Experimental Corynebacterium pseudotuberculosis infection in lambs

Lambs were inoculated IV with 3.2 X 10(3) colony forming units (CFU) to 3.2 X 10(6) CFU of Corynebacterium pseudotuberculosis from a 6-hour broth culture supplemented with 0.1% sorbitan monooleate. After 28 days, multiple abscesses were observed in the lungs and lymph nodes. The number of abscesses in the lungs correlated with the inoculation dose. Two lambs given 10(5) CFU and 10(6) CFU died. Multiple abscesses occurred in other lambs given 10(6) CFU to 10(4) CFU and few abscesses occurred in lambs given 10(3) CFU. Corynebacterium pseudotuberculosis was isolated from lung abscesses, inoculation site abscesses, and lymph node abscesses, but not from normal tissues. Because this procedure consistently induced abscesses in the lungs, we believe it will be a suitable challenge system for studies on the prevention of caseous lymphadenitis in sheep.     

A seroepidemiological investigation of Corynebacterium pseudotuberculosis infection in sheep flocks in southern Norway

Corynebacterium pseudotuberculosis causes caseous lymphadenitis, a disease primarily affecting sheep and goats which occurs as a chronic infection characterized by suppurative lymphadenitis (Lovell 1959, Marsh 1965, Jubb & Kennedy 1970, Ayers 1977, Addo & Eid 1978). The disease has an insidious nature and a high prevalence, resulting in large economic losses due to condemnation of carcasses and organs at slaughter (Turner 1973).     

Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infections in goats

An ELISA was developed for the diagnosis of Corynebacterium pseudotuberculosis infections in goats. A bacterial whole cell extract was used as solid-phase antigen, and serum from a culture-positive animal served as the internal reference standard. The well-to-well and assay-to-assay variations were determined to be 12.7 and 33.0%, respectively. A cut off value was determined by parallel testing of 142 sera (112 ELISA-positive, 30 ELISA-negative) in a Western blot, and the correlation between both tests was highly significant (K=0, 93). In addition, the reliability of the ELISA for the detection of infected herds was proven in a double blind study testing 910 sera from 74 goat herds.     

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％。     

Effect of the interval between shearing and dipping on the spread of Corynebacterium pseudotuberculosis infection in sheep

OBJECTIVE: To determine whether the spread of Corynebacterium pseudotuberculosis infection to sheep in dips could be controlled by increasing the time between shearing and dipping. DESIGN: A controlled treatment trial where only the time between shearing and dipping was varied. ANIMALS AND PROCEDURE: One hundred and ninety-five sheep were found to be negative for C. pseudotuberculosis exposure by assay of CLA toxin antibody, were divided into four treatment groups. Each was shorn at either 0, 2, 4 or 8 weeks before dipping in a solution containing C. pseudotuberculosis. Blood samples were taken 6 weeks after dipping and sheep were slaughtered 12 weeks after dipping. A fifth smaller group of 14 sheep shorn 26 weeks before dipping, was also exposed to C. pseudotuberculosis and was slaughtered with the other sheep. RESULTS: The occurrence of caseous lymphadenitis abscesses did not differ between groups or with sheep shorn 26 weeks before dipping. The proportion of sheep that seroconverted to the C. pseudotuberculosis toxin and cell wall ELISA was larger in sheep dipped immediately after shearing than in sheep in the other groups. CONCLUSIONS: Delaying dipping until 8 weeks after shearing did not decrease the C. pseudotuberculosis infection rate due to dipping. Sheep dipped immediately after shearing developed higher concentrations of antibody to C. pseudotuberculosis than sheep when dipping occurred between 2 and 8 weeks and later after shearing.     

Corynebacterium pseudotuberculosis infection in goats. V. Relationship between the infection and lesions resulting from vaccination against paratuberculosis

In 2 goat herds, one infected with Gorynebacterium pseudo-tuberculosis and one free from the infection,, goats were examined for superficial swellings on the shoulder and chest. All animals in this study had been vaccinated against paratuberculosis before the age of 4 weeks. The vaccine had been applied subcutaneously behind the shoulder. Twenty-two of 40 (55 %) and 31 of 45 (69 %) goats had such lesions in the infected and non-infected herds, respectively. The difference between the herds was not significant, P > 0.05.Swellings found behind the shoulder in 19 goat carcasses derived from 4 herds in which G. pseudotuberculosis infection occurred were examined bacteriologically. No bacteria could be isolated from such lesions in 15 animals, while G. pseudotuberculosis in pure culture was isolated from 3 carcasses, and a mixed bacterial flora from the re-maining carcass. Bacteria could not be isolated from lesions situated behind the shoulder in 7 carcasses from 3 herds free from G. pseudo-tuberculosis infection.It is concluded that most swellings on the shoulder and chest in goats were granulomas resulting from vaccination against paratuber-culosis.     

Corynebacterium pseudotuberculosis infection in adult ewes by inoculation in the external ear

Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, was studied in ewes to devise a reliable method of induced infection that would help to understand mechanisms by which abscesses develop in lymph nodes or internal organs, especially the lungs. Fifteen adult ewes, from a flock where the occurrence of caseous lymphadenitis was unknown, were inoculated SC in the auricle with various doses of a virulent strain of C pseudotuberculosis. Ewes inoculated with at least 10(8) viable corynebacteria develop a strong local reaction that was apparent 4 hours after challenge exposure and lasted 7 to 14 days. Of these ewes, all developed at least 1 abscess and 3 developed multiple abscesses in lymph nodes draining the inoculated auricle and in the lungs. All infected ewes developed a strong antibody response against C pseudotuberculosis exotoxin, which persisted for 3 months. Of 9 ewes infected with 1.2 X 10(8) corynebacteria, 3 developed lesions because of field strains of C pseudotuberculosis and had an increased capacity to limit the dissemination of challenge-inoculum bacteria by developing only one abscess in the draining lymph node (parotid or prescapular lymph node). Seemingly, inoculation in the external ear was a suitable method for obtaining a reproducible infection and indicated that a primary infection might protect against reinfection by limiting dissemination of C pseudotuberculosis.     

Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera

Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA). However, none are commercially available in the UK. It was therefore necessary to develop a new, economic ELISA for use in a research project studying the epidemiology of CLA in UK sheep. The ELISA with its diagnostic qualities is presented. The ELISA was developed using sonicated C. pseudotuberculosis and optimised to detect total antibody or IgG class antibody in serum. Receiver operating characteristic (ROC) curves were obtained and the area under the ROC curve was used to compare the sensitivity and specificity of the two ELISAs. Both versions of the ELISA were evaluated on a panel of 150 positive reference sera and 103 negative reference sera. Using the test at 100% specificity, the sensitivity of detection of total antibody was 71% (95% confidence interval 63-78%), and the sensitivity of detection of IgG antibody to C. pseudotuberculosis was 83% (76-89%), which compares favourably with other reported ELISA tests for CLA in sheep. The sensitivity of the IgG antibody assay may be higher because of the greater affinity of IgG class antibodies compared with the IgM antibodies also detected by the total antibody ELISA. The results of ROC analysis indicated that the IgG isotype ELISA was more accurate than the total antibody ELISA. The efficiency of the test was greatest when serum samples were run in a dilution series than when any single serum dilution was used. The ELISA is considered to be suitable for application in field studies of CLA in UK sheep.     

Corynebacterium pseudotuberculosis infection and lymphadenitis (taloa or mala) in the camel

Summary Pure cultures ofCorynebacterium pseudotuberculosis were obtained from 11 cases of lymphadenitis (known locally as taloa or mala) in camels. Camel isolates produced typical taloa in camels experimentally inoculated subcutaneously at the base of the external ear with 10¹⁰ colony forming units. A sheep strain ofC. pseudotuberculosis inoculated into camels produced a local abscess at the site of inoculation but did not produce taloa. Re-infection of camels recovered from experimental inoculation did not produce taloa suggesting the possibility of the development of a vaccine against lymphadenitis in camels.

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Infeccion PorCorynebacterium Pseudotuberculosis Y Linfadenitis (Taloa O Mala) En El Camello

Resumen Se obtuvieron cultivos puros deCorynebacterium pseudotuberculosis a partir de 11 casos de linfadenitis (conocida localmente como taloa o mala) en camellos. La inoculación subcutánea en camellos en la base de la oreja de 10¹⁰ unidades formadoras de colonias procedentes de un cultivo obtenido de camellos infectados causó taola. Sin embargo, una cepa ovina deC. pseudotuberculosis causó un abceso en el lugar de la inoculación pero no produjo taola en camellos. La reinfección de los camellos que se habían recuperado de la inoculación experimental no causó taola, lo que sugiere la posibilidad de desarrollar una vacuna frente a la linfadenitis caseosa del camello.

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Infection ACorynebacterium Pseudotuberculosis Et Lymphadenite (Taloa Ou Mala) Chez Le Chameau

Résumé On a obtenu des cultures pures deCorynebacterium pseudotuberculosis à partir de 11 cas de lymphadénite (dénommée localement taloa ou mala) chez des chameaux. L'inoculation expérimentale des isolements camelins faite en infection sous-cutanée à la base de l'oreille externe avec 10¹⁰ unités formant des colonies a provoqué un taloa typique. Une souche ovine deC. pseudotuberculosis inoculée à des chameaux n'a conduit qu'à la formation d'un abcès local au point d'inoculation mais pas au taloa. La réinfection des chameaux guéris d'une inoculation expérimentale ne provoque pas de taloa, ce qui permet d'augurer la possibilité de développement d'un vaccin contre la lymphadénite cameline.

     

The spread of Corynebacterium pseudotuberculosis infection to unvaccinated and vaccinated sheep

SUMMARY The decrease in the prevalence of Corynebacterium pseudotuberculosis after two generations of vaccination against the disease it causes, was used to estimate the rate of control of caseous lymphadenitis (CLA). Three groups of 150 sheep, of which 50 in each group were artificially infected with C pseudotuberculosis and 100 in each group were uninfected sheep, were run separately for 40 months and shorn 5 times to promote the spread of CLA. One lot of 50 infected sheep and 2 lots of 100 uninfected sheep were vaccinated against CLA. The rate of spread of CLA was recorded. Sheep vaccinated against CLA and naturally exposed to infection had a 74% lower infection rate than unvaccinated sheep. Sheep vaccinated against CLA and exposed to only vaccinated infected sheep had a 97% lower infection rate. Unvaccinated sheep had a 76% infection rate, with 77% of the transmission occurring at the 4th and 5th shearings, without any discharging CLA abscesses being observed. This study supports the view that in Australian wool producing flocks, CLA spreads mainly from sheep with discharging lung abscesses to sheep with shearing cuts. Vaccinated sheep infected with CLA have 96% fewer lung abscesses compared with unvaccinated infected sheep and are therefore less likely to spread this disease to other sheep .     

Histopathology of the early phase during experimental Corynebacterium pseudotuberculosis infection in lambs.

Histological responses during experimental Corynebacterium pseudotuberculosis infection in lambs were investigated in parotid lymph nodes for ten days following inoculation. Lambs were infected by the subcutaneous route into the right eyelid with a virulent strain of C. pseudotuberculosis. Multiple microscopic acute abscesses, predominantly infiltrated with polymorphonuclear (PMN) leucocytes, were seen in the right parotid lymph node on the 1st day post-inoculation (PI). This massive PMN infiltration coincided with a peripheral blood granulocytosis. On day 3 PI, an influx of histiocytes was observed, while the microabscesses became confluent. From day 3 to day 10 PI, these lesions became enlarged and transformed into typical pyogranulomas with a central necrosis and a peripheral mantle of mononuclear cells composed of macrophages, epithelioid cells and lymphocytes; these histological changes were associated with a bacterial dissemination limited to the superficial lymph nodes. A lymphoid hyperplasia with prominent germinal centers was observed in the draining lymph nodes from day 3 PI. These results illustrate the dual role of granulomatous lesions in chronic bacterial infections: although they limit bacterial dissemination, the granulomas do not impair the persistence of infectious organisms in the host, leading to focal tissue damage.     

Evaluation of temporal and spatial clustering of horses with Corynebacterium pseudotuberculosis infection

OBJECTIVE: To determine whether horses with Corynebacterium pseudotuberculosis infections that were examined at a veterinary medical teaching hospital between July 1, 1992, and June 30, 1994 had patterns of temporal or spatial clustering. ANIMALS: 134 case and 800 control horses randomly selected from all non-case horses admitted during the study period. PROCEDURES: Admission date and geographic location were determined. Scan, Cuzick & Edwards', and Knox tests were applied to determine whether case horses had patterns of temporal or spatial clustering. RESULTS: For all windows > or = 3 days (134 case horses) and > or = 7 days (subset of 69 case horses), results of the Scan test were significant. Results of Cuzick & Edwards' test were significant for all data sets. A significant spatial cluster of case horses was observed for October, November, and December 1992. Results of the Knox test were significant for temporal intervals between 7 and 56 days and spatial intervals between 4.3 and 6.5 km. Higher Knox(x) proportions were observed for temporal intervals of 0 to 7, 8 to 14, 22 to 28, and 29 to 35 days. CONCLUSIONS: Significant spatial and temporal clustering of horses with C pseudotuberculosis infection was detected. CLINICAL RELEVANCE: Analysis of the results strongly indicates that this disease is directly or indirectly (ie, short distance and time) transmitted. In addition, data analyses indicated an incubation period of 3 to 4 weeks. The disease could be transmitted through horse-to-horse contact or from infected to susceptible horses via insects, other vectors, or contaminated soil.     

Experimental Corynebacterium pseudotuberculosis infection in lambs: kinetics of bacterial dissemination and inflammation

Infection and pyogranulomas induced by Corynebacterium pseudotuberculosis were experimentally reproduced in lambs. In two separate experiments, bacterial multiplication and dissemination were studied in 30 male lambs inoculated subcutaneously into the right ear with 1.1 or 1.5 X 10(8) viable C. pseudotuberculosis strain 19R. Infected lambs were necropsied at various times until the 28th day following inoculation. After a transient hyperthermia and a strong local inflammatory reaction, an abscess developed in the right ear from postinoculation day (PID) 6; it enlarged until PID 14 and stabilized thereafter and was associated with adenopathy of lymph nodes draining the head. Three acute phase indicators of inflammation were followed in 14 out of 30 lambs; plasma levels of copper and haptoglobin increased rapidly following inoculation whereas zinc levels decreased. The peaks were reached from PID 1 to 5, and thereafter the values came back slowly to the baseline. Antibodies against C. pseudotuberculosis exotoxin increased from PID 5 and reached a plateau on PID 21. Bacterial dissemination, assessed by the number of infected organs per lamb, was maximal on PID 16 and then stabilized until the end of the experiment. Lungs were infected in seven out of 18 lambs necropsied on PID 28. These results demonstrate a significant relationship between the clinical score of superficial lymph nodes or inoculation site and the infection level of these organs, and an early localization of pyogranulomatous lesions in regional lymph nodes. The subsequent development of the disease was related to the enlargement of these lesions and, in some animals, to a bacterial dissemination from primary sites of infection in the right prescapular lymph node and in the lung.     

Corynebacterium pseudotuberculosis infection in goats. III. The influence of age

Serological examinations for Gorynebacterium pseudotuberculosis in-fection were carried out in 14 known positive herds and in 1 herd that had beeni recently established through purchase of animals from different herds. Serum samples were collected sequentially on up to 4 occasions from animals during their first year of life. In most herds, these animals were examined clinically for superficial swellings once or twice during the same period.The adult goats in 8 infected herds were examined both clinically and serologically. Age distribution was similar in each of these herds. All serum samples were examined for antibodies against Gorynebac-terium pseudotuberculosis in both the bacterial agglutination test (BAT) and the hemolysis inhibition test (HIT).The proportion of kids which were seropositive in both tests de-creased to zero at 4 months of age. At the age of 11–12 months, the proportion of seropositive animals was about 50 % in the BAT and 30 % in the HIT.When examined when housed for the winter at the age of about 8 months (mean age), the prevalence of animals with superficial swellings was 7 %. At the age of about 1 year (mean age), 29 % of the examined animals had such lesions. In the recently established herd, only a few of the yearlings were seropositive.Titre values in BAT and HIT increased until 6 years of age. Anti-body titre values were significantly lower in yearlings than in older goats in both tests, P < 0.005. No significant difference in the propor-tion of goats with superficial swellings was seen at the different ages.     

Corynebacterium pseudotuberculosis infection in goats. VIII. The effect of vaccination against experimental infection

The effect of an inactivated vaccine against C. pseudotuberculosis infection was tested on castrated male kids from a herd free from caseous lymphadenitis. The animals were divided into 3 groups with 8 animals in each. Group 1 was immunized with crude filtrated C. pseudotuberculosis toxoid and whole killed organisms, while Group 2 in addition was given levamisole. The kids were vaccinated twice at an interval of 4 weeks. Group 3 consisted of unvaccinated animals. All groups were challenged subcutaneously with live bacteria 4 weeks after the last vaccination. Unvaccinated animals showed the most severe course of illness after challenge. Development of abscesses in the regional lymph nodes (Inn. subiliaci) was significantly more common in unvaccinated than in vaccinated kids at necropsy 2 months after challenge. There was, however, no such difference between the vaccinated groups, and there was no difference between any of the groups as regards abscess formation at the inoculation site. In each of the 2 vaccinated groups, there was a titre rise following vaccination in the hemolysis inhibition test, whereas no such rise was seen in the bacterial agglutination test. The titre values in both tests increased significantly after challenge in all the groups, the increase being most rapid in the vaccinated animals. The present investigation indicates that development of caseous lesions in lymph nodes in goats, following subcutaneous inoculation with C. pseudotuberculosis, can be reduced by an inactivated vaccine containing whole organisms and crude toxin.