لنفادنیت، کورینه باکتریوم سودوتوبرکلوزیس، نشخوارکنندگان کوچک، تنوع میکروبی، ایران

1.Introduction
Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is a major bacterial disease affecting small ruminants globally, leading to significant economic losses through reduced wool and milk production, reproductive challenges, premature culling, carcass condemnation, and occasional mortality. This gram-positive, facultative intracellular, non-spore-forming, non-capsulated, non-motile pleomorphic bacterium uses a potent phospholipase D (PLD) exotoxin and a mycolic acid-rich cell wall to evade host defenses and cause tissue necrosis [1-5]. In Iran, where small ruminants are critical to rural livelihoods, CLA impact is substantial yet poorly documented [6, 7].
CLA typically presents as enlarged superficial lymph nodes (e.g. submandibular, parotid, prescapular, prefemoral, popliteal, supramammary) and visceral lesions in organs such as the liver, lungs, and kidneys [8]. Lesions are characterized by necrotizing, purulent inflammation with caseous cores [9]. Diagnosis relies on bacterial culture, though chronic lesions often yield few viable bacteria, complicating detection [10]. Biochemical tests and molecular methods, such as polymerase chain reaction (PCR), improve confirmation, despite variability in results [11, 12].
Recent studies have expanded CLA’s epidemiological scope. Research by de Sá et al. (2023) and Almeida et al. (2024) highlights co-infections with pathogens like Staphylococcus spp. and Trueperella pyogenes, alongside environmental triggers such as shearing and overcrowding [13, 14]. Genomic analyses reveal strain diversity, influencing virulence and vaccine response [5, 15]. Emerging evidence also suggests zoonotic potential, with human cases linked to occupational exposure [16]. This study investigates CLA prevalence, clinical features, and bacteriological profile in Khorasan Razavi Province, Northeast Iran, to inform regional control strategies and contribute to global understanding.

2. Materials and Methods
The study covered 15 small ruminant flocks in Khorasan Razavi Province, Northeast Iran, comprising 4,733 animals (4,640 sheep, 93 goats). Clinical examinations identified lymphadenitis cases, documenting age, sex, affected lymph nodes, lesion size, and consistency (e.g. firm, caseous, liquefied). Pus samples were collected from 10–25% of affected animals per flock (102 total), using manual restraint, 70% alcohol disinfection, and a 16-gauge sterile syringe. Samples were stored near ice packs and transported to Ferdowsi University of Mashhad’s microbiological laboratory within 6 hours.
Samples were inoculated onto Columbia blood agar (with 5% sheep blood) and MacConkey agar, incubated at 37 °C for 48–72 hours under aerobic conditions, and inspected for colony morphology. Subcultures purified isolates as needed. Smears underwent Gram staining and microscopic analysis (1000×magnification), followed by biochemical tests: Catalase, oxidase, urease, motility, and fermentation (glucose, maltose, sucrose). Suspected C. pseudotuberculosis isolates were confirmed via synergistic hemolysis with Rhodococcus equi [2, 11]. 
Descriptive statistics calculated prevalence by flock, species, sex, and age group. Confidence intervals (95% CI) were computed for prevalence estimates using the Wilson score method. Pearson correlation coefficients assessed the relationship between flock size and prevalence. Chi-square tests evaluated associations between lymphadenitis prevalence and categorical variables (sex, age group, lymph node site). All statistical analyses were performed using SPSS (version 27.0; IBM Corp., Armonk, NY, USA), with the significance level set at P<0.05. 

3. Results
3.1. Descriptive outcome
Lymphadenitis prevalence across the study area was 11.59% (95% CI, 10.58%, 12.66%), with flock-specific rates ranging from 0% to 28.57% (Table 1).

No significant linear relationship was found between flock size and prevalence (r=-0.018, P=0.23).
Affected lymph nodes included submandibular (51.35%), retropharyngeal (18.02%), parotid (15.32%), prescapular (9.01%), superficial cervical (3.60%), and others (inguinal, facial, etc., 2.70%) (Figures 1 and 2, Table 2).

A chi-square test showed significant variation in lymph node site distribution (P<0.001).
Lesions averaged 2–5 cm in diameter, with 80% exhibiting caseous consistency and 15% showing liquefaction, indicative of chronicity. Females were more affected (66.67%) than males (33.33%) (P<0.001), possibly due to management practices like milking or shearing exposure. Age distribution peaked at 2–3 years (38.24%), followed by <1 year (33.33%), 1–2 years (24.51%), and >3 years (3.92%) (P<0.001) (Table 3, Figure 3).

3.2. Microbiological results
Bacteria were isolated in 56.86% of samples, with 43.14% sterile. C. pseudotuberculosis was isolated in 19.6% (20/102) of samples, forming small, dry, white colonies with β-hemolysis on Columbia blood agar. Other isolates included Coryneform bacteria (14.7%), Actinobacillus spp. (7.8%), Actinomyces spp. (4.9%), Trueperella pyogenes (3.9%), mixed bacteria (2.9%), Actinobacillus lignieresii (2.0%), Micrococcus spp. (2.0%), Acinetobacter spp. (1.0%), Staphylococcus saprophyticus (1.0%), Escherichia coli (1.0%), and Yersinia spp. (1.0%) (Table 4, Figure 4).

3.3. Epidemiological insights
Sheep showed a slightly higher prevalence (8.62%, 95% CI, 7.84%, 9.46%) than goats (8.60%, 95% CI, 4.43%, 15.99%), though the small goat sample (n=93) limits robust comparison (P=0.99). Flock size showed no significant correlation with prevalence (r=-0.018, P=0.23), suggesting transmission dynamics beyond density (Figure 5).

The submandibular focus (51.35%) may reflect regional feeding practices (e.g. prickly forage) or shearing injuries, differing from prescapular dominance reported elsewhere [12].

4. Discussion
This study confirms CLA as a significant concern in Khorasan Razavi’s small ruminant populations, with an overall prevalence of 11.59% (95% CI, 10.58%, 12.66%), affecting 8.62% of sheep and 8.60% of goats. The flock-specific prevalence range (0–28.57%) aligns with global patterns but varies from other Iranian studies. For instance, Zavoshti et al. (2015) reported a higher abattoir-based prevalence of 12.60–20.08% in Iranian sheep [17], likely capturing subclinical cases missed in our clinical inspections. Globally, Said et al. (2015) reported 5.1% in North African sheep [18], Nuttall et al. (2018) found 0.2–7.14% in New Zealand [19], and Guimarães et al. (2015) noted a serological prevalence of 70.9% in Brazil [20], highlighting diagnostic method influences. Our clinical prevalence is moderate compared to these, possibly due to regional differences in husbandry or detection methods.
The predominance of submandibular lymph node involvement (51.35%) contrasts with studies reporting prescapular or parotid dominance, such as Cetinkaya et al. (2016) in European flocks [12] or Kuria and Ngatia (1990) in Kenya [21]. This may stem from local practices, such as shearing injuries or thorny forage exposure, which facilitate bacterial entry at submandibular sites. The significant lymph node site variation (P<0.001) underscores the need to consider regional management practices in CLA epidemiology.
The peak incidence in the 2–3-year age group (38.24%) aligns with shearing-related transmission, as noted by Paton et al. (1994) [22], with a significant age effect (P<0.001). The decline in older animals (>3 years, 3.92%) likely reflects culling practices, consistent with Silva et al. (2018) [4]. The higher prevalence in females (66.67%, P<0.00) may result from prolonged herd retention for milking or breeding, increasing exposure risks compared to males, a pattern also observed by Guimarães et al. (2015) [20].
Bacteriological analysis identified C. pseudotuberculosis in 19.6% of samples, consistent with its role as the primary CLA pathogen [23-25]. However, the diverse microbial profile, including Actinobacillus spp. (7.8%), Trueperella pyogenes (3.9%), and novel isolates like Acinetobacter spp. and Yersinia spp. (1.0% each), suggests a complex etiology. This mirrors findings by de Sá et al. (2023) and Almeida et al. (2024), who reported multi-pathogen dynamics in CLA lesions [13, 14]. The presence of A. lignieresii raises concerns about cross-species transmission, as noted by Rodriguez et al. (2025) [5]. The high sterility rate (43.14%) exceeds reports from acute cases (e.g. 20% in Martins et al., 2024 [15]), likely due to chronic lesion encapsulation or sampling limitations, as described by Costa et al. (2017) [10]. Compared to Magdy et al. (2017) in the Middle East, where C. pseudotuberculosis dominated (26.92%) [8], our lower isolation rate may reflect regional strain differences or diagnostic challenges.
The lack of correlation between flock size and prevalence (r=-0.018, P=0.23) contrasts with studies like Hajtos et al. (2017), which linked larger flocks to higher CLA rates due to crowding [25]. This discrepancy suggests that transmission in Khorasan Razavi Province is driven more by husbandry practices (e.g. shearing, feeding) than flock density. The zoonotic potential, though rare, is concerning given reports of human cases [16], particularly for shepherds and shearers in this region.
These findings highlight CLA’s economic and welfare impacts in northeast Iran, necessitating integrated control strategies. Compared to Iran’s national data (e.g. Zavoshti et al., 2015 [17]), our prevalence is lower, possibly due to clinical versus abattoir-based detection. Globally, our microbial diversity aligns with emerging multi-pathogen models [13, 14], but the high sterility rate suggests a need for advanced diagnostics like real-time PCR or metagenomics, as recommended by Cetinkaya et al. (2016) [12]. Recombinant PLD vaccines, tested by Martins et al. (2024) [15], and CRISPR-based strain typing [27] offer promising solutions but are under utilized in Iran. Enhanced biosecurity, targeting shearing and environmental reservoirs, is critical to reducing CLA’s burden, aligning with global trends toward precision epidemiology.

Ethical Considerations
Compliance with ethical guidelines
All applicable international, national, and institutional guidelines for the care and use of animals were followed.

Data availability
The data supporting this study’s findings are available upon request from the corresponding author.

Funding
This study was financially supported by Ferdowsi University of Mashhad, Mashhad, Iran (Grant No.: 24721).

Authors' contributions
Conceptualization, study design, statistical analysis, review and editing: Gholamreza Mohammadi; Data acquisition: Anoosh Firozeh; Project administration, technical, and material support, data analysis and interpretation: Gholamreza Mohammadi and Mehrnaz Rad; Writing the original draft: All authors. 

Conflict of interest
The authors declared no conflict of interest.

Acknowledgements
We gratefully acknowledge the support from Ferdowsi University of Mashhad, Mashhad, Iran.

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