M.S. Maddadi, M. Hassanzadeh, M. Bozogmehrifard, H. Shojaei, A. Yazdani, V. Karimi and A. Barin 1

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1 Internatinal Jurnal f Pultry Science 9 (3): , 2010 ISSN Asian Netwrk fr Scientific Infrmatin, 2010 Study n Tw Inculatin Ruts f Salmnella enteritidis in Abilities t Clnize in Internal Organs and t Cntaminate f Eggs in Briler Breeder Hens M.S. Maddadi, M. Hassanzadeh, M. Bzgmehrifard, H. Shjaei, A. Yazdani, V. Karimi and A. Barin 1 Faculty f Veterinary Medicine, University f Tehran, Tehran, Iran 2 Bablkenar Pure Line Prject, Iran 10 6 Abstract: Tw grups f chickens were inculated rally and intravenusly with 10 and 10 CFU S. enteritidis rganisms cnsequently. Heavier infectin f liver spleen, caecum, small intestines, infundibulumvules and clac-vagina f chickens that inculated rally were bserved. In intravenusly inculated grup high infectin f liver-spleen and clac- vagina were nticed. In ral grup egg prductin were mre decreased and fecal shedding was higher than intravenusly grup. Key wrds: S. enteritidis, ral, intravenus, chicken, egg prductin INTRODUCTION Salmnellsis is ne f the mst imprtant fd-brne diseases. The Wrld Health Organizatin (WHO) has reprted 1.3 billin cases per year f acute gastrenteritis due t nn-typhid salmnellsis with 3 millin fatal cases (Gmez et al., 1997). In 2006, a ttal f 165, 023 cnfirmed cases f human salmnellsis were reprted in the Eurpean Unin. In this reprt, the prevalence S. enteritidis was identified 62.5% and S. Typhimurium was 12.9%. The verall Eurpean Unin prevalence f Salmnella in table eggs was 0.8% in 2006 and >90% f all egg islates were S. enteritidis whereas, S. enteritidis was the mst cmmn sertype (52.3%) in the laying flck envirnment (EFSA, 2007). The persistence f this rganism in pultry huse envirnments pses a cntinuing threat f infectin fr laying hens (Davies and Breslin, 2003; Kinde et al., 2004; Lapuz et al., 2008). Additinally, there is suggestin that S. enteritidis has sme intrinsic characteristics that allw a specific interactin with either the reprductive rgans f laying hens r the egg cmpnents (Gantis et al., 2009). In pultry, an imprtant step in salmnella pathgenesis is bacterial entry in the epithelial cells f the intestinal tract, especially the caeca (Desmidt et al., 1996). Salmnella actively stimulates its wn uptake int epithelial cells by inducing cytskeletn rearrangements and membrane ruffling (Finlay and Falkw, 1989). These mrphlgical changes are triggered by prteins secreted f Salmnella int the cytsl f the epithelial cells via a type III secretin system (TTSS) encded by genes f the Salmnella pathgenicity island 1 (SPI-1) (Mills et al., 1995; Darwin and Miller, 1999). Several regulatry prteins that are invlved in Salmnella invasin have been characterized (Lucas and Lee, 2000). The key regulatr f SPI-1 is hila, a transcriptinal activatr encded n SPI-1 that regulates the expressin f the SPI-1 secretin system as well as many f its secreted effectrs (Bajaj et al., 1995). Oral infectin f hens with S. enteritidis has led t the invasin f a variety f internal rgans, including the vary and viduct (Gast and Beard, 1990) and prduced spradic egg cntaminatin fr several weeks (Gast and Hlt, 2000; Okamura et al., 2001a). The clnizatin f reprductive tissues in infected laying hens is a pivtal stage in the prductin f cntaminated eggs that can transmit S. enteritidis infectins t ffspring (Gast et al., 2009; Okamura et al., 2001a,b). Egg cntaminatin is caused by penetratin thrugh the eggshell by S. enteritidis cntained in feces after the egg is cvered by the shell (Messens et al., 2005; De Reu et al., 2006). The secnd pssible rute is by direct cntaminatin f ylk r albumen riginating frm the infectin f reprductive rgans with S. enteritidis befre the egg is cvered by the shell (Timney et al., 1989; Keller et al., 1995; Miyamt et al., 1997; Okamura et al., 2001a,b). The lcatin f S. enteritidis depsitin in a develping egg (ylk r albumen) is likely a cnsequence f which regins f the laying hen s reprductive tract are clnized (Bichler et al., 1996; Gast and Hlt, 2000; Humphrey et al., 1991). Base f these, aim f study trail was t establish a mdel infectin f S. enteritidis in laying hens in which the internal rgans e.g. digestive r reprductive systems culd becme infected and cnsequently the incidence f cntaminated eggs culd be studied. Therefre, hens were inculated intravenusly and rally. Different tissue samples were taken fr salmnella recvery. Crrespnding Authr: M.S. Maddadi, Faculty f Veterinary Medicine, University f Tehran, Tehran, Iran 254

MATERIALS AND METHODS Bacterial strain: S. enteritidis phage type 4, strain NIDO 76Sa88 Nalr (parent strain) was used in this experiment, btained frm Ghent University, Belgium.

2 MATERIALS AND METHODS Bacterial strain: S. enteritidis phage type 4, strain NIDO 76Sa88 Nalr (parent strain) was used in this experiment, btained frm Ghent University, Belgium. The nalidixic acid resistant strain is well-characterized (Desmidt et al., 1996; Van Immerseel et al., 2002). Hens: Fifty 26-week-ld briler breeder hens were selected frm an Arian Grand Parent farm that is under strict cntrl fr Salmnella and ther infectius diseases. They were free f any apparent disease thrughut the grwing and laying perids. Hens were divided int tw grups. Befre starting f the experiment clacal swabs were taken frm all hens and checked fr Salmnella infectin, t cnfirm that animals were Salmnella-free. Hens randmly divided in tw grups f 25 birds. First 6 grup was inculated intravenusly (IV) with 10 CFU f S. enteritidis 76Sa88 Nalr parent strain bacteria, using 0.1 ml f PBS and secnd grup hens were inculated by ral (OR) rute in the crp, using a plastic tube with Clny Frming Units (CFU) f same bacteria in a vlume f 1 ml f PBS, as reprted previusly (Barrw and Lvell, 1991). At days 2, 7, 14, 21 and 35 pst-inculatin, tw hens per grup were euthanized and pst-mrtem examinatins were carried ut. Fr bacterial analysis samples were taken frm different parts f digestive (caecal, small intestine, liver- spleen) and reprductin (infundibulum-vules, magnum, isthmus, claca-vagina) systems separately. Clacal swabs were taken n same days and examined fr S. enteritidis. Every 10 eggs were pled and cultured. Bacterilgical analysis: Swabs frm clacae were placed in 5 ml selenite cysteine brth and after 24 h incubatin in 37 C, were cultured n Salmnella- Shigella (SS) agar plates. Suspected clnies were cultured in Triple Sugar Irn Agar (TSI) and urea brth tubes. Samples f internal rgans were hmgenized and 10-fld dilutins made in PBS. Fr each dilutin 100 µl inculated n SS agar plates with 20 µg/ml nalidixic acid. After vernight incubatin (37 C) the number f CFU/g tissue was determined by cunting the bacterial clnies (Bhez et al., 2006). Fr samples which were negative after titratin, pre-enrichment and enrichment was perfrmed in selenite cysteine brth. Samples that were negative after titratin but psitive after Salmnella 1 enrichment were presumed t cntain 10 CFU/g rgans. Samples that were negative after enrichment were presumed t have 0 CFU/g. The mean CFU/g tissue was calculated fr each grup. On experimental daily basis, every 10 eggs were pled tgether int sterile hney jars and cntents mixed and hmgenized by shaking the jars. These were incubated at 37 C fr 48 h and then plated nt the antibitic cntaining SS agars. RESULTS Fllwing f inculatin, prductivity decreased t a lw level and that was mre prnunced in ral grup (Table 1). Whether this was a result f S. enteritidis infectin r simply frm handling and inculatin is unclear. Table 1: Egg prductin after infectin f hens by rally (OR) r intravenusly (IV) with Salmnella enteritidis Weekly pst inculatin OR IV 1 43/25* 52/ /23 63/ /21 64/ /19 60/ /17 57/17 *Daily percentage f egg prductin/number f hens Pic. 1: Inspissated vary and misshapen fllicles in a bird that autpsied in secnd weeks pst infectin In ral grup, at the secnd and third weeks, birds that necrpsizied had sme inspissated vary and misshapen fllicles. S. enteritidis was islated frm these rgans and frm the small intestines, caecum, and viduct (Picture 1). Figure 1 summarizes the detectin f S. enteritidis by bacterial islatin frm the different parts f digestive system. S. enteritidis was islated frm different parts f gastrintestinal thrughut the sampling times, but the majrity f detectin was frm the hens which inculated rally cmpared t the IV grup birds. In the ral grup, the highest recvery rates f S. enteritidis were made frm caecum at 2, 7 and 14 dpi, while in the grup f IV infectius hens, the highest S. enteritidis recvery bserved frm the liver-spleen tissues at 2 and 7 dpi (Fig. 1). Hwever, the ttal recvery f S. enteritidis frm different parts f reprductin system was lwer than t the digestive system but the majrity f islates frm this system were btained in IV grup cmpared than t thse f OR grup hens (Table 2). In the reprductin system, the highest recvery f S. enteritidis was 255

3 10 Fig. 1: Cmparisn f salmnella cunts (lg CFU/g) in different parts f digestive system in tw grups f hens that inculated rally (OR) r intravenusly (IV) inculated with S. enteritidis OR = ral IV: Intravenus SI = Small intestine C = Caecum L and S = Liver and Spleen dpi = days pst infectin perfrmed frm the clac-vagina and thereafter infundibulum-vules tissues. Egg pl cultures were psitive at 7, 14 and 21 dpi in ral grup whereas it was psitive at 2, 14 and 35 dpi fr IV inculated hens (Table 2). Clacal swabs were 64% psitive at 2dpi and decreased t 23.5% at 35 dpi in ral grup, as there was 56% psitive at 2 dpi in intravenus inculated birds but decreased t 17.6% at 35 dpi (Table 2). DISCUSSION Natural infectin f pultry by Salmnella ccurs via ral rute and salmnella clnize the intestinal tract with the crp and ceca being the primary sites f clnizatin (Brwnwell et al., 1970; Serjadi et al., 1981; Stavric, 1987; Impey and Mead, 1989). In the present study, cntaminatin f gastrintestinal rgans in OR grup was higher than t IV grup. Additinally, recvery f S. enteritidis frm caecum f ral grup was higher than IV grup. The ceca have been recgnized as the regin fr the mst frequent recvery f Salmnella after ral infectin. Okamura (2001b) explained that after IV inculatin, S. enteritidis culd keep bacteremia and remained persistently in the liver and ceca t a high degree. In this study caecum was infected with S. enteritidis when hens inculated via intravenusly t. It culd be cnsidered that the ceca may cntaminate frm the liver by the gallbladder secretin. As bserved, S. enteritidis recvery frm infundibulumvules and clac-vagina were mre appeared in cmparisn with magnum and isthmus. On the ther hand, the clnizatin f S. enteritidis in vary and prevulatry fllicles f IV grup were clearly higher than ral grup that cnfirmed the previus reprts (De Buck et al., 2004; Gantis et al., 2006). In the majrity f these studies in laying hens, a higher frequency f vary clnizatin is reprted, cmpared with the frequency f recvery frm ther sectins f the viduct (De Buck et al., 2004; Gast et al., 2007). Because, it is strngly believed that S. enteritidis must interact with the cellular cmpnents f the prevulatry fllicles. The extensive permeability f the vascular endthelia bserved in the vary may cntribute t the high clnizatin rate at this site (Griffin et al., 1984). Oviduct infectin in IV grup appeared t be the result f haematgenus spread (Barrw and Lvell, 1991) and in ral grup it is generally believed that clnizatin f the reprductive rgans is a cnsequence f systemic spread f Salmnella frm the intestine (Vazquez-Trres et al., 1999). Eggs cntents pl culture results were nt cnsistent at different days but it seems at a time that salmnella were islated frm infundibulum-vule r clac-vagina, these cultures wuld be psitive. Table 2: Recvery and cunts (lg 10 CFU/g) f Salmnella enteritidis frm different parts f reprductin system and the clacal swaps in tw grup hens that inculated rally (OR) and intravenusly (IV) with Salmnella enteritidis 2 dpi 7 dpi 14 dpi 21dpi 35 dpi Reprductin system OR IV OR IV OR IV OR IV OR IV Infundibulum-vules Magnum Isthmus Clac-vagina Eggs cntents culture Clacal swaps 64% 56% 26% 52% 33% 29% 37% 26% 24% 18% 256

4 Numerus studies have als been perfrmed t De Buck, J., F. Van Immerseel, F. Haesebruck and R. investigate the effect f the inculatin rute n the prductin f cntaminated eggs (Miyamt et al., 1997;Gast et al., 2002). While Gast et al. (2002) reprted that ral, aersl and intravenus inculatins led t similar frequencies f egg cntaminatin. Miyamt et al. (1997) bserved a higher cntaminatin rate when birds were inculated intravenusly and intravaginally. Our data indicated that mst parts f digestive system infected when birds inculated rally, as may bserve in the natural cnditins in the field. Whereas, the majrity f S. enteritidis recvered frm reprductive system was in intravenusly grup. This indicates that the main rut the cntaminatin f reprductive system might be thrugh the systemic infectius, as was reprted previusly. Hwever, under the varius cnditins and rutes by which chickens might becme infected by S. enteritidis phage type 4, eggs are mre likely t becme cntaminated during passage thrugh the claca and/r as a result f varian infectin. ACKNOWLEDGMENT This research was funded by the research cmmittee, University f Tehran, Faculty f Veterinary Medicine. REFERENCES Bajaj, V., C. Hwangand and C.A. Lee, hila is a nvel mpr/ txr family member that activates the expressin f Salmnella Typhimurium invasin genes. Ml. Micrbil., 18: Barrw, P.A. and M.A. Lvell, Experimental infectin f egg laying hens with Salmnella enteritidis phage type 4. Avian Pathl., 20: Bichler, L.A., V. Kabambi, D. Nagaraja and D.A. Halvrsn, Salmnella enteritidis in eggs, clacal swab specimens and internal rgans f experimentally infected white leghrn chickens. Am. J. Vet. Res., 57: Bhez, L., R. Ducatelle, F. Pasmans, N. Btteldrn, F. Haesebruck and F. Van Immerseel, Salmnella enterica servar Enteritidis clnizatin f the chicken caecum requires the HilA regulatry prtein. Vet. Micrbil., 116: Brwnwell, J.R., W.W. Sadler and M.J. Fanelli, Rle f the ceca in intestinal infectin f chickens with Salmnella typhimurium. Avian Dis., 14: Darwin, K.H. and V.L. Miller, Mlecular basis f the interactin f Salmnella with the intestinal mucsa. Clin. Micrbil. Rev., 12: Davies, R. and M. Breslin, Observatins n Salmnella cntaminatin f cmmercial laying farms befre and after cleaning and disinfectin. Vet. Rec., 152: Ducatelle, Effect f type-1 fimbriae f Salmnella enteric sertype Enteritidis n bacteremia and reprductive tract infectin in laying hens. Avian Pathl., 33: De Reu, K., K. Grijspeerdt, W. Messens, M. Heyndrickw, M. Uyttendaele, J. Debevere and L. Herman, Eggshell factrs influencing eggshell penetratin and whle egg cntaminatin by different bacteria, including Salmnella enteritidis. Int. J. Fd Micrbil., 112: Desmidt, M., R. Ducatelle, F. Haesebruck, P.A. De Grt, M. Verlinden, R. Wijffels, M. Hintn, J.A. Bale and V.M. Allen, Detectin f antibdies t Salmnella enteritidis in sera and ylks frm experimentally and naturally infected chickens. Vet. Rec., 138: EFSA, The cmmunity summary reprt n trends and surces f znses, zntic agents, antimicrbial resistance and fdbrne utbreaks in the Eurpean Unin in EFSA J., 130: Finlay, B.B. and S. Falkw, Cmmn themes in micrbial pathgenicity. Micrbil. Rev., 53: Gantis, I., R. Ducatelle, L. Timbermnt, F. Byen, L. Bhez, F. Haesebruck and F. Van Immerseel, Oral immunizatin f laying hens with live vaccine strains f TAD Salmnella vace and TAD Salmnella vact reduces internal egg cntaminatin with Salmnella enteritidis. Vaccine., 24: Gantis, I., R. Ducatelle1, F. Pasmans, F. Haesebruck, R. Gast, T.J. Humphrey and F. Van Immerseel, Mechanisms f egg cntaminatin by Salmnella enteritidis, FEMS Micrbil. Rev., J., pp: Gast, R.K. and C.W. Beard, Prductin f Salmnella Enteritidis-cntaminated eggs by experimentally infected hens. Avian Dis., 34: Gast, R.K. and P.S. Hlt, Influence f the level and lcatin f cntaminatin n the multiplicatin f Salmnella enteritidis at different strage temperatures in experimentally inculated eggs. Pult. Sci., 79: Gast, R.K., J. Guard-Petter and P.S. Hlt, Characteristics f Salmnella enteritidis cntaminatin in eggs after ral, aersl and intravenus inculatin f laying hens. Avian Dis., 46: Gast, R.K., R. Guraya, J. Guard-Buldin, P.S. Hlt and R.W. Mre, Clnizatin f specific regins f the reprductive tract and depsitin at different lcatins inside eggs laid by hens infected with Salmnella enteritidis r Salmnella Heidelberg. Avian Dis., 51:

5 Gast, R.K., J. Guard-Buldin, R. Guraya and P.S. Hlt, Effect f Prir Passage Thrugh Laying Hens n Invasin f Reprductive Organs by Salmnella enteritidis. Int. J. Pult. Sci., 8: Gmez, M., Y. Mtarjemi, S. Miyagawa and F.R. Kaferstein, Fd-brne salmnellsis. Wrld Health Stat. Q., 50: Griffin, H.D., M.M. Perry and A.B. Gilbert, Ylk frmatin. Physilgy and Bichemistry f the Dmestic Fwl (Freeman BM, Ed)., pp: Academic Press, Lndn. Humphrey, T.J., A. Whitehead, A. Gawer, A. Henley and B. Rwe, Numbers f Salmnella enteritidis in the cntents f naturally cntaminated hen s eggs. Epidemil Infect., 106: Impey, C.S. and G.C. Mead, Fate f salmnellas in the alimentary tract f chicks pretreated with a mature caecal micrflra t increase clnizatin resistance. J. Appl. Bacteril., 66: Keller, L.H., C.E. Bensn, K. Krtec and R.J. Eckrade, Salmnella enteritidis clnizatin f the reprductive tract and frming and freshly laid eggs f chickens. Infect Immun., 63: Kinde, H., D.M. Castellan, P.H. Kass, A. Ardans, G. Cutler, R.E. Breitmeyer, D.D. Bell, R.A. Ernst, D.C. Kerr, H.E. Little, D. Willughby, H.P. Riemann, J.A. Snwdn and D.R. Kuney, The ccurrence and distributin f Salmnella enteritidis and ther servars n Califrnia egg laying premises: a cmparisn f tw sampling methds and tw culturing techniques. Avian Dis., 48: Lapuz, R., H. Tani, J. Sasai, K. Shirta, H. Kath and E. Baba, The rle f rf rats (Rattus rattus) in the spread f Salmnella enteritidis and S. infantis cntaminatin in layer farms in eastern Japan. Epidemil. Infect., 136: Lucas, R.L. and C.A. Lee, Unravelling the mysteries f virulence gene regulatin in Salmnella Typhimurium. Ml. Micrbil., 36: Messens, W., K. Grijspeerdt and L. Herman, Eggshell penetratin by Salmnella: a review. Wrld Pult. Sci. J., 61: Mills, D.M., V. Bajaj and C.A. Lee, A 40 kb chrmsmal fragment encding Salmnella Typhimurium invasin genes is absent frm the crrespnding regin f the Escherichia cli K-12 chrmsme. Ml. Micrbil., 15: Miyamt, T., E. Baba, T. Tanaka, K. Sasai, T. Fukata and A. Arakawa, Salmnella enteritidis cntaminatin f eggs frm hens inculated by vaginal, clacal and intravenus rutes. Avian Dis., 41: Okamura, M., Y. Kamijima, T. Miyamt, H. Tani, K. Sasai and E. Baba, 2001a. Differences amng six Salmnella servars in abilities t clnize reprductive rgans and t cntaminate eggs in laying hens. Avian Dis., 45: Okamura, M., T. Miyamt, Y. Kamijima, H. Tani, K. Sasai and E. Baba, 2001b. Differences in abilities t clnize reprductive rgans and t cntaminate eggs in intravaginally inculatedhens and in vitr adherences t vaginal explants between Salmnella enteritidis and ther Salmnella servars. Avian Dis., 45: Serjadi, A.S., S.M. Stehman, G.H. Sneyenbs, O.M. Weinack and C.F. Smyser, Sme measurements f prtectin against paratyphid Salmnella and Escherichia cli by cmpetitive exclusin in chickens. Avian Dis., 25: Stavric, S., Micrbial clnizatin cntrl f chicken intestine using defined cultures. Fd Technl., 41: Timney, J.F., H.L. Shivaprasad, R.C. Baker and B. Rwe, Egg transmissin after infectin f hens with Salmnella enteritidis phage type 4. Vet. Rec., 125: Van Immerseel, F., J. De Buck, I. De Smet, J. Mast, F. Haesebruck and R. Ducatelle, Dynamics f immune cell infiltratin in the caecal lamina prpria f chickens after nenatal infectinwith a Salmnella enteritidis. Dev. Cmp. Immunl., 26: Vazquez-Trres, A., J. Jnes-Carsn, A.J. Baumler, S. Falkw, R. Valdivia, W. Brwn, M. Le, R. Berggren, W.T. Parks and F.C. Fang, Extraintestinal disseminatin f Salmnella by CD18- expressing phagcytes. Nature, 401:

5.1. What do we need to know before we start planning a canine rabies control programme?

5.1. What do we need to know before we start planning a canine rabies control programme? 5.1. What d we need t knw befre we start planning a canine rabies cntrl prgramme? Yu need t knw abut: The epidemilgy f rabies in yur area The reservir species in yur area Hw rabies is transmitted. This