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 .     

An improved Corynebacterium pseudotuberculosis vaccine for sheep

Extensive experiments in mice confirmed that the immunogenicity of a Corynebacterium pseudotuberculosis vaccine could not be significantly improved with the use of various adjuvants. Immunity against C. pseudotuberculosis likewise could not be enhanced by incorporating various immunostimulants into the vaccine or by the use of live vaccines. However, a combination of aluminium hydroxide gel and saponin as adjuvant did have a beneficial effect. This vaccine was tolerated better, and a smaller dose apparently protected sheep more effectively against intralymph node challenge than the currently available alum-precipitated vaccine.     

Rapid detection of Corynebacterium pseudotuberculosis in clinical samples from sheep

Corynebacterium pseudotuberculosis, a Gram-positive bacterium is the causative agent of caseous lymphadenitis (CLA), a chronic disease of sheep, goats and other warm blooded animals. In the present study, a total of 1,080 sheep reared under semi-intensive system on organized farms situated in the semi arid tropical region of Rajasthan, India, was clinically examined. Pus samples from superficial lymph nodes of 25 (2.31 %) adult sheep showing clinical lesions similar to CLA were collected for laboratory analyses. On the basis of morphological, cultural and biochemical characteristics 12 (48 %) bacterial isolates from pus identified it as C. pseudotuberculosis. A polymerase chain reaction (PCR) assay targeting Putative oligopeptide/dipeptide ABC transporter, nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase coenzyme F420-dependent and proline iminopeptidase (PIP) genes of C. pseudotuberculosis was developed that showed 14 pus samples as positive. All C. pseudotuberculosis isolates were also found positive for these genes in the PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products that showed 98–100 % homology with published sequences available in the NCBI database. The present study shows the incidence of CLA as 2.31 %, 1.1 % and 1.29 % based on clinical, bacterial culture and direct pus PCR assay, respectively. The PCR assay was rapid, specific and as significant as bacterial culture in detecting bacteria directly in the clinical pus samples. The PCR assay developed in the study can be applied for the diagnosis and control of CLA. Furthermore, the assay can also be applied to detect C. pseudotuberculosis in various clinical samples.     

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.     

An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge

Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA.     

Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by PCR

The present study was carried out to estimate the prevalence of caseous lymphadenitis (CL) in sheep and goats slaughtered at the local abattoir in Elazig province located in the east of Turkey, between September and December 2000. A total of 2046 sheep and 2262 goat carcasses were examined during the study period and 118 abscessed lymph nodes, 89 from sheep and 29 from goats, were collected. Corynebacterium spp. strains were isolated from 81.4% of the abscesses, giving an overall prevalence of 2.2%. The prevalence was 3.5 and 1.1% in sheep and goats, respectively. PCR on DNA extracted from 96 suspicious isolates, using a pair of Corynebacterium pseudotuberculosis-specific primers, was positive for 93. Although cross-reaction with C. ulcerans, a human/bovine species, was observed, the PCR assay used in this study may successfully be applied for the diagnosis of CL in goats and sheep as an alternative to conventional methods, owing to its advantages of specificity and speed.     

Serological response of cattle after vaccination and challenge with Brucella abortus

New and currently used serological procedures were evaluated using sera from cattle that were challenged with B. abortus S544 (S544) after vaccination with either B. abortus S19 (S19) or B. abortus 45/20 (S45/20) as calves or adults. In animals vaccinated with S19, titres to the indirect haemolysis test (IHLT) rose more slowly, declined more rapidly and involved fewer animals than did titres to the complement fixation test (CFT). In animals vaccinated with S45/20 the rough antigen complement fixation test (RCFT) showed persistent titres. At slaughter the IHLT and CFT were found to be more specific and more sensitive than the Rose Bengal Plate Test (RBPT) and Serum Agglutination Test (SAT) in the detection of cattle infected with B. abortus.     

Effect of levamisole on immunity to Corynebacterium pseudotuberculosis in mice and sheep

The stimulating effect of levamisole on immunity to Corynebacterium pseudotuberculosis in mice was marginal, while no enhancement of immunity could be detected in sheep. The conclusion was reached that, as levamisole has no potentiating effect on immunity to C. pseudotuberculosis in normal sheep, it is of no practical value as an immunostimulant in this instance.     

Serological response of cattle, sheep and goats in Kenya vaccinated with killed Brucella melitensis strain H38 adjuvant vaccine

The antibody responses of cattle, sheep and goats to the Brucella melitensis H38 adjuvant vaccine were monitored by serum (tube) agglutination, complement fixation and Rose Bengal plate tests. High and persisting antibody titres were induced by a single dose of this vaccine in all three species. It would be difficult to classify reactors by commonly used diagnostic tests in a control or an eradication programme using the H38 adjuvant vaccine.     

Diagnosis of Corynebacterium pseudotuberculosis infections in sheep, using an enzyme-linked immunosorbent assay

Lambs infected with Corynebacterium pseudotuberculosis (ovis) by injecting suspensions of live bacteria into the wool-free area of the axilla developed antibody against cell wall antigens and antitoxin, as measured by the enzyme-linked immunosorbent assay. The toxin was a better antigen for measuring infection than was the cell wall antigen. The enzyme-linked immunosorbent assay appeared to be as sensitive as the antihemolysin inhibition test for detecting antitoxin and was easier to perform.     

Infection with Corynebacterium pseudotuberculosis in five alpacas

Among the population of an alpaca breeding farm, 5 alpacas (22 days to 14 months old) developed focal swellings in the subcutaneous tissues of the head or neck. Infection with Corynebacterium pseudotuberculosis was confirmed on the basis of results of microbial culture of abscess material and a serum hemolysis inhibition assay to detect C. pseudotuberculosis toxin. The dams of the affected alpacas were seronegative for C. pseudotuberculosis toxin. The affected alpacas underwent surgical excision of the abscesses and were isolated from herdmates for 90 days; treatment was successful, and no other alpacas in the herd became infected. Common risk factors for sources of infection in the affected alpacas included housing in a maternity barn and a pasture. Also, the infection potentially originated from new alpacas introduced into the herd during the preceding 3 months. Infection with C. pseudotuberculosis should be considered as a differential diagnosis for camelids with peripheral lymphadenopathy or abscesses in subcutaneous tissues.     

The response of sheep vaccinated with irradiated Trichostrongylus colubriformis larvae to impulse and sequential challenge with normal larvae

Sheep aged 9–10 months were vaccinated with three doses of 20 000 irradiated Trichostrongylus colubriformis larvae at fortnightly intervals. This gave 97–99% protection against subsequent “impulse” challenge with 40 000 normal larvae or “sequential” challenge with 2 000 larvae/week-day for 4 weeks.The sheep were protected against both types of challenge when worms from the immunizing infections were present at the time of challenge, and when these worms were first removed by anthelmintic treatment.     

Serological response of chickens either vaccinated or artificially infected with Haemophilus paragallinarum

The serological response of chickens either vaccinated or artificially infected with Haemophilus paragallinarum (Hpg) serovar A or C was investigated using both a specific hemagglutinin (HA) antigen and a common HA antigen. With Hpg serovar A, both vaccinated and artificially infected chickens produced hemagglutination-inhibition (HI) antibodies to Hpg serovar-specific and Hpg common HA antigens. Most chickens vaccinated with Hpg serovar C had detectable HI antibodies to both types of HA antigen by 3 weeks postvaccination, after which titers gradually declined. In contrast, most chickens artificially infected with serovar C produced HI antibodies to only the common HA antigen; very few of these chickens produced HI antibodies to the serovar-specific HA antigen.     

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).