Dose Determination for Acute Salmonella Infection in Pigs

Pigs were exposed to various levels of Salmonella enterica subsp.enterica serovar Typhimurium by either intranasal inoculationor by subjecting them to a contaminated environment. More than10³ salmonellae were required to induce acute Salmonella infection.These results indicate that intervention against acute Salmonellainfection in lairage may be more readily achieved than previouslythought.

INTRODUCTION

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Introduction

Pigs frequently harbor Salmonella spp. subclinically, allowingthe organism to be transmitted among pigs prior to slaughter(4, 6, 7, 10). Transmission of Salmonella from pigs with subclinicalinfections to naïve pigs during transportation and lairagehas been proposed to be a major source of Salmonella introductioninto the food chain (5, 10). Events immediately prior to slaughterhave been shown to correlate with an increased rate of Salmonellaisolation from pig carcasses (5, 6) and from pork products (2,8, 12).

Previous work in our laboratory has shown that numerous Salmonellaserovars are capable of acutely infecting both alimentary andnonalimentary tract tissues within 3 h after intranasal inoculation(9). Acute Salmonella infection has been shown to occur in market-weightpigs after they have been rooting in an environment contaminatedat doses comparable to those reported in lairage (<10⁶ salmonellae)(4).

The objectives of this study were to determine the minimum doserequired to induce acute Salmonella infection in pigs by intranasalinoculation with Salmonella enterica subsp. enterica serovarTyphimurium (trials 1 and 2) and to evaluate acute Salmonellainfection in pigs exposed to a contaminated environment containingvarious levels of Salmonella (trials 3 and 4).

Isolation rooms.

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Isolation rooms.

Prior to pig arrival, drag swab samples of the rooms were preenrichedin buffered peptone water (Becton Dickinson, Difco), selectivelyenriched in Rappaport Vassiliadis broth (Becton Dickinson, Difco),and selectively plated on xylose lysine deoxycholate (XLD) agar(Becton Dickinson, Difco). Salmonella suspect colonies werethen transferred to differential biochemical media as previouslydescribed (9).

Animals.

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Animals.

Crossbred pigs, 10 to 14 days old, were randomly assigned toone of three principal groups (five animals per group) or toa negative control group. The pigs were acclimatized for 7 to14 days in isolation rooms and given water and irradiated feed(Harlan Teklad, WI) ad lib. During acclimatization, rectal swabsand pooled pen fecal samples (3) were obtained to verify thepigs to be free of detectable Salmonella.

Salmonella.

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Salmonella.

The challenge strain, S. enterica subsp. enterica serovar Typhimuriumstrain HL 10969, was derived from nalidixic acid-resistant strain ${chi}$ 4232, which was genetically modified to produce green fluorescentprotein as previously described (1). The isolate was selectedfor increased acute infection virulence determinants by inoculatingthe organism into pigs and reisolating it from the ileocecallymph node 3 h later.

Strain HL 10969 was grown to late log phase in Luria-BertaniMiller broth (Becton Dickinson, Difco) and centrifuged at 5,000rpm for 15 min at 5°C. The cell pellet was washed in phosphate-bufferedsaline and centrifuged two additional times. Following the thirdcentrifugation step, the cell pellet was resuspended in phosphate-bufferedsaline containing 20% glycerol and frozen at –80°C.

Challenge/necropsy. (i) Intranasal challenge (trials 1 and 2).

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Challenge/necropsy. (i)...

Strain HL 10969 was removed from the freezer and serially dilutedto 4.5 x 10⁵, 4.2 x 10³, and 4.8 x 10¹ (trial 1) or 2.8 x 10⁷,2.8 x 10⁵, and 2.8 x 10³ (trial 2) organisms per ml, as determinedby viable plate counts. Animals were intranasally inoculatedas previously described (9).

(ii) Contaminated-environment challenge (trials 3 and 4).

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(ii) Contaminated-environment...

During acclimatization, feces were collected and stored at 4°Cuntil the day of challenge. Five days prior to challenge, thepooled feces were verified to be free of Salmonella by preenrichment,selective enrichment, and selective plating techniques. Twelvehours before challenge, approximately 1 liter of physiologicalsaline was added for every 1,500 g of feces. The feces weremixed using an electric mixer (Hamilton Beach/Proctor SilexInc., NC) on setting 1 for 2 min. The feces were then placedinto bowls for each Salmonella dilution, and the appropriatenumbers of salmonellae were added to obtain 5.2 x 10⁴, 5 x 10²,and 2.5 x 10¹ (trial 1) or 4 x 10⁶, 4.1 x 10⁴, and 9.1 x 10²(trial 2) organisms per g of feces as determined by direct platecounts. Twenty-five grams of the spiked feces was applied toeach square foot of the challenge environment.

Animals were euthanized 3 h following challenge. Approximately5 g each of tonsil, mandibular lymph node, thymus, lung, liver,spleen, colon contents, ileocecal lymph node, diaphragmaticmuscle, and ileum; 20 g of cecum contents; and 20 ml of bloodwere aseptically collected for the isolation of Salmonella.

Salmonella isolation.

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Salmonella isolation.

Tissue samples were collected and processed as previously described(9), except for the selective plating techniques. For the selectiveplating process, samples were plated onto XLD agar containing50 µg of nalidixic acid per ml of agar. Tissues from thenegative control animals were processed identically to thoseof the principal groups, except they were plated for isolationonto XLD agar without nalidixic acid. XLD plates were placedunder UV light and observed for fluorescence typical of thegreen fluorescent protein-containing challenge strain. The presenceor absence of Salmonella in tissues was recorded.

Calculations.

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Calculations.

Fifty percent infectious dose calculations were done based onthe Reed-Muench equation (http://www.fao.org/DOCREP/005/AC802E/ac802e00.htm).

Salmonellae were not isolated from the environmental drag swabs.All rectal swabs and pooled pen fecal samples taken during acclimatizationwere negative for Salmonella. All alimentary and nonalimentarytissues from the negative control animals (trials 1, 2, 3, and4) were culture negative for Salmonella at necropsy. The resultsfor intranasal and contaminated-environment challenge are presentedin Tables 1 to 3.

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TABLE 1. Incidence of Salmonella infection following intranasal challenges of pigs with various levels of Salmonella serovar Typhimurium

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TABLE 3. Fifty percent infective dose calculations for acute Salmonella infections following intranasal challenge

These experiments establish a minimum dose of Salmonella neededfor acute infection of both alimentary and nonalimentary tissuesof swine. An intranasal challenge dose of greater than 10³ salmonellaeis required to infect both alimentary and nonalimentary tissues.The ingestion of Salmonella from contaminated environments containingmore than 10³ salmonellae per gram of feces induces acute infectionof both the alimentary and nonalimentary tissues.

Regarding the movement of swine from farm to slaughter, a varietyof conditions, such as the number of pigs shedding Salmonellainto the environment, length of time in lairage, animal age,breed, concurrent disease status, and stress encountered duringtransportation and lairage, may influence the minimum dose ofsalmonellae required to induce acute infection in pigs. However,these results suggest that reduction of acute Salmonella infectionmay be readily achievable by simple environmental sanitation.

In Denmark, a Salmonella control program exists in which farmsare categorized into groups of low, medium, or high Salmonellaprevalence (13). This program mandates that pens are washedbetween group changes, and farms with low prevalence have transport,lairage, and slaughter separate from those with high and mediumprevalence. These prophylactic techniques have decreased acuteinfection, as evidenced by decreased Salmonella isolation frompig cecal contents and slaughter carcasses (2, 11), possiblyby reducing the numbers of salmonellae below the minimal doseneeded to cause acute Salmonella infection.

Thus, our results are consistent with the experience of theDanish Salmonella control program in that reducing the levelof environmental Salmonella is adequate for minimizing acuteinfection by the organism.

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TABLE 2. Incidence of Salmonella infection following contaminated-environment challenges of pigs with various levels of Salmonella serovar Typhimurium

ACKNOWLEDGMENTS

We thank PIC (Franklin, Kentucky), the Biotechnology Researchand Development Corporation, and the Tri-State Food Safety Consortiumfor their financial support.

FOOTNOTES

* Corresponding author. Mailing address: Iowa State University, Department of Animal Science, Room 11, Kildee Hall, Ames, IA 50011. Phone: (515) 294-1664. Fax: (515) 294-5294. E-mail: hharris{at}iastate.edu.

REFERENCES

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