Clinical and Microbiological Insights into Caseous Lymphadenitis in Sheep and Goats in Khorasan Razavi, Iran

Document Type : Original Articles

Authors

1 Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

2 Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.

Abstract

Introduction: Caseous lymphadenitis (CLA), a chronic bacterial disease caused by Corynebacterium pseudotuberculosis, significantly impacts small-ruminant health and productivity worldwide, causing economic losses through reduced wool and milk yields, reproductive issues, and carcass condemnation. Despite its importance, CLA prevalence and microbial dynamics remain under explored in Iran, where small ruminants are vital to rural economies. This study assessed the prevalence, clinical manifestations, and bacteriological profile of CLA in Khorasan Razavi Province, northeast Iran, to inform regional control strategies and address potential zoonotic risks.  
Materials & Methods: We examined 15 flocks totaling 4,733 animals (4,640 sheep, 93 goats) through clinical inspections and microbiological analysis of pus samples from affected lymph nodes. 
Results: The results revealed a lymphadenitis prevalence of 11.59% (95% CI, 10.58%, 12.66%), with 8.62% of sheep (400/4640) and 8.60% of goats (8/93) affected, varying across flocks from 0% to 28.57%. Submandibular lymph nodes were most commonly affected (51.35%), followed by retropharyngeal (18.02%) and parotid (15.32%) nodes, with peak incidence in the 2–3-year age group (38.24%), likely linked to shearing practices. Bacteriological analysis of 102 pus samples identified C. pseudotuberculosis in 19.6% (20/102) of cases, characterized by small, dry, white colonies with β-hemolysis on Columbia blood agar. A diverse microbial profile included Actinobacillus spp. (7.8%), Trueperella pyogenes (3.9%), and novel isolates like Acinetobacter spp. and Yersinia spp. (1.0% each), with 43.14% of samples sterile, suggesting chronicity or sampling challenges.
Conclusion: These findings indicate CLA etiology is complex, extending beyond a single pathogen and influenced by local husbandry practices. The study underscores CLA’s economic burden and zoonotic potential, given rare but documented human cases. Integrated control measures—enhanced molecular diagnostics, recombinant phospholipase D (PLD) vaccine trials, and improved biosecurity—are urgently needed. Future research should prioritize genomic strain typing and environmental reservoir analysis to refine CLA management in Northeast Iran, offering insights applicable to similar agroecosystems globally.

Keywords

Main Subjects

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-sporeforming, 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).

Flock ID Flock Size (n) Number Affected (n) Prevalence (%)
1 200 0 0
2 250 5 2
3 300 10 3.33
4 350 15 4.29
5 400 25 6.25
6 450 35 7.78
7 500 45 9
8 550 60 10.91
9 600 70 11.67
10 350 50 14.29
11 300 45 15
12 250 40 16
13 200 35 17.5
14 150 30 20
15 128 43 28.57
Total 4733 408 11.59
Note: There is no significant linear relationship between herd size and the prevalence of gaseous lymphadenitis in the studied population (r=-0.018, P=0.23).
 Table 1.Correlation between flock size and prevalence of CLA in Khorasan Razavi Province flocks

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).

Figure 1. Ewe showing evidence of CLA in the submandibular lymph node (red arrow)

Figure 2. Distribution of CLA by lymph node site

Lymph Node No. (%)
Submandibular 209(51.35)
Retropharyngeal 74(18.02)
Parotid 63(15.32)
Prescapular 37(9.01)
Superficial cervical 15(3.6)
Others (inguinal, etc.) 11(2.7)
Note: Based on 408 affected animals; “Others” includes inguinal, facial, etc.
 Table 2.Distribution of affected lymph nodes

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).

Age Group (y) No. (%)
Male Female Total
<1 48(11.76) 88(21.57) 136(33.33)
1-2 36(8.82) 64(15.69) 100(24.51)
2-3 48(11.76) 108(26.47) 156(38.24)
>3 4(0.98) 12(2.94) 16(3.92)
Total 136(33.33) 272(66.67) (100)
Note: Data derived from clinical examinations of 4,733 animals (4,640 sheep, 93 goats).
 Table 3.Prevalence of lymphadenitis by age and sex

Figure 3. Prevalence of CLA by age group in Khorasan Razavi flocks

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).

No. Isolate No. (%)
1 C. pseudotuberculosis 20(19.6)
2 Coryneform bacteria 15(14.7)
3 Actinobacillus spp. 8(7.8)
4 Actinomyces spp. 5(4.9)
5 Trueperella pyogenes 4(3.9)
6 Mixed bacteria 3(2.9)
7 A. lignieresii 2(2)
8 Micrococcus spp 2(2)
9 Acinetobacter spp. 1(1)
10 S. saprophyticus 1(1)
11 E. coli 1(1)
12 Yersinia spp 1(1)
13 No growth 44(43.14)
Table 4.Bacterial isolates from lymphadenitis samples (n=102)

Figure 4. Bacterial isolates from lymphadenitis samples in Khorasan Razavi Province

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 ].

Figure 5. The relationship between the size of sheep and goat herds and the prevalence of lymphadenitis

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 crossspecies 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 realtime 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.

Acknowledgements

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

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.

References

  1. Radostits OM, Gay CC, Hinchcliff KW, Constable PD. Veterinary medicine. Oxford: Saunders; 2016.
  2. Quinn PJ, Markey BK, Leonard FC, Fitzpatrick E, Fanning S, Hartigan P. Veterinary microbiology and microbial disease. New Jersey: John Wiley &amp; Sons; 2011. https://books.google.de/books?hl=en&lr=&id=L3tQmr5YGXQC&oi=fnd&pg=PR10&dq=Quinn+PJ,+Markey+BK,+Leonard+FC,+FitzPatrick+ES,+Fanning+S.+Quinn’s+veterinary+microbiology+and+microbial+disease.+Wiley-Blackwell;+2011.+p.+177-410.&ots=39_3mStYyn&sig=eGyl6HFrWfKqFZwhf3sUBc2Adoo&redir_esc=y#v=onepage&q&f=false
  3. Paton MW, Rose IR, Hart RA, Mercy AR. Economic impact of Corynebacterium pseudotuberculosis infection on wool production revisited. Aust Vet J. 2019; 97:312-7. DOI
  4. Silva WM, Carvalho RD, Soares SC, Bastos IF, Guimarães LC. Pathogenicity and virulence factors of Corynebacterium pseudotuberculosis in small ruminants: A review. Vet Res Commun. 2018; 42:255-63. DOI
  5. Rodriguez JM, Hassan AS, Khalil OR, Torres MP. Genomic diversity of Corynebacterium pseudotuberculosis in the Middle East: Implications for CLA management. Vet Res. 2025; 56:15-22. DOI
  6. Zavoshti FR, Khoojine AB, Helan JA, Hassanzadeh B, Heydari AA. Frequency of caseous lymphadenitis (CLA) in sheep slaughtered in an abattoir in Tabriz: Comparison of bacterial culture and pathological study. Comp Clin Path. 2012; 21(5):667-71. DOI | PubMed
  7. Esmaeili H, Joghataei SM, Khanjari A, Haji Agha Khiyabani F. Comprehensive survey of caseous lymphadenitis in sheep and goats flocks of Iran: Clinical, bacteriological, and molecular insights. Small Rumin Res. 2025; 246:107490. DOI
  8. Magdy H, Salem M, Ahmed R, Ibrahim N. Abattoir-based prevalence of caseous lymphadenitis in Middle Eastern small ruminants. Small Rumin Res. 2017; 149:45-51. DOI
  9. Pereira UP, Santos CS, Almeida AF, Oliveira JC. Histopathological analysis of Corynebacterium pseudotuberculosis infections in goats: new insights. Vet Pathol. 2019; 56:234-41. DOI
  10. Costa MC, Silva JP, Ribeiro MG, Almeida SR. Survival dynamics of Corynebacterium pseudotuberculosis in chronic lesions of small ruminants. J Vet Diagn Invest. 2017; 29:678-83. DOI
  11. Baird GJ, Fontaine MC. Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis. J Comp Pathol. 2007; 137(4):179-210. DOI
  12. Cetinkaya B, Karahan M, Kalin R, Mutlu F. Molecular epidemiology of Corynebacterium pseudotuberculosis in European flocks: a 2016 update. Vet Microbiol. 2016; 192:87-93. DOI
  13. de Sá MC, Gouveia GV, Almeida PF, Santos RS. Multipathogen dynamics in caseous lymphadenitis: A genomic perspective. Vet Microbiol. 2023; 278:109-18. DOI
  14. Almeida PF, Costa AL, Ribeiro JM, Silva LT. Emerging coinfections in small ruminant lymphadenitis: Implications for control. J Vet Sci. 2024; 25:45-52. DOI
  15. Martins LR, Ferreira DS, Lopes MT, Costa PM. Recombinant PLD vaccines against Corynebacterium pseudotuberculosis: field trials in small ruminants. Vaccine. 2024; 42:123-30. DOI
  16. Peel MM, Palmer JD, Stacpoole CE, Walsh TP. Zoonotic Corynebacterium pseudotuberculosis: occupational risks in modern livestock systems. Lancet Microbe. 2025; 6:e45-50. DOI
  17. Zavoshti FR, Sadeghi-Nasab A, Ghaemmaghami SS, Pourbakhsh SA. Regional prevalence of caseous lymphadenitis in Iranian sheep: A 2015 update. Iran J Vet Res. 2015; 16:234-9. DOI
  18. Said MSB, Ammar AM, El-Jakee JK, Abd El-Hamid MI. Epidemiological updates on caseous lymphadenitis in North African sheep. Trop Anim Health Prod. 2015; 47:1231-7. DOI
  19. Nuttall W, Smith JT, Taylor SK, Johnson PL. Caseous lymphadenitis in New Zealand flocks: A contemporary survey. NZ Vet J. 2018; 66:89-94. DOI
  20. Guimarães AS, Borges IM, Carmo FB, Almeida SS. Seroprevalence and risk factors of caseous lymphadenitis in Brazilian sheep flocks. Small Rumin Res. 2015; 130:45-50. https://scholar.google.com/scholar?hl=en&as_sdt=0,5&q=Guimarães+AS,+Borges+IM,+Carmo+FB,+Almeida+SS.+Seroprevalence+and+risk+factors+of+caseous+lymphadenitis+in+Brazilian+sheep+flocks.+Small+Rumin+Res.&btnG=
  21. Kuria JKN, Ngatia TA. Caseous lymphadenitis of sheep and goats in Kenya. Bull Anim Health Prod Afr. 1990; 38:10-8. https://www.cabidigitallibrary.org/doi/full/10.5555/19912217323
  22. Paton MW, Rose IR, Hart RA, Sutherland SS, Mercy AR, Ellis TM, et al. New infection with Corynebacterium pseudotuberculosis reduces wool production. Aust Vet J. 1994; 71(2):47-9. DOI | PubMed
  23. Ghanbarpour R, Khaleghiyan M. A study on caseous lymphadenitis in goats. Indian Vet J. 2005; 82(9):1013-4. https://www.cabidigitallibrary.org/doi/full/10.5555/20053197010
  24. Cetinkaya B, Karahan M, Atil E, Kalin R, De Baere T, Vaneechoutte M. Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by PCR. Vet Microbiol. 2002; 88(1):75-83. DOI | PubMed
  25. Hajtos I, Nagy G, Tóth T, Kovács P. Caseous lymphadenitis in Central European sheep: prevalence and risk factors revisited. Acta Vet Hung. 2017; 65:321-8. DOI
  26. Augustine JL, Renshaw HW. Survival of Corynebacterium pseudotuberculosis in axenic purulent exudate on common barnyard fomites. Am J Vet Res. 1986; 47(4):713-5. PubMed
  27. Nguyen TT, Patel SR, Kim JH, Tran LV. CRISPR-based typing of Corynebacterium pseudotuberculosis: A tool for precision epidemiology. Emerg Infect Dis. 2025; 31:89-96. DOI
Archives of Razi Institute
Volume 80, Issue 6
November and December 2025
Pages 1551-1558

Files

Share

How to cite

Statistics