caseous lymphadenitis (CL). The extent and severity of lesions were quantitated on the basis of a lesion score, derived from an examination of the carcass (peripheral lymphoid tissue) and viscera (including internal lymphoid tissue) at the time of slaughter. The overall prevalence of C pseudotuberculosis-positive CL lesions in 104 sheep was 31.7%. The cell wall ELISA detected antibodies in 96.9% (32/33) of sheep with C pseudotuberculosis-positive CL lesions. The exotoxin ELISA detected antibodies in 84.8% (28/33) of positive sheep in the same group. Both ELISA resulted in a high number of apparent false-positives, with 64.7% and 49.2%, respectively, positive optical density (OD) values in sheep with no gross CL lesions and no apparent C pseudotuberculosis infection. There was no significant relationship between the extent of lesion development (lesion score) and OD values in both cell wall (r = 0.472) and exotoxin (r = 0.464) ELISA. Similarly, there was no significant relationship between the titer of antitoxin antibodies, as measured by the hemolysis inhibition test, and the extent of disease. These investigations indicate that those ELISA that use crude C pseudotuberculosis antigens are of questionable utility in the field, where C pseudotuberculosis infection is endemic in many sheep populations. Furthermore, these studies suggest that antibodies that are reactive with components of C pseudotuberculosis and that develop in response to infection may have little impact on the recovery of the host.     

A comparison of four serological tests for the diagnosis of caseous lymphadenitis in sheep and goats

A double antibody sandwich ELISA (ELISA A) developed for the detection of Corynebacterium pseudotuberculosis infection in sheep and goats was modified to improve its sensitivity. To establish the sensitivity and specificity of this modified ELISA (ELISA B), sera from 183 sheep and 186 goats were tested using ELISAs A and B. Comparison was also made with two further ELISAs (C and D) developed in Australia that, respectively, detect antibodies to cell wall antigens or toxin.ELISA B had the best performance of the four tests. Its specificity was 98+/-1% for goats and 99+/-1% sheep. Its sensitivity was 94+/-3% for goats and 79+/-5% for sheep. ELISA B will now be tested for use in caseous lymphadenitis eradication and control programmes in The Netherlands. It will also be used in experimental studies of CL in Scotland.     

Serologic and virologic investigations into pestivirus infection in wild and domestic ruminants in the Pyrenees (NE Spain)

An outbreak of disease associated to a border disease virus was described in the Southern chamois (Rupicapra pyrenaica) in Spain in 2002. Sera and/or spleen samples from 57 mouflon, 15 red deer, 21 roe deer, 3 fallow deer, 55 sheep, 32 cattle, and 68 goats sharing the chamois habitat were studied. An antibody ELISA test yielded an inconclusive result in 2 mouflon and positive results in 5 goat sera. Comparative virus neutralization tests were performed on the 2 inconclusive mouflons, 3 of the 5 seropositive goats, 55 sheep and 32 cattle, using 6 pestivirus strains. Positive results were obtained in 1 mouflon, 2 goats, 69% of sheep and 78% of cattle. Virological investigations performed with an antigen ELISA test yielded negative results in 21 goats and 39 mouflons, the result in 1 mouflon being inconclusive. PCR performed on 12 goats and the inconclusive mouflon gave negative results. These results suggested that it is unlikely that chamois BDV is infecting wild and domestic ruminants.     

Comparison of infection rates of Oestrus ovis between sheep and goats kept in mixed flocks

Oestrosis is a nasal myiasis of sheep and goats caused by larvae of the fly Oestrus ovis and can lead to severe clinical signs, which together with the disturbance caused by the adult fly may result into serious economic losses. Infection rates and larval burdens are always higher in sheep than in goats after either natural or artificial infestation. The aim of this study was to compare the host preference of the adult fly O. ovis between sheep and goats in mixed flocks, where they are kept together under the same husbandry conditions and hence, are very similarly exposed to the fly preference. Blood sera samples were collected from a total of 397 sheep and 335 goats, from 43 mixed flocks located at different regions of Greece. Antibodies specific to O. ovis IgG were measured by ELISA. A flock was considered positive when at least one individual was positive, i.e. showed a seropositivity of >or=20% in relation to positive control sera. A total of 193 (48.6%) sheep and 58 (17.9%) goats were found to be seropositive against O. ovis. Thirty-eight (88.4%) out of 43 flocks had at least one seropositive animal. The mean seroconversion against O. ovis in animals from the different flocks was 38.6% and 13.6% for sheep and goats, respectively, whereas the variance of infection within each flock was 0-100%. The mean seropositivity between sheep that were found to be positive or negative was 60.6% and 5.4%, respectively, whereas the corresponding values between goats were 35.2% and 5.2%, respectively. No significant difference in the seroconversion values was noted between flocks from the different areas (P=0.817), whereas a very significant difference was observed between animal species (P=0.001). However, there was no significant difference when seroconversion comparisons were made within samples of the same animals species, sheep or goats from different flocks of all the regions included in the study (P=0.695). The results of this study clearly demonstrate that O. ovis has a widespread distribution in Greece, and the seroprevalence is significantly higher in sheep than goats (P=0.001).     

Diagnostic performance of PCR and ELISA on blood and milk samples and serological survey for small ruminant lentiviruses in central Spain

The diagnostic performance of an ELISA for the detection of antibodies to the small ruminant lentiviruses (SRLVs) maedi-visna virus and caprine arthritis-encephalitis virus in milk and corresponding blood samples was evaluated in 50 sheep. The agreement between ELISA results in blood and milk was 90 per cent, and the κ value was 0.79. In addition, a serological survey in the central zone of Spain was performed using milk samples from 413 animals (250 sheep and 163 goats) from 12 flocks/herds. All flocks/herds had some animals that were positive for SRLV. Among the animals, 60.0 per cent of the sheep and 8.0 per cent of the goats tested were seropositive. Each sample was also tested using a PCR technique, which increased the percentage of positive animals detected. Using a combination of ELISA and PCR gave a total of 72.2 per cent of sheep and 28.8 per cent of goats positive for SRLV.     

Evaluation of a modified Rose Bengal test and an indirect enzyme-linked immunosorbent assay for the diagnosis of Brucella melitensis infection in sheep

A modified Rose Bengal test (mRB) and an indirect ELISA (iELISA) with Protein G as the conjugate, were evaluated for the diagnosis of Brucella melitensis infection in unvaccinated sheep with a known bacteriological status, and their diagnostic efficacy was compared with that of the standard Rose Bengal (RB) and Complement Fixation (CF) tests used in the current eradication campaign in EU countries. All tests showed 100% specificity when testing the sera from 212 Brucella-free sheep. When testing the sera from 219 Brucella melitensis culture-positive sheep, both the mRB and iELISA tests were more sensitive (98.6% and 96.8%, respectively) than the RB and CF tests (95.0% and 92.7%, respectively). These results were similar when testing the sera from 181 animals belonging to infected flocks but found bacteriologically negative, suggesting that the mRB or iELISA tests could advantageously replace the current RB procedure used as the screening test.     

Synergistic hemolysis-inhibition titers associated with caseous lymphadenitis in a slaughterhouse survey of goats and sheep in Northeastern Brazil

A survey of caseous lymphadenitis was conducted at a goat and sheep slaughterhouse in Northeastern Brazil One hundred and fifty-eight goats and 43 sheep were examined for the presence of abscesses, with bacterial culturing of purulent material to define the etiological agent. Blood was collected simultaneously for determination of serological titer via the synergistic hemolysis-inhibition test which measures antibodies to an exotoxin of Corynebacterium pseudotuberculosis. Thirteen and nine-tenths percent of the goats had abscesses, with a high proportion having mediastinal or pulmonary lesions (9.5%). Two sheep had abscesses, both with internal organ involvement. Corynebacterium pseudotuberculosis was the most frequently isolated organism. Of 22 goats with abscesses, 20 were positive via the synergistic hemolysis-inhibition test. Both of the sheep with abscesses had positive synergistic hemolysis-inhibition titers. The proportion of serological reactors was greater than the proportion of animals with abscesses. The synergistic hemolysis-inhibition test may be detecting subclinically infected animals.     

The association of sub-clinical paratuberculosis with the fertility of Greek dairy ewes and goats varies with parity

Our cross-sectional study investigated the association of sub-clinical Mycobacterium avium subsp. paratuberculosis (MAP) infection with failing to produce a live offspring the season of lambing/kidding (November 2001 to January 2002) before testing (in April-May 2002), in four dairy-sheep and/or goat flocks in Greece (369 animals >or=1.5-year-old). From each selected animal 10 ml of blood and 10 g of feces from the rectum were obtained. The harvested sera were tested for antibodies to MAP with a commercial ELISA test kit; the feces were cultured on Herrold's egg-yolk medium supplemented with mycobactin J and antibiotics. An animal was considered sub-clinically infected when found either seropositive or culture positive. The true prevalence of sub-clinically infected animals, adjusted for the sensitivity and specificity of the parallel testing, was 14% (0.1-28%) and 35.9% (9.2-62.7%) in sheep and goats, respectively. The association of fertility of sheep and goats with sub-clinical paratuberculosis was investigated in random-effects logistic models. Sub-clinically infected animals (compared to uninfected) had OR for live offspring the previous year of 5.4 for parity <4, OR=0.05 for parity >6, and a non-significant OR for the middle parity category.     

羊伪结核棒状杆菌的分离鉴定及ELISA检测方法的建立

从疑似山羊干酪性淋巴结炎羊的肩前淋巴结脓肿内分离到2株菌,经鉴定均为羊伪结核棒状杆菌。利用羊伪结核棒状杆菌标准菌株(ATCC19410株)制成外毒素作为检测抗原,通过酶标抗体、阳性血清、外毒素抗原最适浓度的选择试验,确定适当的抗原、抗体和酶标抗体稀释浓度,建立了间接ELISA方法用于检测抗体。用建立的ELISA方法检测100份待检羊血清,其中阳性血清4份,阳性检出率为4％。     

Serodiagnosis of inapparent caseous lymphadenitis in goats and sheep, using the synergistic hemolysis-inhibition test

The synergistic hemolysis-inhibition (SHI) test, a serologic test for the detection of infection with Corynebacterium pseudotuberculosis, was applied to serum samples from 196 goats and 76 sheep, including animals both with and without C pseudotuberculosis abscesses. Fifty-one of 52 (98%) goats and 27 of 28 (96%) sheep with abscesses caused by C pseudotuberculosis had seropositive titers. Seropositivity continued on subsequent samplings, even after superficial lesions were completely healed. The SHI test may detect subclinically infected animals, as well as animals with clinically recognizable lesions. Of the animals without abscesses, 53 of 186 (28%) goats and 4 of 41 (10%) sheep were seropositive. Either the SHI test is lacking in specificity or these titers are a reflection of a past or a current infection without any grossly visible abscesses.     

Antibody to Pseudomonas pseudomallei exotoxin in sheep exposed to natural infection

Specific antibody to Pseudomonas pseudomallei exotoxin was detected in sheep sera exposed to natural infection. An enzyme-linked immunosorbent assay (ELISA) was used. Serum antitoxin was present in 49.3% of sera obtained from a flock of sheep naturally exposed to P. pseudomallei infection. Among these sera, 17.0% gave titers of 10,000. In contrast, serum antitoxin was present in only 6.0% of sera collected from sheep kept on a melioidosis-free farm. The ELISA reactivity of all positive sera could be completely absorbed with purified P. pseudomallei exotoxin. Similarly, preincubation of the exotoxin-coated wells with specific antiserum inhibited the ELISA reactivity of sheep sera. The results indicate that exotoxin is produced in vivo during infection by P. pseudomallei.     

Enteritis in sheep, goats and pigs due to Yersinia pseudotuberculosis infection

The features of naturally occurring Yersinia pseudotuberculosis serotype III infections in 16 sheep, one goat and 3 pigs, and Y. pseudotuberculosis serotype I infections in 3 goats, are described. Affected animals usually had diarrhoea and were in poor condition or emaciated. A number were moribund or dead when submitted for necropsy. Thickening of the caecal and colonic mucosa was the only gross lesion attributable to Y. pseudotuberculosis infection, with liver or other visceral abscesses not being seen. Characteristic microabscesses were demonstrated in the intestinal mucosa of 10 sheep, one goat and one pig infected with Y. pseudotuberculosis serotype III and one goat infected with Y. pseudotuberculosis serotype I. Sheep, goats and pigs dosed orally with Y. pseudotuberculosis serotype III, the serotype isolated most commonly from these species, developed intestinal infection. In sheep and pigs, infection was accompanied by diarrhoea. Haematological changes and specific antibodies were elicited in all 3 species in response to infection. Microabscesses were seen in the intestinal mucosa of all experimentally exposed animals. The occurrence of field cases and the results of experimental exposure confirm that Y. pseudotuberculosis serotype III is an enteropathogen of sheep, goats and pigs. The association of Y. pseudotuberculosis serotype I with lesions in a goat, indicates that this bacterium may also be a pathogen of this species. It is concluded that Y. pseudotuberculosis serotype III is an enteric pathogen of a wide range of ungulate species including cattle, buffalo, deer, antelopes, sheep, goats and pigs. Serotypes I and II, while having a more restricted host range, are probably also pathogens of ungulates and, in particular, deer, antelopes and goats.     

A simplified double immunodiffusion technique for detection of Corynebacterium ovis antitoxin

A method is described for obtaining yields of Corynebacterium ovis exotoxin sufficiently high to allow use of unconcentrated supernatant and serum as the reactants in a double immunodiffusion test. C ovis supernatant with a haemolytic titre or rabbit dermal necrosis titre of 1:16,394 or greater produced readily detectable precipitation lines with specific antitoxin in the range of concentrations commonly found in sera from cases of natural C ovis infection. When used as a diagnostic screening test, sera from 32 confirmed cases of caseous lymphadenitis in sheep and goats were positive whereas sera from 16 sheep free of caseous lymphadenitis were negative. Sera from 10 out of 16 kids which had been suckled by mothers from an affected flock of goats were positive.     

The use of a microagglutination assay for the detection of antibodies to Corynebacterium pseudotuberculosis in naturally infected sheep and goat flocks

Two goat flocks comprising 326 animals and four sheep flocks comprising 343 animals, all with a previously recognized problem of abscesses due to Corynebacterium pseudotuberculosis, were examined for the presence of abscesses and antibody titers to C. pseudotuberculosis as detected by direct microagglutination assay. In sheep there was a strong positive relationship between age and titer (p less than 0.0001). However, the relationship in goats between age and titer could not be determined due to a strong interaction between flock and age. When the relationship between abscesses and titer was examined, it was found that goats with abscesses had higher titers than those that did not (p less than 0.05), whereas there was no difference in titer between sheep with abscesses and those without (p = 0.5753). The sensitivity of the microagglutination test was poor to good for both species (52.3% for goats and 89.7% for sheep). The specificity of the test was fair to poor (64.9% for goats and 21.7% for sheep). Given a disease prevalence of 13.5% for goats and 8.5% for sheep the predictive value of the positive test was very poor (18.9% for goats and 9.6% for sheep) but the predictive value of the negative test was good to excellent (89.7% for goats and 95.8% for sheep). The poor specificity of the test and therefore the positive predictive value may be due in part to the criterion of classification of presence of disease, i.e. presence of an abscess at the time of sampling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of three enzyme-linked immunosorbent assays for serologic diagnosis of contagious agalactia in sheep.

Serologic diagnosis of ovine contagious agalactia (Mycoplasma agalactiae) with the enzyme-linked immunosorbent assay (ELISA) developed by Agence Fran?aise de Sécurité Sanitaire des Aliments (AFSSA) may produce a few false-positive (FP) and false-negative (FN) results. When the prevalence of disease is low, these erroneous results may generate problems for eradication schemes. To prevent this, 2 commercial ELISAs were compared with the AFSSA ELISA. Flocks of known status were selected and classified into 4 categories: true positive (TP), FP, true negative (TN), and FN; 20 sheep per flock were submitted for blood sampling. A flock was considered positive when at least 1 out of 20 sera was positive or 2 sera were doubtful. In the flock, the diagnostic sensitivity of the 3 kits was very good (100%), and the diagnostic specificity showed an improvement from 46% (AFSSA test) to 88% and 92% (commercial tests). Considering individual animals, very few positive ewes were detected within TN or FP flocks; the proportion of positive ewes varied greatly from one kit to another (48% to 82%) within TP flocks. The kinetics of antibody response in sheep experimentally infected with various field strains of M. agalactiae were quite similar with all 3 ELISAs. The agreement between the 3 tests, assessed using the kappa value, varied from moderate to good (respective values of 0.56, 0.61, and 0.86). The 2 commercial ELISAs showed better performances, probably because of a superior analytical sensitivity, and are a good alternative for the serodiagnosis of contagious agalactia in sheep.     

Presence of antibodies to bovine viral diarrhea-mucosal disease virus (border disease) in sheep and goat flocks in Quebec.

A seroepidemiological study of border disease was conducted in sheep and goats in various areas of Quebec. Sera of 10% of animals of selected flocks were collected and specific antibodies against bovine viral diarrhea- mucosal disease were tested by seroneutralization. Results show that 10.9% and 16% of sheep and goats respectively gave a positive reaction. The lower serological prevalence was found in sheep flocks of the Sherbrooke area (5.4%) while the highest percentage of positive sera was observed in the Quebec area (24.7%). The prevalence in goats varied according to areas (2.6 to 45.5%). No relation was observed in seropositive animals between age, sex, breed and the presence of abortions in the flocks. Our serological results indicate that border disease is probably present in Quebec sheep and goat flocks but that the clinical diagnosis of this disease is not well established.     

Seroprevalence of, and risk factors for, peste des petits ruminants in sheep and goats in Northern Jordan

Peste des petits ruminants (PPR) is an economically important disease that affect sheep and goat industry in Asia and Africa. In this study, we investigated the seroprevalence, and risk factors, of PPR in sheep and goat flocks from five different governorates (Irbid, Jarash, Ajloun, Mafraq and Zarka) located in Northern Jordan. Serum samples from 929 and 400 sheep and goats, respectively, corresponding to 122 sheep flock and 60 goats flock were collected. Seroprevalence was determined using PPR competitive ELISA. Health status and management information were collected using a semi-structured pre-tested questionnaire. The individual true prevalence of PPR in sheep and goats was 29 and 49%, respectively. The flock level true prevalence of PPR was 60 and 74% in sheep and goats, respectively. In both sheep and goat flocks, large flock size, visiting live animals market and inadequate veterinary services were identified as risk factors for PPR seropositivity. Mixed (sheep and goats) raising was identified as a risk factor for PPR seropositivity in sheep flocks only.     

Antigen specificity of antibody responses to Corynebacterium pseudotuberculosis in naturally infected sheep with caseous lymphadenitis.

Constituents of Corynebacterium pseudotuberculosis were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of sonicated whole bacterial cells and ether-extracted cells revealed more than 35 bands in silver-stained gels. SDS-PAGE analysis of concentrated culture filtrates with exotoxin activity demonstrated more than 15 bands. Sera from sheep with C. pseudotuberculosis-induced disease of variable severity were used to probe immunoblots of electrophoresed ether-extracted cells and culture filtrates. Twenty or more corynebacterial molecules, ranging in molecular weight from 20 to 112 kDa, in ether-extracted cells were recognized by antibodies in the sera of naturally exposed sheep with positive ELISA titers. These sera also recognized up to six molecules, ranging from, 20 to 68.1 kDa, on immunoblots of ammonium sulfate-concentrated culture filtrate. There was no apparent relationship between the stage of disease and the response to specific corynebacterial antigens in these animals.     

Specificity of absorbed ELISA and agar gel immuno-diffusion tests for paratuberculosis in goats with observations about use of these tests in infected goats

OBJECTIVE: To determine the specificity of serological tests that are currently used in veterinary diagnostic laboratories in Australia for detection of Mycobacterium avium subsp paratuberculosis infection in goats. DESIGN: A laboratory study. PROCEDURE: Four tests were studied, comprising AGID with M. a. paratuberculosis antigen derived from cattle isolates of caprine or bovine origin, the EMAI caprine Johne's disease absorbed ELISA and the CSL PARACHEK Johne's absorbed EIA. The specificities of AGID and ELISA for paratuberculosis (Johne's disease) were estimated after examining a panel of 1000 serum samples collected from goats in Western Australia, a region free of paratuberculosis. In addition a comparison was made of test performance in a small number of paratuberculous goats from New South Wales using sera from two archival collections. RESULTS: The specificity of the AGID tests was 100% while the specificities of the two absorbed ELISA were 99.7 to 99.8% at appropriate positive-negative cut-offs. Based on testing the small sample of sera from infected goats, the absorbed ELISA tests detected about twice as many goats with Johne's disease as the AGID. Each test detected paratuberculous animals regardless of whether infection was caused by cattle or sheep strains of M. a. paratuberculosis. CONCLUSIONS: Both ELISA and AGID tests for paratuberculosis have high specificity and can be used in a market assurance program without risk of generating large proportions of false positive test results. However, the results suggested the ELISA is more sensitive for detection of infected goats and should be used in preference to the AGID. The two formats of ELISA evaluated in this study have similar characteristics and could be used in paratuberculosis control programs for the goat industries, but further data on sensitivity would increase confidence in their application.     

Diagnosis of psoroptic sheep scab with an improved enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of specific antibodies against crude Psoroptes antigen. The diagnostic sensitivity was 93.7% in 191 sheep with clinical signs associated with mange. These animals originated from 29 flocks in which psoroptic mites were detected. All of 59 sheep infested with Psoroptes ovis were seropositive. Additionally, in 49% of 70 clinically unaffected sheep originating from P. ovis-infested flocks, specific antibodies could be detected, suggesting that asymptomatic infestations can be diagnosed by serology. The specificity of the ELISA was 96.5% as determined with 254 sheep originating from 44 flocks without clinical mange. Cross-reactivity in a low range was detected with selected sera of sheep with clinical chorioptic or forage mite infestations. Four sheep seroconverted 2 weeks after experimental P. ovis infestation, i.e. 2 weeks before clinical signs became obvious. After successful doramectin treatment of 14 sheep with naturally acquired P. ovis infestation, the ELISA values declined slowly but remained positive in seven cases beyond 17 weeks.