Pathomicrobial studies on Salmonella Gallinarum ... - Veterinary World

Veterinary World, EISSN: 2231-0916Available at www.veterinaryworld.org/Vol.6/Oct-2013/6.pdfRESEARCH ARTICLEOpen Access<strong>Pathomicrobial</strong> <strong>studies</strong> on Salmonella Gallinarum infectionin broiler chickensDivya Kumari, S.K. Mishra and Deepika LatherDepartment of Veterinary Pathology, College of Veterinary Science,Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana 125 001, IndiaCorresponding author: Divya Kumari, email: divyasworld19@gmail.comReceived: 02-05-2013, Revised: 17-06-2013, Accepted: 17-06-2013, Published online: 08-08-2013doi: 10.14202/vetworld.2013.725-729How to cite this article: Kumari D, Mishra SK and Lather D (2013) <strong>Pathomicrobial</strong> <strong>studies</strong> on Salmonella Gallinaruminfection in broiler chickens, Veterinary World 6(10): 725-729.AbstractAim: To conduct detailed pathomicrobial <strong>studies</strong> on Salmonella Gallinarum infection in broiler chickens.Materials and Methods: Bacteriological and pathological <strong>studies</strong> were conducted on 134 dead poultry birds collected from23 different farms suspected to be infected with S. Gallinarum.Results: Mortality pattern revealed that maximum mortality occurred in 1-2 week aged birds. Out of 23 Salmonella isolates,19 samples were identified as S. Gallinarum (9, 12) and 4 samples as Salmonella Enteritidis (9, 12: gm). Isolates were found tobe most sensitive to Polymyxin B (100%). The present study also showed re-emergence of chloramphenicol sensitivity(83.33%). Pathological lesions observed were bronze discolouration of liver, splenomegaly and necrotic foci on liver, spleenand heart. Microscopically, liver and spleen revealed aggregation of heterophils, lymphocytes and macrophages, nonsuppurativemyocarditis, fibrinous pericarditis, interstitial nephritis, necrotic enteritis and serofibrinous pneumonia.Conclusion: It was concluded that S. Gallinarum 9, 12 was the main serotype causing Salmonellosis in poultry birds.Polymyxin B was the most sensitive drug (100%) for Salmonella infection along with re-emergence of chloramphenicolsensitivity for Salmonella (83.33%) infection.Key words: broiler, drug sensitivity, pathology, Salmonella GallinarumIntroduction[7]. Mahajan et al. [8] studied major diseases affectingFowl typhoid, an acute septicaemic disease of broiler chickens during 1987-1990 in Haryana andavian species that is caused by Salmonella Gallinarum observed that fowl typhoid was the one that caused[1] affects all age groups of chickens. Morbidity is highmaximum mortality (10.54%) and accounted for theamong all age groups of the birds, whereas mortalitysecond highest number of outbreaks (198) after E. colimay range widely from 10% to 90% [2]. Maintaining a(266). Control of fowl typhoid is difficult [9] due todisease free status is a challenging exercise due to theendemicity of the disease, facultative intracellularrapid expanding nature of the industry. This is indicatednature of causative organism, both vertical [10] andby the fact that a number of Salmonella outbreakshorizontal [11] modes of transmission, presence ofreported in the world are a result of injudiciouscarrier stage and multiple drug resistance. Theintroduction of infected birds [3]. Thus, poultrywidespread and indiscriminate use of antibiotics in theindustry is facing great setbacks due to frequenttreatment of poultry diseases has lead to an increase inthe number of resistant Salmonella strains isolatedoutbreaks of salmonellosis [4]. Since its discovery,[12]. Antimicrobial resistance is a global public healthmany efforts have been made to control and prevent theconcern [13].occurrence in commercial poultry farming. However,Thus, the present study aimed to evaluate theoutbreaks of Salmonellosis still remain a serious economicpathological and bacteriological findings of fowlproblem in countries where control measures are nottyphoid and to understand their antibiogram.efficient or in those areas where the climatic conditionsfavour the environmental spread of these microbes [5]. Materials and MethodsThe economic losses are chiefly due to morbidity, Postmortem examination was conducted on 134mortality, reduced growth rate, reduced feed conversion dead poultry carcasses received from 23 differentefficiency, drop in egg production, decreased fertility farms and were divided into three groups on the basis ofand hatchability [6]. The endemicity of the disease has their age i.e. Group I (0-1 wks), Group II (1-2 wks) andbeen established in India including Haryana where the Group III (2-3 wks) to study the mortality pattern. Theinfection was first detected in Gurgaon area in 1981 study was conducted in the Department of VeterinaryPathology, College of Veterinary Science, Lala LajpatRai University of Veterinary and Animal Sciences,Hisar, Haryana. Gross pathological changes likeswollen, congested liver with bronze discoloration,Copyright: The authors. This article is an open access article licensedunder the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/2.0) which permitsunrestricted use, distribution and reproduction in any medium,provided the work is properly cited.Veterinary World, EISSN: 2231-0916 725

Available at www.veterinaryworld.org/Vol.6/Oct-2013/6.pdfTable-1. In-vitro chemotherapeutic drug sensitivity (% sensitivity) of Salmonella spp. isolated from visceral organs andheart bloodDrug% sensitivity to Salmonella spp.Amikacin 83.33Ampicillin 75.00Amoxycillin 91.66Cefixime 83.33Chloramphenicol 83.33Doxycycline Hydrochloride 91.66Ciprofloxacin 66.66Furoxone 83.33Cefoperazone 75.00Colistin 83.33Co-Trimoxazole 75.00Enrofloxacin 91.66Gentamycin 66.66Amoxycillin & Sulbactam 91.66Nalidixic acid 16.66Amoxyclav 75.00Ofloxacin 75.00Polymyxin B 100Streptomycin 75.00Oxytetracycline 83.33Isolation and characterization: Heart blood and tissuesamples collected aseptically were inoculated inoRappaport enrichment medium and incubated at 43 Cfor 24 hours. After 24 hours, inoculation was done onMacConkey's Lactose agar (MLA), Brilliant Greenagar (BGA), Salmonella Shigella Agar (SSA) andXylose Lysine Deoxycholate agar (XLD). Primaryidentification of the growth was done by colonymorphology and Gram's staining. The pure cultureswere subjected to various biochemical tests for furthercharacterization [14]. Isolates were subjected to invitrodrug sensitivity test using 20 antimicrobials by thedisc diffusion method as suggested by Bauer et al. [15].Serotyping of the isolates: Organisms positive forSalmonella spp. were sent to National Salmonella andEscherichia Centre (NSEC), Central ResearchInstitute, Kasauli, Himachal Pradesh, India for furtherserotyping.Pathological findings: All the organs and tissues of thecarcasses were examined critically for gross changesduring postmortem examination and the lesionsobserved were recorded. The formalin fixed tissueswere processed and stained using routine haematoxylinand eosin staining method [16].Results and Discussionenlarged spleen, multiple necrotic foci on spleen andliver; and multiple white nodules on heart with distortionin shape suggest that carcass can be suspected to beinfected with Salmonella Gallinarum. Clinical signsand history regarding vaccination was collected fromowners which showed that infected birds exhibitedacute illness, ruffled feathers, inappetance, difficulty inbreathing, reluctance to move and watery to mucoidyellowish diarrhoea which further confirms ourassumption of Salmonellla Gallinarum infection. Thebirds were vaccinated against Marek's disease (on 0day), Newcastle disease (at 5 days and 22 days) andInfectious bursal disease (at 14 days) and weremaintained under standard managemental practices.During postmortem examination, blood from thehearts of carcasses was collected with help of sterilizedsyringes for bacteriological examination. Differentorgans were examined critically for gross lesions andwere collected under aseptic conditions for bacterio-logical isolations. Tissue pieces of heart, liver, lung,spleen, intestine, pancreas, proventriculus, bursa offabricius and kidney were collected in a 10% bufferedformalin for histopathological examination.Mortality pattern: Age-wise mortality due to Salmonellaspp. revealed maximum mortality in group II followedby group I (Fig. 1). These results are in agreement withprevious reports [17]. It is likely due to the fact thatchicks are not fully immunocompetent when they arebelow 2 weeks of age because of a lower percentage ofCD4+CD8- in the thymus; CD4-CD8+ andCD4+CD8+ in the spleen [18] as well as due to absenceof protection from maternal antibodies at 1-2 weeks ofage.Figure-1. Age-wise distribution of mortality (%) in poultry due toSalmonella infectionSalmonella characterization: Out of 23 Salmonellaisolates sent for serotyping, 19 samples were identifiedas Salmonella Gallinarum (9,12) and 4 samples asSalmonella Enteritidis (9,12: gm). SalmonellaGallinarum and Salmonella Enteritidis share acommon immunodominant surface antigen (O9), suchVeterinary World, EISSN: 2231-0916 726

Available at www.veterinaryworld.org/Vol.6/Oct-2013/6.pdfFigure-2. Bronze discoloration withenlargement of liver (Salmonella Gallinaruminfection)Figure-3. Liver: Showing large area of coagulativenecrosis, surrounded by leucocytes.(Salmonella Gallinarum infection) H & E x 33Figure-4. Necrotic nodule on heart(Salmonella Gallinarum infection)Figure-5. Heart: Severe myocarditis withfragmented myocardial muscle fibres alongwith infiltration of lymphocytes and heterophils(Salmonella Gallinarum infection)H & E x 33Liver: Grossly, the liver appeared swollen, congestedalong with bronze discoloration (Fig.2). Microscopi-cally, hepatitis characterized by leucocytic infiltrationat perivascular areas along with hydropic vacuolationin hepatocytes, multiple necrotic foci was noticed withKupffer cell hyperplasia. Few areas displayed necrosisof hepatocytes, with focal aggregation of heterophils,lymphocytes and macrophages (Fig.3). Similardegenerative, necrotic and infiltrative lesions havebeen reported earlier [28-30].that S. Enteritidis preinfected poultry were protectedagainst colonization with Salmonella Gallinarum. Thisshows that coexistence of S. Gallinarum and S.Enteritidis in poultry prompts competition as a result ofthe shared immunodominant O9-antigen whichgenerates cross-immunity [19]. Several published<strong>studies</strong> in other countries have reported antimicrobialresistance in Salmonella [20-22]. Antibiogram patternsof isolates in the present investigation showed varyingdegree of sensitivity to the chemotherapeutic agentstested (Table-1). Salmonella spp. were found to bemost sensitive to polymyxin B (100%) which was inagreement with the findings of Kavitha et al. [23]followed by amoxicillin, amoxicillin sublactam,enrofloxacin, doxycycline hydrochloride (91.66%). Inour study, Salmonella spp. was found to be resistant tonalidixic acid (84%). The Salmonella isolates in Indiafrom 1996-99 and 2001 were reported to be 100%chloramphenical sensitive and sensitivity of 79% wasalso reported in 2000 [24]. The present study alsoobserved chloramphenical sensitivity of around83.33%; similar findings have been reported byTaddele et al. [25]. This re-emergence of chloramphenicolsensitivity could be attributed to the limited use of thisantimicrobial during the last decade in India [26].Highest sensitivity of Salmonella to polymyxin Bmight be due to the cationic action of polymyxin B oncell membrane of susceptible bacteria where it causesFigure-6. Spleen: Showing secondaryfollicles and multiple necrotic areas(Salmonella Gallinarum infection) H & E x 33Figure-7. Intestine: Necrotic enteritischaracterized by presence of necrotic massalong with desquamated epithelial cells andleucocytes in the lumen (SalmonellaGallinarum infection) H & E x 33bleb formation on cell wall and morphological changesin the cytoplasm [27]. The varying degree of resistanceand sensitivity to chemotherapeutic drugs has alsobeen reported by Sujatha et al. [28]. High prevalence ofnalidixic acid resistance among Salmonella Gallinarumwas also reported by previous workers [25]. In thepresent study, the resistance to above mentionedantibiotics might be attributed mainly to the frequentand indiscriminate use of these antibiotics fortreatment, prophylaxis and as feed supplements.Pathological <strong>studies</strong>: During necropsy examinationcarcasses of the Salmonella infected birds appearedjaundiced.Heart: The cardiac lesions consisted of mild tomoderate congestion and hemorrhage. In few cases,multiple white nodules with distorted shapes wereobserved on the heart (Fig.4). HistopathologicallyVeterinary World, EISSN: 2231-0916 727

severe degeneration and fragmentation of myocardialmuscle fibres (non suppurative myocarditis) was observedin most of the cases with leucocytic infiltration (Fig.5).Fibrinous pericarditis with infiltration of heterophils,lymphocytes and macrophages was also observed insome cases. Similar findings have been reported byMsoffe et al., [31].Available at www.veterinaryworld.org/Vol.6/Oct-2013/6.pdfbursal follicles along with interfollicular fibrosis. Lossof lymphoid tissue from follicles and degeneration ofbursa of fabricius has been reported by Garren andBarber [33]. These workers suggested that changes inbursa resulted from adverse physiological conditionslike anorexia, dehydration, anaemia etc.Poventriculus: The proventriculitis was characterizedby congestion, mucosal degeneration along withinfiltration of heterophils and lymphocytes in mucosaextending up to serosal layer in some cases. Mucosalglands were atrophied due to leucocytic infiltration in afew cases. There was degeneration of proventricularglands and lumen was filled with detached epithelialmass. The microscopic changes in proventriculus wereSpleen: There was splenomegaly along with multiplenecrotic foci on the surface. Microscopically, there wassevere depletion of lymphoid cells in white pulp alongwith reticuloendothelial cell hyperplasia (Fig.6).Besides this in a few cases, a number of secondarylymphoid follicles was noticed. Similar histopathologicalchanges including focal necrosis, reticuloendothelial cell hyperplasia and secondary lymphoid similar to those reported by Rao et al. [34].follicles have been reported by Shivaprasad [30].ConclusionIntestine: Grossly, catarrhal enteritis was evident insome cases characterized by thick slimy mucusexudate on mucosal surfaces in the lumen of intestine.Histopathologically there was desquamation ofmucosal epithelium resulting in denuded villi andlumen was filled with necrotic mass (Fig.7). Secretoryglands were atrophied at some places due to severeinfiltration of heterophils and mononuclear cells.Goblet cell hyperplasia and focal fibroblasticconnective tissue proliferation between the glands waspresent in many cases. Giannella [32] reported thatSalmonella induced diarrhoea is multifactorial. Theonset of fluid secretion is preceded by a massive influxof inflammatory cells leading to release of prostaglandinsthat stimulate intestinal adenylcyclase mediatedfluid secretion.Pancreas: In pancreas, there was mild congestion andhemorrhages along with mild degenerative changes.Leucocytic infiltration in acinar cells and interlobularconnective tissue was present in most of the cases. Thehistopathological changes in affected birds were incongruence with the observations of Prasanna et al.[29].Lungs: Lungs were highly congested with pneumoniclesions in most of the cases. Microscopically moderatecongestion and hemorrhages were observed withpresence of RBCs in the alveoli (red hepatization). Atplaces, there was presence of serofibrinous exudate inalveoli and interlobular septa. Similar lesions havebeen reported by Shivprasad [30].S. Gallinarum 9, 12 was mainly involved incausing Salmonellosis in poultry birds tested in thepresent study. In some of the cases S. Enteritidis wasalso isolated. Polymyxin B was the most sensitive drug(100%) for treating Salmonella infections. Surprisingly,our study also revealed that Salmonella infections arealso highly sensitive to chloramphenicol .Recommendations for farm ownersBecause polymyxin B was found to be the mostsensitive drug for Salmonella infection in the presentstudy, we recommend the drug for treatment ofsalmonellosis.Authors’ contributionsDK and SKM participated in the preparation ofexperimental design and the facilities of the research,drafted the manuscript. DL revised the final draft ofmanuscript. All authors read the final manuscript.AcknowledgementsThe authors express their gratitude to the Dean,College of Veterinary Science, Lala Lajpat RaiUniversity of Veterinary and Animal Sciences, Hissar,Haryana for providing the facilities and fund forconducting this experiment.Competing interestsThe authors declare that they have no competing interests.References1. Priyantha, M.A.R (2012) An Overview: Vaccination toKidneys: Grossly, kidneys were enlarged with control fowl typhoid in Commercial layers, Sri Lanka.prominent lobulation and necrotic foci on the surface. Wayamba Journal of Animal Science, Pp.- 23-25.2. Latife Beyaz, Ayhan Atasver, Fuat Aydin, K. SemihMicroscopically, glomeruli were contracted alongwith Gumssoy and Secil Abay (2010) Pathological and clinicaldegenerative changes in the renal tubular epithelium. findings and tissue distribution of Salmonella GallinarumSimilar degenerative and infiltrative changes in infection in turkey poults. Turk. J. Vet. Anim. Sci., 34(2): 101-kidneys of birds affected with fowl typhoid have been 110.3. Meeusen, E.N.T., Waker, J., Peter, A., Pastoret, P.P. anddescribed by Shivaprasad [30].Jungersen, G. (2007) Current status of Veterinary vaccine.Bursa of fabricius: Grossly, only mild congestion was Clinical Microbiology Review. Pp.- 489-510.4. Fatma, A.G., El-Gohary, A.H., El-Bably, M.A. andobserved in bursa of Fabricius. Histopathological Mohamed, A.A. (2012). Vitro antibiotic sensitivity ofchanges include mild depletion of lymphoid tissue in isolated strains of salmonella and E. coli from poultry farm.Veterinary World, EISSN: 2231-0916 728

Available at www.veterinaryworld.org/Vol.6/Oct-2013/6.pdfCompandium of 7th Int. Sci. Conf., Mansoura, 28-30 August. Journal of Veterinary Sciences. 2(1): 33-36.Pp.- 191-199.20. Pokharel P, Rai SK, Karki G, Katuwal A, Vitrakoti R. and5. Barrow, P.A. and Freitas Neto, O.C. (2011) Pullorum disease Shrestha SK (2009) Study of enteric fever and antibogram ofand fowl typhoid--new thoughts on old diseases: a review. Salmonella isolates at a teaching hospital in KathmanduAvian Pathol. 40 (1): 1-13. Valley. Nepal Med. Coll. J., 11(3): 176-178.6. Mamta, S.K. Mishra, and Deepika Lather (2010) 21. Wei, L.S. and Wee, W. (2011) Antibiogram and heavy metalAmeliorating effect of tulsi (ocimum sanctum) leaf powder resistance pattern of Salmonella spp. Isolated from wildon pathology of salmonella gallinarum infection in broiler Asian Sea Bass (Lates calcalifer) from Tok Bali, Kelantan,chickens. Haryana Vet. 49: 6-10. Malaysia. Jordan J. Biological Sci., 4(3): 125-128.7. Anon (1980-81) Annual progress report of disease 22. Selvaraj, R., Das, R., Ganguly, S., Ganguli, M., Dhanalainvestigationlaboratory, Gurgaon, Department of Veterinary kshmi, S. and Mukhopadhayay, S.K. (2010) Characteri-Public Health and Epidemiology, Haryana Agricultural zation and antibiogram of Salmonella spp. from poultryUniversity, Hisar. specimens. J. Microbiol. Antimicrobials, 2(9): 123-126.8. Mahajan, N.K., Jindal, N. and Kulshrestha, R.C. (1994) 23. Kavitha, R., Chaturvedi, V.K. and Pandey, K.D. (2008)Major broiler diseases in some parts of Haryana. Indian J. Plasmid profile of Salmonella Weltevereden isolates ofAnim. Sci. 64: 1118-1122.lizard and goat origin. Journal of Veterinary Public Health,9. Soncine, R.A. and Back, A. (2001) Salmonella Enteritidis 6(2): 117-120.em aves: erradicação ou controle por vacinação. In: 24. Mandal, S., Mandal, M.D. and Pal, N.K. (2004) ReducedConferência Apinco de Ciência e Tecnologia Avícolas, minimum inhibitory concentration of chloramphenicol forCampinas. Anais, São Paulo: FACTA, 1: 21-30. Salmonella enterica serovar typhi. Indian J. Med. Sci., 58:10. Paiva, J.B., Penha, F., Anguello, Y.M.S., Siva, M.D., Gardin, 16-23.Y., Resende, F., Berchieri, A. and Sestsi, L. (2009) Efficacy 25. Taddele, M.H., Rathore, R. and Dhama, K. (2012)of several Salmonella vaccination programme againstAntibiogram assay of Salmonella Gallinarum and otherexperimental challenge with Salmonella Gallinarum inSalmonella enteric serovars of poultry origin in India. Asiancommercial brown breeder hens. Brazilian Journal ofJ. of Animal and Veterinary Advances. 7(4): 309-317.Poultry Science, 11(1): 65-72.26. Mohanty, S., K., Renuka., Sood. S., Das, B.K. and Kapil, A.11. Cox, N.A., Bailey, J.S. and Berrang, M.E. (1996) Extent of(2006) Antibiogram pattern and seasonality of SalmonellaSalmonellae contamination in breeder hatcheries, Poultryserotypes in north Indian tertiary care hospital. Epidemiol.Science, Champaign, 70: 416-418.Infect., 134: 961-966.12. Enabulele, S.A., Amune, P.O. and Aborisade, W.T. (2010)27. Daugelavicius, R., Bakiene, E. and Bamford, D.H. (2000)Antibiograms of Salmonella isolates from poultry in OviaNorth East local government area Edo State, Nigeria. Agric.Stages of polymyxin B interaction with E. coli cell envelope.Biol. J. North Am., 1(6): 1287-1290.Antimicrobial agents and chemotherapy. 44(11): 2969.13. Ahmed, M.M., Rahman, M.M., Mahbub, K.R. and28. Sujatha, K., Dhanalakshmi, K. and Rao, A.S. (2003)Wahiduzzaman, M. (2011) Characterization of antibioticIsolation and characterization of Salmonella Gallinarumresistant Salmonella spp isolated from chicken eggs of from chicken. Indian Vet. J. 80: 473-474.Dhaka city. J. Sci. Res., 3: 191-196.29. Prasanna, K., Somvanshi, R. and Paliwal, O.P. (2001)14. Carter, G.R. (1984) Diagnostic procedures in Veterinary Experimental fowl typhoid and pullorum disease infection inthBacteriology and Mycology, 4 edn. Charles C. Thomas, chicken: Histopathological and ultrastructural <strong>studies</strong> ofSpringfield, Illinois, U.S.A.small intestine and liver. Indian J. Vet. Path. 21: 18-20.15. Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Truck, M. 30. Shivprasad, H L. (2000) Fowl typhoid and Pullorum disease.(1966) Antibiotic susceptibility testing by standardizing Sci Tech. Rev.19: 405-424.single disc method. Am. J. Clin. Path. 45: 493.31. Msoffe, P.L.M., Minga, U.M., Mtambo, M.M.A., Gwakisa,16. Luna L.G. (1968) Manual of histologic staining methods of P.S. and Olsen, J.E. (2006) Differences in resistance tordthe Armed forces Institute of Pathology. 3 edn. Mc Graw hill Salmonella enterica serovar Gallinarum infection amongBook Company, New York.33-37.indigenous local chicken ecotypes in Tanzania. Avian17. Kumar, T., Mahajan, N.K. and Rakha, N.K. (2010) Pathol. 35(4): 270-276.Epidemiology of fowl typhoid in Haryana, India. World 32. Giannella, R.A. (1979) Importance of intestinalPoultry Science Journal. 66: 503-510.inflammatory reaction in Salmonella- mediated intestinal18. Erf, G.F, Walter G Bottje, Tina K Bersi (1998) CD4, CD8 and secretion. Infect. Immun. 23: 140-145.TCR defined T-cell subsets in thymus and spleen of 2- and 7- 33. Garren, H.W. and Barber, C.W. (1955) Endocrine andweek old commercial broiler chickens, Vet Immunol lymphatic glands changes occurring in young chickens withImmunopatho, 62(4): 339 - 348.fowl typhoid. Poult. Sci. 34: 1250-1258.19. Lee, Y.J., Kang, M.S., Woo, Y.K., Mo, I.P. and Tak, R.B. 34. Rao, S.B.V., Naraya, D.R., Ramnani and Das, J. (1952)(2001) Competitive exclusion against Salmonella Avian salmonellosis: Studies on Salmonella Gallinarum.Gallinarum of Salmonella Enteritidis infected chickens. Indian J. Vet. Sci. 22: 199-208.********Veterinary World, EISSN: 2231-0916 729