Corynebacterium pseudotuberculosis and Caseous Lymphadenitis in Sheep and Goats

Etiology and Taxonomy

Corynebacterium pseudotuberculosis is a Gram-positive, facultative intracellular, pleomorphic rod that is the causative agent of caseous lymphadenitis (CLA) in sheep and goats. The bacterium is classified within the genus Corynebacterium, which belongs to the phylum Actinobacteria. It is characterized by a high G+C content in its genomic DNA and a mycolic acid-containing cell wall that confers acid-fastness upon staining. Two biovars are recognized based on nitrate reduction capability: biovar ovis (nitrate negative) and biovar equi (nitrate positive). Biovar ovis is the primary cause of CLA in small ruminants, while biovar equi is more frequently associated with ulcerative lymphangitis in horses and other species [1, 2]. Whole genome sequencing and comparative genomics have identified specific biomarkers for biovar ovis, enabling precise molecular differentiation [2]. The bacterium produces a potent exotoxin, phospholipase D (PLD), which is a major virulence factor responsible for the characteristic pathology of CLA [3].

Epidemiology

Caseous lymphadenitis is a globally distributed disease with significant economic impact on sheep and goat industries. The disease is endemic in many regions, including Australia, New Zealand, South America, Africa, and parts of Europe and Asia. Herd-level true seroprevalence studies have demonstrated substantial variation across populations. For example, a study in the goat population of Poland reported a true seroprevalence of 12.3% at the herd level, indicating widespread subclinical infection [4]. In South Korea, a pilot study on Korean native goats revealed a prevalence of 8.7%, with a higher occurrence in older animals and no significant sex predilection [5]. A comparative study of ovine and caprine strains on Czech farms found that biovar ovis isolates from sheep and goats were genetically similar, suggesting cross-species transmission is possible [6]. The bacterium has also been isolated from cattle, camels, and wildlife such as roe deer, indicating a broader host range than previously appreciated [7, 8, 9]. Genomic diversity studies using comparative population genomics have revealed host-specific genetic signatures, with distinct clades associated with small ruminants versus equids [1]. Transmission occurs primarily through direct contact with purulent material from ruptured abscesses, contaminated fomites, and environmental persistence. The bacterium can survive for extended periods in soil and on equipment, facilitating indirect transmission within and between flocks.

Clinical Signs

The clinical presentation of CLA in sheep and goats is characterized by the formation of encapsulated abscesses in superficial and internal lymph nodes. External abscesses are most commonly observed in the parotid, submandibular, retropharyngeal, and prescapular lymph nodes. These abscesses are firm, non-painful, and gradually enlarge over weeks to months. They may eventually rupture, discharging a thick, greenish-white, non-odorous pus that is highly infectious. Internal abscesses, which are often clinically silent, can develop in the mediastinal, bronchial, mesenteric, and iliac lymph nodes, as well as in parenchymatous organs such as the lungs, liver, and kidneys. In sheep, chronic weight loss, reduced wool production, and decreased milk yield are common sequelae. Respiratory signs, including coughing and dyspnea, may occur when pulmonary lymph nodes are involved. In goats, the disease can also present with mastitis, orchitis, and, rarely, neurological signs if abscesses impinge on the spinal cord. A case series described granulomatous omentitis in multiple sheep associated with both Trueperella pyogenes and C. pseudotuberculosis, highlighting the potential for mixed infections [10]. An unusual presentation involving a suspected Bartholin gland cystic-like structure in a Nigerian dwarf doe has also been reported [11].

Pathology

The hallmark lesion of CLA is a pyogranulomatous abscess. Macroscopically, these abscesses are well-encapsulated and contain concentric layers of inspissated, caseous pus, often described as having an "onion ring" appearance on cut section. The capsule is composed of fibrous connective tissue, and the inner lining is a layer of epithelioid macrophages and multinucleated giant cells. The central core consists of necrotic debris, degenerate neutrophils, and bacterial cells. The pathogenesis is driven by the exotoxin phospholipase D (PLD), which is a sphingomyelinase that targets host cell membranes. Recent research has elucidated that PLD targets mitochondrial sphingomyelin and induces NLRP3-GSDMD axis-mediated pyroptosis in macrophages, a critical mechanism for bacterial survival and dissemination [3]. This pyroptotic pathway facilitates the release of pro-inflammatory cytokines and promotes the formation of the characteristic abscess. The bacterium also expresses other virulence factors, including a superoxide dismutase (SodC) that is responsible for oxidative stress resistance and is essential for full pathogenicity [12]. The mismatch uracil DNA glycosylase (Mug) is maintained in the genome and exhibits affinity for uracil, suggesting a role in DNA repair and survival within the host [13]. In vitro studies using MDBK cell lines have demonstrated cytopathic effects after adhesion and internalization of biovar ovis, confirming the bacterium's ability to invade and damage epithelial cells [14].

Diagnostics

Accurate diagnosis of CLA is essential for implementing control measures. A combination of clinical examination, bacteriological culture, molecular methods, and serological assays is recommended.

Clinical and Postmortem Examination

External abscesses in characteristic lymph node locations are highly suggestive of CLA. However, differential diagnoses include abscesses caused by other pyogenic bacteria such as Trueperella pyogenes, Staphylococcus aureus, and Streptococcus spp. Postmortem examination reveals the characteristic encapsulated abscesses in internal lymph nodes and organs. A comprehensive pathological study of slaughtered sheep, goats, and cattle confirmed the presence of typical CLA lesions in multiple species [7].

Bacteriological Culture

Isolation of C. pseudotuberculosis from pus samples is the gold standard for diagnosis. The bacterium grows on standard media such as sheep blood agar and brain heart infusion agar, forming small, dry, cream-colored colonies after 24 to 48 hours of incubation at 37 degrees Celsius in an atmosphere of 5% carbon dioxide. The colonies are catalase positive, urease positive, and reduce nitrate (biovar equi) or do not (biovar ovis). The synergistic hemolysis test (CAMP test) with Rhodococcus equi is used to confirm PLD production.

Molecular Diagnostics

Polymerase chain reaction (PCR) assays targeting the PLD gene are widely used for rapid and specific detection. A high-resolution melting (HRM) method has been developed and validated for differentiating ovis and equi biovars, providing a rapid, closed-tube genotyping tool [15]. PCR-based DNA fingerprinting methods, including random amplified polymorphic DNA (RAPD) and repetitive element PCR (rep-PCR), have been used for genotyping isolates and assessing genetic diversity [16]. Whole genome sequencing has revealed unforeseen genetic diversity and is increasingly used for epidemiological investigations and biomarker discovery [17]. A genome-wide association study has identified genetic markers associated with antibody response to C. pseudotuberculosis in sheep, which could inform breeding for resistance [18].

Serological Assays

Serological tests detect antibodies against PLD and other bacterial antigens. Commercial enzyme-linked immunosorbent assay (ELISA) kits are available and are useful for herd-level screening. A novel approach using oral fluid as a material for serological diagnostics has been evaluated in goats, offering a non-invasive sampling method for large-scale surveillance [19]. The specificity and sensitivity of serological tests can vary, and interpretation at the individual animal level requires caution due to the potential for cross-reactivity with other corynebacterial species.

Treatment

Antimicrobial therapy for CLA is generally unrewarding due to the thick fibrous capsule surrounding abscesses, which limits drug penetration, and the intracellular location of the bacterium. Surgical drainage and removal of abscesses are the primary treatment options for superficial lesions. However, this approach carries a risk of environmental contamination and requires strict biosecurity measures to prevent spread. Antimicrobial susceptibility profiles of C. pseudotuberculosis isolates have been characterized. A study from the Cankiri region of Turkiye reported that isolates were susceptible to penicillin, amoxicillin-clavulanic acid, and ceftiofur, but resistance was observed to tetracycline and erythromycin [20]. The antibacterial efficacy of nalidixic acid has been investigated using structural, proteomic, and metabolomic approaches, revealing potential targets for novel therapeutic agents [21]. Silver nanoparticles have demonstrated antibacterial activity against multidrug-resistant C. pseudotuberculosis isolates, and biogenic silver nanoparticles have shown activity against both planktonic and biofilm-associated bacteria [22, 23]. Probiotic approaches, such as Lactobacillus acidophilus, have been shown to protect against infection by regulating macrophage autophagy and maintaining gut microbiota homeostasis in a murine model [24].

Control and Prevention

Control of CLA is based on a combination of biosecurity, culling, and vaccination.

Biosecurity and Management

Strict biosecurity measures are essential to prevent introduction and spread. These include quarantine of new animals, segregation of infected animals, and rigorous hygiene practices. Shearing, tattooing, and other procedures that break the skin should be performed with disinfected equipment. Infected animals should be culled or isolated, and their abscesses should be managed to minimize environmental contamination.

Vaccination

Vaccination is a key component of CLA control programs. Several vaccine formulations have been developed and evaluated. A bacterin-toxoid vaccine using a Korean isolate has been developed and shown to protect goats against CLA [25]. An inactivated vaccine has also been evaluated in South Korea across various animal models [26]. Recombinant protein vaccines have been a major focus of research. Multi-epitope recombinant chimeras and inactivated Escherichia coli expressing chimeric genes of C. pseudotuberculosis have been tested as vaccines [27]. The recombinant protein CP01850, adjuvanted with fish oil from Rhamdia quelen or lipid extracts from Iridea cordata and Sacorpletis skottibergii, has elicited protective immune responses in mice and sheep [28, 29, 30]. Immunization with recombinant proteins rPTS, rRBN, and rCP40 has also been evaluated in mice and goats [31]. A bioinformatic approach has identified B and T cell epitopes of PLD and CP40 proteins for peptide-based vaccine design [32]. A novel strategy using Mycobacterium bovis BCG expressing CP40 or CP09720 proteins has shown promise in protecting mice after challenge [33]. A live attenuated vaccine using a sodC-deleted mutant has been shown to provide immunity in mice [12]. Autogenous vaccines have been developed for use in dairy cattle outbreaks, demonstrating the adaptability of vaccine strategies across host species [34].

Genetic Selection

Genome-wide association studies have identified genetic markers associated with antibody response to C. pseudotuberculosis, suggesting that selective breeding for increased resistance may be a viable long-term control strategy [18].

flowchart TD
    A[Clinical Suspicion of CLA], > B{External Abscess Present?}
    B, >|Yes| C[Palpate and Aspirate Pus]
    B, >|No| D[Serological Screening ELISA]
    C, > E[Bacteriological Culture and PCR]
    D, > F{Seropositive?}
    F, >|Yes| G[Confirm with PCR on Swab or Tissue]
    F, >|No| H[Monitor and Retest]
    E, > I{PLD Gene Detected?}
    I, >|Yes| J[Confirm C. pseudotuberculosis]
    I, >|No| K[Consider Other Pathogens]
    J, > L[Biovar Differentiation via HRM or Nitrate Test]
    L, > M[Biovar ovis]
    L, > N[Biovar equi]
    M, > O[Implement Control Measures]
    N, > P[Assess Host Range and Epidemiology]
    O, > Q[Cull or Isolate Infected Animals]
    O, > R[Vaccinate Flock]
    O, > S[Enhance Biosecurity]

References

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