BACKGROUND: Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis (CLA), a chronic and very common disease in sheep and goats, which can lead to severe economic losses in the livestock industry.OBJECTIVES: This study aims to investigate the prevalence of CLA in sheep in Kurdistan province of Iran using phenotypic and molecular methods, and assess the antibiotic resistance of isolated Corynebacterium pseudotuberculosis.METHODS: In this study, from September to March 2022, 270 samples of skin abscesses were collected from sheep in livestock farms of Kurdistan province. Immediately, using the cold chain system, the samples were transferred to the microbiology laboratory of the Faculty of Medicine at Kurdistan University of Medical Sciences. Identification of isolates was done using biochemical tests and polymerase chain reaction (PCR) method. The antibiotic resistance of the isolates was examined using the Kirby-Bauer disk diffusion method.RESULTS: Based on biochemical tests, out of 270 samples, 82 suspected to have Corynebacterium pseudotuberculosis. Out 82 samples, the presence of bacteria was confirmed in 76 samples by the PCR. The antibiotic sensitivity test showed that the isolates had high sensitivity to doxycycline and ceftriaxone and high resistance to streptomycin and kanamycin.CONCLUSIONS: The CLA has a high prevalence in sheep in Kurdistan province. According to high resistance rate of Corynebacterium pseudotuberculosis to streptomycin and kanamycin, it recommended to avoid treatment of CLA cases with these antibiotics.

BACKGROUND: Corynebacterium pseudotuberculosis and Trueperella pyogenes are two important pyogenic bacteria that cause many annual economic losses worldwide. Currently, antibiotic resistance of these bacteria is on the rise. Early detection of infection with these bacteria is important for controlling the infections caused by these two bacteria.OBJECTIVES: This study aimed to investigate the contamination of cattle cutaneous abscesses with  Corynebacterium pseudotuberculosis and Trueperella pyogenes in five large cattle farms around Tehran and propose an accurate method for a rapid detection of these two bacteria.METHODS: Out of 60 cows involved in cutaneous abscesses in the summer of 2018, sterile sampling was performed to diagnose the bacterial agent that caused the abscess. Bacteriological examination of the samples was performed using standard biochemical reactions and polymerase chain reaction using specific primers.RESULTS: Of the 60 samples studied, 25 % (15.60) were isolated as Corynebacterium pseudotuberculosis and 20 % (12.60) as Trueperella pyogenes. In 55 % of the samples (33.60), both bacteria were present simultaneously. All the samples detected using biochemical reactions were confirmed by the polymerase chain reaction.CONCLUSIONS: Corynebacterium pseudotuberculosis and Trueperella pyogenes are the main causes of cutaneous abscess in cattle farms around Tehran. Because the accurate diagnosis of the cause of abscesses is very important for effective treatment, polymerase chain reaction, based on 16S-23S rDNA and 16S rRNA, can be used to rapidly and accurately detect these bacteria in the early stages of the infection.

BACKGROUND: Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable losses for herd owners. Phospholipase D (PLD) is a potent exotoxin produced by C. pseudotuberculosis and it has been considered as the major virulence factor for this bacterium, possibly contributing to the spread of the bacteria from the initial site of infection to secondary sites within the host. Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. OBJECTIVES: The aim of this study was the cloning and expression of the PLD and HSP60 genes of C. pseudotuberculosis, used subsequently to evaluate the protectivity of these recombinant proteins for vaccine development against this bacterium. METHODS: PLD and HSP60 genes were cloned into pMAL-c2X vector and recombinant plasmids construct was transformed to DH5 strain of E. coli. Expression of the proteins was shown by SDS-PAGE and accuracy of the cloned genes was confirmed by nucleotide sequence analysis. RESULTS: The transformed E. coli strain DH5 expressed PLD and HSP60 proteins effectively. The expressed fusion protein was found almost entirely in the soluble form. CONCLUSIONS: In the following studies the immunogenicity and protectivity of these recombinant proteins against C. pseudotuberculosis infections can be assessed.

Corynebacterium pseudotuberculosis phospholipase D (PLD) significantly affected viability of ovine neutrophils after 24 hours' exposure, This effect was more marked in cells that ingested PLD emulsified in oil. Treatment of neutrophils with PLD significantly (P < 0.05) reduced the ability of these cells to migrate toward activated sheep serum. The PLD was not chemotactic, but it activated normal sheep serum, producing factors that were chemotactic for neutrophils.

Corynebacterium pseudotuberculosis phospholipase D (PLD) significantly affected viability of ovine neutrophils after 24 hours' exposure, This effect was more marked in cells that ingested PLD emulsified in oil. Treatment of neutrophils with PLD significantly (P < 0.05) reduced the ability of these cells to migrate toward activated sheep serum. The PLD was not chemotactic, but it activated normal sheep serum, producing factors that were chemotactic for neutrophils.

A double-antibody sandwich ELISA for detection of antibodies directed against the exotoxin of Corynebacterium pseudotuberculosis, the cause of caseous lymphadenitis (CL) in small ruminants, was developed. A concentrated exotoxin was used. For interpretation of ELISA results, these sera were tested: sequentially obtained sera of C pseudotuberculosis-inoculated goats and sheep that were monitored for 68 weeks; sequentially obtained sera from 80 goats of 3 flocks with CL; sera from 652 goats of 7 flocks without CL; sera from 160 sheep of 4 flocks without CL; and 2,265 caprine and 208 ovine sera submitted for diagnostic testing. Data regarding the infection status and history of 10,454 of the 23,302 animals were collected after testing; most of these were goats that had been part of a CL control program. Specificity and sensitivity of the ELISA were nearly 100%. Subsequently, 31,978 animals from which no data on infection status of flocks had been collected were then tested. It was concluded that the ELISA is a useful diagnostic test for CL eradication programs. Sera with doubtful or inconclusive ELISA results were examined by use of immunoblot analysis. Proteins from C pseudotuberculosis culture supernatant were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Six proteins with molecular mass of 68, 65, 39, 38, 31, and 29 kDa reacted with sera from goats and sheep with experimentally induced or naturally acquired infection. Immunoblot analysis was valuable in classifying sera with doubtful or inconclusive results by ELISA.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA) a contagious and infectious disease in smallruminants. This report describes a case of CLA in a Boer cross goat infected with Corynebacterium pseudotuberculosis. The manifestation of abscess in the superficial lymph node presented as a mass ofapproximately 5 cm by 5 cm in diameter at the base of the ear. The mass was solid and non-movable upon palpation. The mass was incised and the caseous material was aspirated and sent to the bacteriologylaboratory where Corynebacterium pseudotuberculosis was isolated. Thewound was cleaned and flushed with diluted chlorhexidine and iodine solutions daily. This procedure aids in the complete healing of the wound after a few weeks. However, the agent of the disease persistsin the animal in its entire life and culling is recommended to prevent the spread of the disease in the herd.

Caseous lymphadenitis (CIA) caused by Corynebacterium pseudotuberculosis is a worldwide disease of sheep and goats and is characterized by development of pyogranulomas in lymph nodes and lungs. Control of this disease by vaccination remains controversial, although toxoid vaccines are now commercially available in some countries. To determine the efficacy of acquired immunity to control CLA, the effect of primary infection on subsequent challenge exposure was investigated. Adult seronegative ewes were primarily inoculated with a streptomycin-sensitive strain of C pseudotuberculosis on the external part of the left ear and thereafter challenge-exposed by inoculation of the streptomycin-resistant strain 19R in the right ear. This protocol indicated that primary infection with at least 10(7) viable bacteria induced strong protection against subsequent challenge exposure; the ewes with primary infection did not develop lesions as a result of challenge exposure, whereas immune-naive ewes developed numerous pyogranulomas in the right car, in lymph nodes draining the inoculation site, and in the lungs. However, ewes with primary infection remained carriers of the disease as a result of primary inoculation. These results offer experimental support for development of more effective vaccination to control CLA, in sheep and goats, and this model indicates that animals with primary infection can be used as positive controls for protection when testing a candidate vaccine against CLA.

Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistance plasmid from C diphtheriae. Corynebacterium pseudotuberculosis cultivated in brain-heart infusion broth was washed 3 times with water, and resuspended to a final concentration of about 5 X 10(13) colony-forming units/ml. An electroporator constructed in our laboratory incorporated an electrode with 0.8-mm interelectrode gap, using disposable spectrophotometer cuvettes as containers for electroporation. The pNG2 was prepared in Escherichia coli and 4 to 16 microgram of pNG2 DNA was mixed with 400-microliter amounts of cell suspension in prechilled cuvettes. After incubation on ice for 5 to 10 minutes, the mixture was electroporated at field strengths of up to 18 kV/cm, mixed with 1.5 ml of brain-heart infusion broth, and incubated at 37 C for 2 hours with agitation. Aliquots were then plated on brain-heart infusion blood agar with 15 microgram of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of approximately 4 X 10(4) transformants/microgram of pNG2 DNA. Most total transformants and most transformants per microgram of pNG2 were generated at a field strength of 18 kV/cm. When the concentration of pNG2 DNA was varied, the average total number of transformants increased through a concentration of 30 microgram/ml, but the efficiency of transformation was highest at the lowest DNA concentration. Transformants contained unmodified pNG2.

Caseous lymphadenitis (CLA) is a chronic and contagious disease of sheep and goats caused by Corynebacterium (C.) pseudotuberculosis. A four-year-old female Saanen dairy goat was submitted to the Animal Disease Diagnostic Center at National Veterinary Research and Quarantine Service. The clinical signs of the goat were emaciation, abortion and quadriplegia. The multifocal nodules of lymph nodes were encapsulated and filled with whitish caseous contents on the cut surface. Histopathologically, lymph nodes displayed suppurative and necrotizing granulomas. Caseous necrosis was diffusely observed in the center of the lymph nodes. Gram positive bacilli were shown in the lesions. C. pseudotuberculosis was isolated and confirmed by the biochemical tests and PCR assay. Based on clinical signs, histopathological examination and bacterial isolation, we diagnosed this case as CLA. To our knowledge, this is the first report of CLA in a Saanen dairy goat in Korea.