Listeria monocytogenes: Circling Disease in Ruminants – Association with Silage, Diagnosis, and Public Health

Introduction

Listeria monocytogenes is a Gram-positive, facultative intracellular rod-shaped bacterium responsible for listeriosis in ruminants. The clinical syndrome most frequently recognized by veterinary practitioners is encephalitis, which in sheep and cattle manifests as the classic "circling disease." This article provides an exhaustive review of L. monocytogenes infection in ruminants, focusing on the association with silage feeding, diagnostic approaches, and public health implications. The discussion is grounded in peer-reviewed literature and prioritizes molecular, epidemiological, and pathological perspectives.

Etiology and Taxonomy

Listeria monocytogenes belongs to the genus Listeria within the family Listeriaceae. The species is subdivided into serovars based on somatic (O) and flagellar (H) antigens. Serovars 1/2a, 1/2b, and 4b are most commonly associated with clinical disease in ruminants and humans [1]. The bacterium is psychrotrophic, capable of growth at refrigeration temperatures (4 degrees Celsius), and can survive in a wide pH range (pH 4.5 to 9.6). These attributes allow L. monocytogenes to persist in silage, soil, and fecal matter for extended periods [2].

Key virulence factors include internalins (InlA, InlB), which mediate invasion of epithelial cells; listeriolysin O (LLO), a pore-forming toxin that enables escape from phagosomes; and ActA, which facilitates actin-based intracellular motility and cell-to-cell spread [3]. Lysozyme resistance has been identified as a factor enhancing conjunctival invasion, as demonstrated in a bovine conjunctiva model [4].

Epidemiology and Host Range

Listeriosis is primarily a disease of ruminants, with sheep being more susceptible than cattle and goats. Outbreaks are often associated with the feeding of poor-quality silage, which provides a permissive environment for bacterial proliferation [1, 5]. The bacterium is ubiquitous in the farm environment and can be isolated from soil, water, vegetation, and feces of healthy carrier animals [6, 2].

Fecal shedding dynamics exhibit high day-to-day variability, with both sporadic and outbreak-associated shedding patterns documented [6, 7]. A Markov chain model analyzing fecal shedding dynamics demonstrated that cattle can transition between shedding and non-shedding states stochastically, complicating herd-level surveillance [7].

Whole genome sequencing (WGS) and multilocus sequence typing (MLST) have revealed that certain clonal complexes (CCs) are hypervirulent and overrepresented in clinical cases. Papić et al. [8] demonstrated that subclinical mastitis due to hypervirulent CCs is a significant reservoir on dairy farms. Ribotype diversity among outbreak strains has been documented, with specific ribotypes linked to epizootic clusters [9, 10].

Environmental reservoirs extend beyond silage. Gismervik et al. [11] showed that invasive slugs (Arion vulgaris) can act as vectors, carrying L. monocytogenes in their digestive tracts and potentially contaminating pasture or feed.

Listeria monocytogenes Circling Disease Ruminants Silage: Association and Pathogenesis

The association between silage feeding and listeriosis is well established. Silage with a pH above 4.2, often due to inadequate fermentation, permits the growth of L. monocytogenes [1, 12]. Fenlon et al. [5] documented that silage at primary production and initial processing stages harbored L. monocytogenes at levels sufficient to cause disease when consumed.

In ruminants, ingestion of contaminated silage leads to two primary clinical forms: encephalitis and abortion. The encephalitic form, commonly called circling disease, results from the bacterium ascending the trigeminal nerve after invading the oral mucosa. From the oral cavity, L. monocytogenes enters nerve endings and travels intra-axonally to the brainstem, causing a focal, asymmetric meningoencephalitis. The rhombencephalon is the primary target, explaining the characteristic neurological signs.

Laven and Lawrence [13] described an outbreak of iritis and uveitis in dairy cattle associated with supplementary feeding of baleage, illustrating that ocular manifestations can accompany silage-associated listeriosis. Welchman et al. [14] reported ocular disease in fallow deer linked to silage feeding, further supporting the role of the conjunctival route.

Clinical Signs

Clinical presentation varies by species and form of disease.

Encephalitic Form (Circling Disease): In sheep and cattle, the incubation period ranges from 2 to 6 weeks after exposure. Initial signs include depression, anorexia, and pyrexia. As the infection progresses, ipsilateral cranial nerve deficits become apparent. These include:

• Facial nerve (CN VII) paralysis: drooping ear, drooling saliva.
• Vestibular signs: head tilt, circling toward the affected side.
• Trigeminal nerve (CN V) involvement: reduced mastication and jaw tone.
• Nystagmus and strabismus (CN III, IV, VI).

In advanced cases, recumbency, opisthotonos, and seizures occur. Mortality is high in untreated animals [15, 1].

Abortive Form: Abortion typically occurs in the last trimester without preceding clinical signs. The fetus may be autolyzed, and placentitis with multifocal necrotic lesions is observed [16].

Mastitis: Subclinical mastitis due to L. monocytogenes is increasingly recognized. Papić et al. [8] found a high occurrence of subclinical mastitis due to hypervirulent CCs on a dairy farm, with bacterial shedding in milk representing a zoonotic risk.

Ocular Form: Iritis, uveitis, and keratoconjunctivitis have been reported in cattle and deer fed silage or baleage [13, 14].

Pathology

Gross Lesions: In the encephalitic form, gross brain lesions are often absent or subtle. In some cases, areas of malacia and hemorrhage are visible in the brainstem, pons, and medulla oblongata.

Histopathology: Microscopic examination reveals a characteristic microabscessative meningoencephalitis with perivascular cuffing by mononuclear cells. Foci of neutrophilic infiltration (microabscesses) are scattered throughout the neuropil. Gram-positive rods are sometimes visible within phagocytes or extracellularly.

In aborted fetuses, the liver contains multifocal necrotic foci (miliary necrosis), and the placenta shows suppurative placentitis with Gram-positive bacilli.

Diagnosis

Definitive diagnosis requires isolation or molecular detection of L. monocytogenes from brain tissue, fetal stomach contents, placenta, or milk.

Bacteriological Culture: Selective enrichment media, such as Oxford agar or PALCAM agar, are used for isolation. Cold enrichment at 4 degrees Celsius for several weeks may increase recovery from contaminated samples [12]. Phage typing and pyrolysis mass spectrometry were historically used for strain discrimination [17, 12].

Molecular Diagnostics: Real-time PCR targeting the hlyA gene (encoding listeriolysin O) or the iap gene (encoding invasion-associated protein) offers high sensitivity and specificity. Barkallah et al. [18] developed a locked nucleic acid (LNA) probe-based real-time PCR assay for ruminant samples, demonstrating improved specificity over conventional PCR.

Multiplex PCR can differentiate L. monocytogenes from other Listeria species and identify serovars. ERIC-PCR (enterobacterial repetitive intergenic consensus PCR) has been used to assess genetic diversity and clonal relationships among strains, as demonstrated by Elsayed et al. [19] in an epidemiological study of dairy cattle.

Whole Genome Sequencing: WGS provides the highest resolution for epidemiological tracing. Papić et al. [20] retrospectively investigated listeriosis outbreaks in small ruminants using WGS-based typing and demonstrated that core genome MLST (cgMLST) can discriminate outbreak-associated strains from sporadic isolates. Whitman et al. [16] used WGS to link environmental and clinical L. monocytogenes strains in an abortion outbreak in beef heifers.

Serological Assays: ELISA-based detection of antibodies against listeriolysin O and internalin A has been used for seroepidemiological studies. Boerlin et al. [3] applied these antigens in a study of Swiss dairy cows, finding that seropositivity correlated with farm-level risk factors such as silage feeding practices.

Hematological and Biochemical Analysis: Hematological examination of infected cattle reveals neutrophilia and lymphopenia. Munir et al. [15] reported significant changes in hematological parameters in dairy cows with listeriosis, including decreased hemoglobin and packed cell volume, consistent with a systemic inflammatory response.

Diagnostic Decision Tree

flowchart TD
    A[Animal with neurological signs<br>circling, head tilt, CN deficits], > B{History of silage feeding?}
    B, Yes, > C[Collect brainstem, CSF,<br>and conjunctival swabs]
    B, No, > C
    C, > D[Gram stain & culture<br>on selective media]
    D, > E[Positive for<br>L. monocytogenes?]
    E, Yes, > F[Confirm by<br>hlyA-targeted real-time PCR]
    E, No, > G[PCR on brain tissue<br>or fetal stomach contents]
    G, > H[PCR positive?]
    H, Yes, > F
    H, No, > I[Consider differential diagnoses:<br>rabies, pasteurellosis,<br>polioencephalomalacia,<br>cerebrocortical necrosis]
    F, > J[WGS-based typing<br>for epidemiological linkage]
    J, > K[Compare with silage<br>and fecal isolates]
    K, > L[Confirm silage-associated outbreak]

Differential Diagnoses: The differential diagnosis for circling disease includes rabies (especially in cattle), listeriosis, and other causes of brainstem encephalitis such as bovine herpesvirus 5 (BHV-5) and Streptococcus suis infections. Polioencephalomalacia (thiamine deficiency) and cerebrocortical necrosis (lead poisoning) should also be considered, though these typically produce bilateral cortical signs rather than asymmetric brainstem signs.

Treatment

Early intervention is critical. High-dose penicillin G (22,000 to 44,000 IU/kg intramuscularly twice daily for 5 to 7 days) is the treatment of choice. Oxytetracycline and erythromycin are alternative options. Antimicrobial susceptibility testing is recommended given the emergence of multidrug-resistant strains. Elsayed et al. [19] reported a high prevalence of multidrug resistance in L. monocytogenes isolated from Egyptian dairy cattle, with resistance to tetracycline, ampicillin, and erythromycin.

Supportive care includes fluid therapy, nutritional support (tube feeding if dysphagia is present), and nursing care to prevent pressure sores in recumbent animals. Non-steroidal anti-inflammatory drugs (NSAIDs) may reduce brain edema and inflammation.

Control and Prevention

Control strategies focus on silage management, biosecurity, and hygiene.

Silage Management:

• Ensure proper fermentation: target pH below 4.2, moisture content between 60% and 70%.
• Avoid feeding moldy or spoiled silage, particularly to sheep.
• Maintain clean feed bunks and remove uneaten silage regularly.

Herd Biosecurity:

• Isolate sick animals and implement barrier nursing.
• Dispose of aborted fetuses and placentas promptly.
• Implement rodent and slug control programs to reduce environmental contamination [11].

Vaccination: No commercially available vaccine for ruminant listeriosis exists. Autogenous bacterins have been used experimentally but lack demonstrated efficacy.

Culling and Quarantine: Culling of chronic carriers, particularly those with subclinical mastitis, should be considered. Quarantine of newly introduced animals reduces the risk of introducing hypervirulent CCs [8].

Public Health Implications

Listeria monocytogenes is a zoonotic pathogen. Ruminants, particularly dairy cattle, can shed the bacterium in milk and feces, posing a risk to farm workers and consumers. Human listeriosis is a severe foodborne illness causing septicemia, meningitis, and abortion in pregnant women. The silage-to-milk transmission route is well documented; subclinical mastitis due to hypervirulent CCs is a significant public health concern [8]. Pasteurization effectively kills L. monocytogenes in milk, but raw milk consumption remains a risk factor.

Farm workers should wear personal protective equipment when handling aborted materials or sick animals. The one health framework underscores the interconnection between animal health, silage quality, and food safety.

References

[1] Low JC, Donachie W. A review of Listeria monocytogenes and listeriosis. Vet J. 1997. https://pubmed.ncbi.nlm.nih.gov/9125353/

[2] Ivanek R, Gröhn YT, Wiedmann M. Listeria monocytogenes in multiple habitats and host populations: review of available data for mathematical modeling. Foodborne Pathog Dis. 2006. https://pubmed.ncbi.nlm.nih.gov/17199514/

[3] Boerlin P, Boerlin-Petzold F, Jemmi T. Use of listeriolysin O and internalin A in a seroepidemiological study of listeriosis in Swiss dairy cows. J Clin Microbiol. 2003. https://pubmed.ncbi.nlm.nih.gov/12624029/

[4] Warren J, Owen AR, Glanvill A, et al. A new bovine conjunctiva model shows that Listeria monocytogenes invasion is associated with lysozyme resistance. Vet Microbiol. 2015. https://pubmed.ncbi.nlm.nih.gov/25778543/

[5] Fenlon DR, Wilson J, Donachie W. The incidence and level of Listeria monocytogenes contamination of food sources at primary production and initial processing. J Appl Bacteriol. 1996. https://pubmed.ncbi.nlm.nih.gov/8972091/

[6] Ho AJ, Ivanek R, Gröhn YT, et al. Listeria monocytogenes fecal shedding in dairy cattle shows high levels of day-to-day variation and includes outbreaks and sporadic cases of shedding of specific L. monocytogenes subtypes. Prev Vet Med. 2007. https://pubmed.ncbi.nlm.nih.gov/17481754/

[7] Ivanek R, Gröhn YT, Jui-Jung Ho A, et al. Markov chain approach to analyze the dynamics of pathogen fecal shedding-example of Listeria monocytogenes shedding in a herd of dairy cattle. J Theor Biol. 2007. https://pubmed.ncbi.nlm.nih.gov/17092523/

[8] Papić B, Golob M, Kušar D, et al. Source tracking on a dairy farm reveals a high occurrence of subclinical mastitis due to hypervirulent Listeria monocytogenes clonal complexes. J Appl Microbiol. 2019. https://pubmed.ncbi.nlm.nih.gov/31432571/

[9] Wiedmann M, Bruce JL, Knorr R, et al. Ribotype diversity of Listeria monocytogenes strains associated with outbreaks of listeriosis in ruminants. J Clin Microbiol. 1996. https://pubmed.ncbi.nlm.nih.gov/8727881/

[10] Wiedmann M, Czajka J, Bsat N, et al. Diagnosis and epidemiological association of Listeria monocytogenes strains in two outbreaks of listerial encephalitis in small ruminants. J Clin Microbiol. 1994. https://pubmed.ncbi.nlm.nih.gov/8027356/

[11] Gismervik K, Aspholm M, Rørvik LM, et al. Invading slugs (Arion vulgaris) can be vectors for Listeria monocytogenes. J Appl Microbiol. 2015. https://pubmed.ncbi.nlm.nih.gov/25580873/

[12] Vázquez-Boland JA, Dominguez L, Blanco M, et al. Epidemiologic investigation of a silage-associated epizootic of ovine listeric encephalitis, using a new Listeria-selective enumeration medium and phage typing. Am J Vet Res. 1992. https://pubmed.ncbi.nlm.nih.gov/1595962/

[13] Laven RA, Lawrence KR. An outbreak of iritis and uveitis in dairy cattle at pasture associated with the supplementary feeding of baleage. N Z Vet J. 2006. https://pubmed.ncbi.nlm.nih.gov/16751847/

[14] Welchman D, Hooton JK, Low JC. Ocular disease associated with silage feeding and Listeria monocytogenes in fallow deer. Vet Rec. 1997. https://pubmed.ncbi.nlm.nih.gov/9234555/

[15] Munir MZ, Khan JA, Ijaz M, et al. Molecular characterization and hematological analysis of Listeria monocytogenes infection in dairy cows in Punjab (Pakistan). Arch Microbiol. 2022. https://pubmed.ncbi.nlm.nih.gov/35239048/

[16] Whitman KJ, Bono JL, Clawson ML, et al. Genomic-based identification of environmental and clinical Listeria monocytogenes strains associated with an abortion outbreak in beef heifers. BMC Vet Res. 2020. https://pubmed.ncbi.nlm.nih.gov/32087722/

[17] Low JC, Chalmers RM, Donachie W, et al. Pyrolysis mass spectrometry of Listeria monocytogenes isolates from sheep. Res Vet Sci. 1992. https://pubmed.ncbi.nlm.nih.gov/1410821/

[18] Barkallah M, Gharbi Y, Hmani M, et al. Locked nucleic acid probe-based real-time PCR for the diagnosis of Listeria monocytogenes in ruminants. Mol Cell Probes. 2016. https://pubmed.ncbi.nlm.nih.gov/26921518/

[19] Elsayed MM, Elkenany RM, Zakaria AI, et al. Epidemiological study on Listeria monocytogenes in Egyptian dairy cattle farms' insights into genetic diversity of multi-antibiotic-resistant strains by ERIC-PCR. Environ Sci Pollut Res Int. 2022. https://pubmed.ncbi.nlm.nih.gov/35298798/

[20] Papić B, Kušar D, Zdovc I, et al. Retrospective investigation of listeriosis outbreaks in small ruminants using different analytical approaches for whole genome sequencing-based typing of Listeria monocytogenes. Infect Genet Evol. 2020. https://pubmed.ncbi.nlm.nih.gov/31629888/