Pathology, diagnosis, and molecular characterization of salmonellosis in Kadaknath chickens

Introduction

Salmonella is a major zoonotic pathogen affecting both animals and humans (Li Q Wang et al., 2020) [33] . Infections in poultry reduce production and contaminate the food chain, leading to economic losses and public health risks (Syelejmani D et al., 2016). Despite control measures like vaccination (Zhang-Barber L et al., 1999) [37] , Salmonella remains a global challenge, as survivors become permanent carriers (Shivprasad and Barrow, 2008). Over 2,600 serotypes have been identified (Hendriksen et al., 2011) [13] , exhibiting similar genomes but different host ranges. Specifically, S. gallinarum and S. pullorum cause fowl typhoid and pullorum disease, resulting in significant losses in the poultry industry, particularly in China (Soria et al., 2012) [30] . Meanwhile, S. Typhimurium and S. Enteritidis are prevalent food-borne pathogens; S. Typhimurium causes typhoid-like illness, while S. Enteritidis is a leading cause of human gastroenteritis (Vugia DJ et al., 1999). Other serotypes, such as S. Kentucky and S. Newport, are also commonly found in poultry (Long et al., 2016) [18] . Fowl typhoid and Pullorum disease are globally prevalent, particularly in developing nations where antimicrobial resistance is increasing. Free-range systems may elevate infection risks. In India, Salmonellosis is documented across multiple regions. Transmission occurs trans-ovarianly or horizontally via hatcheries and contaminated litter, with wild birds acting as reservoirs. Salmonella causes bacteraemia and septicaemia through cellular invasion and multiplication. Clinical signs in young chicks include high mortality and poor quality, while older birds exhibit anaemia, respiratory distress, and diarrhoea.

Breeding flocks often experience reduced egg production and hatchability. Pathological lesions range from peritonitis and inflamed yolk sacs in chicks to misshapen ovaries in adults, alongside septicaemic indicators like liver enlargement. Diagnosis is based on flock history, clinical presentation, and laboratory culture confirmed by biochemical testing. PCR technology represents an exceptionally rapid and highly sensitive alternative for clinical diagnosis, offering the robust capability to detect and differentiate multiple serovars simultaneously from a single biological sample (Xiong et al., 2018) [35] . To date, there is a significant lack of comprehensive benchmark data or systematic epidemiological studies regarding the prevalence of Pullorum disease and Fowl typhoid specifically within the Kadaknath chicken populations found in the state of Chhattisgarh. As per recent studies, the prevalence of Salmonellosis in Kadaknath farms located in the Durg, Raipur, and Rajnandgaon districts has been observed to be at rates of 9.2%, 7.9%, and 5.2%, respectively (Pradeep et al., 2026) [22] . At present, diagnostic efforts for Salmonellosis in this region rely almost exclusively on traditional, timeconsuming phenotypic methods which often lack the necessary precision. Consequently, there is an urgent and clear necessity to develop and implement a standardized molecular diagnosis protocol to accurately identify Pullorum disease and Fowl typhoid. This study focuses on the detailed molecular characterization of Salmonella pullorum and Salmonella gallinarum species to enhance diagnostic accuracy and disease management. The study was conducted with following objectives:  To examine pathomorphological changes in chicken organs due to Salmonellosis.  Molecular characterization of Salmonella pullorum and Salmonella gallinarum species. Nucleotide sequences were aligned using NCBI multiple alignment tool and compared with Salmonella enterica sequences. The consensus sequence of rat A ROD gene was derived. Rat A ROD gene analysis was done using an online tool. The sequence was uploaded to the ExPASy server (www.expasy.org/tool) for primary structure analysis.

Materials & Methods

Phylogenetic analysis

The consensus sequence of rat A ROD gene of S. gallinarum was used for homology search with BLAST. Multiple sequence alignment was done using Clustal W, and a phylogenetic tree was generated using MEGA X (Kumar et al., 2018) [15] neighbor method (Saitou and Nei, 1987) [23] . All sequences were retrieved from GENBANK for analysis. Accession number from GENBANK was Banklt2529837 BSeq#1 OL871236.

Clinical signs

Total of 210 dead birds showed signs like decreased feed intake, depression, huddling, droopy wings, labored breathing, diarrhea, dehydration, and ruffled feathers, suspected for Salmonellosis. These findings matched Shivaprasad (2000) [28] , who reported similar signs in fowl typhoid and pullorum disease, including depression, somnolence, anorexia, huddling, droopy wings, dehydration, labored breathing, diarrhea, ruffled feathers, weakness, and soiled vent. Barrow et al. (2012) [1] , Soufy et al. (2016) [29] ,

Blackall et al. (2008) [2] , and Haider et al. (2014) [12] also reported similar signs in Salmonellosis-affected chickens.

Gross pathology

Most birds with Salmonellosis showed hepatomegaly, congestion, hemorrhages, and necrotic foci (Fig. 2), consistent with findings by Freitas Neto et al. (2007) [9] , [10] . Characteristic lesions included a friable liver with bronze discoloration and necrotic foci, similar to those reported by Saha et al. (2012) [24] . The spleen was enlarged with congestion and necrotic foci (Fig.

3 (2013) [10] . Intestinal examination revealed congestion, hemorrhages, and enteritis (Fig. 4). The intestines and caeca showed dilation with yellowish exudates (Fig. 4), and chronic cases exhibited typhlitis with necrotic casts, agreeing with several studies ( [11,31,34,14,16,24,8] . Since these clinical and post-mortem signs are not unique to specific Salmonella infections, definitive diagnosis requires organism isolation, serological testing, and PCR assay (OIE, 2012) [21] .

Histopathological examination

Histopathological examination of liver sections from birds affected by Salmonellosis revealed extensive degenerative alterations, including vacuolar degeneration and coagulative necrosis, accompanied by vascular congestion, hemorrhages, and inflammatory cell infiltration. Notable hepatocellular necrosis was characterized by eosinophilic hepatocytes and pyknotic nuclei. These pathological findings are consistent with the lesions reported by Soufy et al. (2016) [29] in broiler chicks challenged with S. gallinarum, as well as observations documented by Hafeji et al. (2001) [11] , Hossain et al. (2006) [14] , Wigley et al. (2005) [34] , and Casagrande et al. (2014) [5] . Similar manifestations were also recorded in chickens infected with S. gallinarum by Prasanna and Paliwl (2003). The virulence of S. gallinarum has been attributed to the presence of a virulence plasmid (Bailey and Scott, 1990). The pathogenesis likely involves initial intestinal localization following ingestion, subsequently progressing to bacteremia and hepatitis. Spleen sections exhibited degeneration, necrosis, and lymphocyte depletion within germinal centers, with certain cases demonstrating reticuloendothelial cell proliferation. These splenic lesions align with the findings of Haider et al. [31] , Wigley et al. (2005) [34] , Hossain et al. (2006) [14] , Saha et al. (2012) [24] , and Fotouh et al. (2014) [8] . Many organisms exhibit resistance to intestinal acidity and proteolytic enzymes, being subsequently shed through feces. Salmonella organisms adhere to mannose-like receptors on the microvilli of epithelial cells. Localized microvilli degeneration at the attachment sites facilitates bacterial entry, thereby breaching the cell surface. Following epithelial penetration, the pathogenicity is contingent upon bacterial multiplication, systemic spread, toxin production, cellular damage, and inflammatory responses.

References

1. Barrow PA, Jones MA, Smith AL, Wigley P. The long view: Salmonella -the last forty years. Avian Pathol. 2012;41:413-420.
2. Blackall PJ, Soriano EV. Avian salmonellosis. In: Diseases of Poultry. 12th ed. Blackwell Publishing, Oxford; 2008. p. 789-803.
3. Berchieri A Jr, Page KL, Smith AL, Barrow PA. Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent, disease-free carriage in chickens. Infect Immun. 2001;69(12):7873-7879.
4. Carli KT, Unal CB, Caner V, Aysegul E. Detection of Salmonellae in chicken faeces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis. J Clin Microbiol. 2001;39(5):1871-1876.
5. Casagrande RA, Wouters AT, Wouters F, Pissetti C, Cardoso MR, Driemeier D. Fowl typhoid (Salmonella Gallinarum) outbreak in Japanese quail (Coturnix coturnix japonica). Avian Dis. 2014;58:491-494.
6. Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;39:783791.
7. Feng Y, Johnston RN, Liu GR, Liu SL. Genomic comparison between Salmonella Gallinarum and Pullorum: differential pseudogene formation under common host restriction. PLoS One. 2013;8(3):e59427.
8. Fotouh A, Gab-Allah MS, Tantawy AA, Soufy H, Nasir SM. Alterations of blood components in broiler chicks experimentally infected with Salmonella Gallinarum. Global Veterinaria. 2014;13:787-793.
9. Freitas Neto OC, Arroyave W, Alessi AC, Fagliari JJ, Berchieri A Jr. Infection of commercial laying hens with Salmonella Gallinarum: clinical, anatomopathological and haematological studies. Brazilian Journal of Poultry Science. 2007;9:133-141.
10. Garcia KO, Berchieri A, Santana AM, Alarcon MF, Freitas NC, Fagliari JJ. Experimental infection of commercial layers with wild or attenuated Salmonella Gallinarum mutant strains: anatomic pathology. Brazilian Journal of Poultry Science. 2013;15(2):91103.
11. Hafeji YA, Shah DH, Joshi BP, Roy A, Prajapati KS. Experimental pathology of field isolates of Salmonella gallinarum in chicken. Indian Journal of Poultry Science. 2001;36:338-340.
12. Haider G, Chowdhary EH, Hossain M. Mode of vertical transmission of Salmonella enterica subsp. enterica serovar Pullorum in chickens. African Journal of Microbiology Research. 2014;8:1344-1351.
13. Hendriksen RS, Vieira AR, Karlsmose S, et al. Global monitoring of Salmonella serovar distribution. Foodborne Pathogens and Disease. 2011;8(8):887-900.
14. Hossain MS, Chowdhury EH, Islam MM, Haider MG, Hossain MM. Avian salmonellosis infection: isolation and identification of organisms and histopathological study. Bangladesh Journal of Veterinary Medicine. 2006;4(1):7-12.
15. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 2018;35:1547-1549.
16. Islam MM, Haider MG, Chowdhury EH, Hossain MM. Seroprevalence and pathological study of Salmonella infections in layer chickens. Bangladesh Journal of Veterinary Medicine. 2006;4(2):79-85.
17. Li Q, Wang X, Xia J, et al. Salmonella-containing vacuole development in avian cells and characteristics of cigR in Salmonella enterica serovar Pullorum replication within macrophages. Veterinary Microbiology. 2018;223:65-71.
18. Long M, Li H, Deng W, et al. Disinfectant susceptibility of different serotypes isolated from chickens and egg production chains. Journal of Applied Microbiology. 2016;121(3):672-681.
19. Newton CR. Nucleic acid substrates. In: PCR: Essential Data. New York: John Wiley and Sons; 1995. p. 24-26.
20. Oliveira SD, Santos LR, Schuch DMT, Silva AB, Salle CTP, Canal CW. Detection and identification of Salmonella from poultry-related samples by PCR. Veterinary Microbiology. 2002;87:25-35.
21. OIE. Fowl typhoid and Pullorum disease. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 7th ed. Paris: OIE; 2012. p. 1-14.
22. Dubey P, Jolhe DK, Ghosh RC, Kumar V, Sonkusale PM. Prevalence, diagnosis and colony characterisation of salmonellosis in Kadaknath chickens. Biological Forum. 2026;18(3):74-80.
23. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 1987;4:406-425.
24. Saha AK, Sufian MA, Hossain MI, Hossain MM. Salmonellosis in layer chickens: pathological features and isolation of bacteria from ovaries and eggs. Journal of Bangladesh Agricultural University. 2012;10:61-67.
25. Thomson NR, Clayton DJ, Windhorst D, et al. Comparative genome analysis of Salmonella Enteritidis International Journal of Advanced Biochemistry Research https://www.biochemjournal.com PT4 and Salmonella Gallinarum 287/91. Genome Research. 2008;18(10):1624-1637.
26. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies using neighbor-joining method. Proceedings of the National Academy of Sciences USA. 2004;101:11030-11035.
27. Sannat C, Patyal A, Rawat N, et al. Characterization of Salmonella gallinarum outbreak in Raigarh, Chhattisgarh. Vet World. 2017;10:144-146.
28. Shivaprasad HL. Fowl typhoid and pullorum disease. Revue Scientifique et Technique Office International des Epizooties. 2000;19:405-424.
29. Soufy H, Gab-Allah MS, Tantawy AA, Fotouh A, Nasir SM. Pathogenesis of experimental Salmonella Gallinarum infection in broiler chicks. Egyptian Journal of Veterinary Science. 2016;47(2):117-131.
30. Soria MC, Soria MA, Bueno DJ. Comparison of culture methods and PCR assays for Salmonella detection. Poultry Science. 2012;91(3):616-626.
31. Syed HR, Khan MS, Hamayun K, Nazir A, Bhatti WM. Incidence and pathology of Salmonella gallinarum infection in chicken. Journal of Animal and Veterinary Advances. 2004;3:175-178.
32. Vugia DJ, Samuel M, Farley MM, et al. Invasive Salmonella infection in the United States. Clinical Infectious Diseases. 2004;38:S149-S156.
33. Wang X, Wang H, Li T, et al. Characterization of Salmonella spp. isolated from chickens in Central China. BMC Veterinary Research. 2020;16:299.
34. Wigley P, Hulme SD, Powers C, et al. Infection of reproductive tract and eggs with Salmonella Pullorum. Infection and Immunity. 2005;73:2986-2994.
35. Xiong D, Song L, et al. Identification and discrimination of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum based on multiplex PCR. Frontiers in Microbiology. 2018;9:1718.
36. Zanetti NS, Carli SD, Souza MN, et al. Molecular detection of Salmonella Gallinarum from poultry farms. Journal of Applied Poultry Research. 2019;28:13351341.
37. Zhang-Barber L, Turner AK, Barrow PA. Vaccination for control of Salmonella in poultry. Vaccine. 1999;17(20-21):2538-2545.