Assessment of Virulence of Pigeon Isolates of Salmonella enterica subsp. enterica Serovar Typhimurium Variant Copenhagen for Humans

Salmonella enterica serovar Typhimurium variant Copenhagen wasisolated from 5 of 152 (3.3%) feral pigeons from the city ofGhent (Belgium) and from 26 pooled fecal samples from 114 pigeonlofts (22.8%). These isolates belonged to phage type (PT) 99.Seven of the pigeon isolates were further compared in vitroto five human variant Copenhagen isolates, 2 isolates of PT208, 1 isolate each of PT 120 and U302, and a nontypeable isolate.No differences in invasiveness in human intestinal epithelialCaco-2 cells were found. The human strains, however, were ableto multiply significantly more inside human THP-1 macrophagesthan the pigeon strains. After inoculation of mice with a pigeonPT 99 strain, high numbers of Salmonella bacteria were shedwith the feces, the internal organs were heavily colonized,and the animals showed severe clinical symptoms resulting indeath. In conclusion, the less-pronounced ability of the pigeonvariant Copenhagen strains to multiply inside human macrophagesthan human strains as well as the lack of human PT 99 isolatesduring 2002, despite the relatively high frequency of this PTin the pigeon population, suggest these strains to be of lowvirulence to humans. However, the high virulence for mice ofthe tested strain implies that rodents may act as reservoirs.

INTRODUCTION

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Introduction

Because of the high numbers of free-roaming and captive pigeonsin Belgium and their well known association with Salmonella,pigeons might be a source of salmonellosis in humans. Pigeonisolates of Salmonella enterica subsp. enterica serovar Typhimuriumvariant Copenhagen are considered a host-adapted lineage ofthe serovar Typhimurium (5, 6, 8). As might be expected fromhost restricted Salmonella strains (3, 10), these pigeon isolatesare highly virulent to pigeons (6, 7). Adaptation to one hostoften coincides with low virulence to other hosts, such as humans(3, 10). It was the aim of the present study to determine thevirulence of pigeon-associated Salmonella strains for humansby using epidemiological data, in vitro enterocyte and macrophagemodels, and an in vivo murine model. The in vitro and in vivomodels have been previously used to assess virulence of Salmonellastrains for humans (1).

MATERIALS AND METHODS

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Materials and Methods

Prevalence of pigeon-adapted variant Copenhagen strains in pigeons and humans. For the pigeon strains, pooled fecal samples from 114 differentpigeon lofts in Belgium and pooled samples from the intestines,livers, and spleens of 152 feral pigeons from the city of Ghent(Belgium) were examined for the presence of Salmonella. Thesamples were enriched overnight in tetrathionate brilliant greenbroth (Merck, Darmstadt, Germany) at 37°C and then streakedonto brilliant green agar (Lab-M, Bury, United Kingdom). Colonieswith characteristics of Salmonella were further identified biochemicallyon the basis of glucose fermentation, sulfur reduction, thepresence of lysine decarboxylation, the absence of lactose fermentation,and urease production. The isolates were serotyped by meansof agglutination tests by using single-factor antisera, andthe phage types (PTs) were determined.

For the human strains, clinical Salmonella isolates were obtainedfrom 194 Belgian laboratories during the year 2002. The strainswere serotyped, and the PTs were determined in 319 (13%) of2,438 serovar Typhimurium isolates. The maximum number (B) ofhuman salmonellosis cases due to an infection with pigeon-adaptedstrains with 95% probability ( ${alpha}$ ) was determined with the followingformula: B = [1 – (1 – ${alpha}$ )^1/n] x {N – [(n –1)/2]}, where N is the total number of serovar Typhimurium strainsand n is the number of serovar Typhimurium strains of whichthe PT was determined.

Invasion of pigeon versus human isolates of variant Copenhagen into Caco-2 cells. Seven pigeon-derived variant Copenhagen PT 99 strains belongingto four different BlnI pulsed-field gel electrophoresis types(6) and five human variant Copenhagen isolates, 2 of PT 208,1 of PT 120, 1 of PT U302, and 1 that was nontypeable, wereused in a gentamicin protection assay. Briefly, overnight bacterialcultures grown in Luria-Bertani broth (37°C) were resuspendedin cell culture medium (Dulbecco's modified Eagle's medium [Gibco,Paisley, Scotland], 10% fetal calf serum, 1% nonessential aminoacids [Gibco]). Confluent monolayers of Caco-2 cells in a 96-wellplate were exposed to 10⁷ CFU of the different human and pigeonSalmonella isolates. The plate was centrifuged for 10 min at37°C and incubated at 37°C and 5% CO₂ for 1 h. The cellswere rinsed five times with Hanks' balanced salt solution (Gibco)and incubated for another hour at 37°C and 5% CO₂ in cellculture medium containing 50 µg of gentamicin (Gibco)/ml.Afterwards, the cells were rinsed five times with Hanks' balancedsalt solution and lysed at room temperature for 10 min with1% Triton X-100 (Acros, Geel, N.J.) in distilled water. Finally,120 µl of 10-fold dilutions was plated on brilliant greenagar, and Salmonella colonies were counted after incubationfor 24 h at 37°C. The invasion assays were performed intriplicate.

Intracellular survival of pigeon versus human isolates of variant Copenhagen inside THP-1 cells. The same strains as described for the Caco-2 invasion assaywere used to compare the pigeon- and human-derived strains forthe ability to survive inside the human macrophage cell lineTHP-1. Inoculation of the THP-1 cells was performed as describedabove, with the exception that at 1 h after the addition of50 µg of gentamicin/ml, the medium was replaced by mediumcontaining 20 µg of gentamicin/ml. At 0, 2, and 6 h afterthis medium replacement, the cells were lysed and the numbersof bacteria were counted. The assays were performed in triplicate.

Cytotoxicity of Salmonella strains for THP-1 cells. The macrophages were exposed to the pigeon and the human strainsas described above. At 6 h postinoculation (p.i.), 20 µlof the cell proliferation agent WST-1 (Roche, Mannheim, Germany)was added to each well and absorbencies were measured at 450nm (Titertek, Helsinki, Finland). The number of cells per wellwas determined by using a standard curve prepared with a dilutionseries of the cell suspension.

In vivo virulence of a pigeon versus human variant Copenhagen strain for mice. A pigeon-derived variant Copenhagen strain with known virulencefor pigeons (DAB69) and a human variant Copenhagen strain (MB2504)were grown for 6 h in Luria-Bertani broth at 37°C. Ten 6-week-oldBALB/c mice were inoculated orally with 10⁴ CFU of one of thevariant Copenhagen strains. The number of Salmonella bacteriawas determined in feces, which were collected daily for 7 days.The clinical health of the animals was assessed daily. At 7days p.i., the mice were humanely killed and the numbers ofCFU of Salmonella per gram of cecum, spleen, and liver weredetermined. If only positive for Salmonella after enrichment,a bacterial count of 10 CFU/g was attributed.

RESULTS

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Results

Prevalence of pigeon-adapted variant Copenhagen strains in pigeons and humans. Variant Copenhagen was isolated from 5 of 152 (3.3%) feral pigeonsfrom the city of Ghent (Belgium) and from 26 pooled fecal samplesfrom 114 pigeon lofts (22.8%). All of the isolates belongedto PT 99.

In 2002, 10,075 human Salmonella isolates were collected inBelgium, of which 2,438 (24.2%) belonged to serovar Typhimurium.Among the serovar Typhimurium isolates, 574 belonged to variantCopenhagen. None of 319 serovar Typhimurium strains (of which42 were variant Copenhagen) belonged to PT 99. The maximum numberof human salmonellosis cases in 2002 due to infection with PT99 strains was estimated to be 21, this is a maximum of 0.87%of the infections with serovar Typhimurium and 0.21% of theSalmonella infections.

Invasion of pigeon versus human isolates of variant Copenhagen into Caco-2 cells. Results of the invasion assays are summarized in Fig. 1. Allof the strains tested were able to invade the Caco-2 cells.No significant differences between human and pigeon strainswere noticed.

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FIG. 1. Average magnitude of invasion ± standard error of the mean of pigeon- and human-derived strains of variant Copenhagen into Caco-2 cells. The results represent the percentages of bacteria recovered from the inoculum.

Intracellular survival of pigeon versus human isolates of variant Copenhagen inside THP-1 cells. All of the strains tested were able to survive and multiplyinside the THP-1 cells. Results are shown in Fig. 2. At 6 hp.i., significantly less Salmonella bacteria were isolated fromcells inoculated with the pigeon strains than from cells inoculatedwith the human strains (P < 0.05, Mann-Whitney U test).

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FIG. 2. Average intracellular survival ± standard error of the mean of pigeon-derived (triangles) and human-derived (diamonds) strains of variant Copenhagen inside THP-1 cells. Intracellular survival is expressed relative to the number of bacteria recovered at 0 h p.i..

Cytotoxicity of Salmonella strains for THP-1 cells. None of the strains induced cytotoxic effects in the THP-1 cellsat 6 h p.i., as measured by the WST-1 assay.

In vivo virulence of a pigeon versus human variant Copenhagen strain for mice. Fecal shedding of the two strains was comparable during thefirst 5 days p.i. (Fig. 3). From day 5 p.i. on, shedding ofthe human strain decreased, whereas shedding of the pigeon strainincreased and reached a plateau phase. Mice inoculated withthe human strain did not show any clinical symptoms. Mice inoculatedwith the pigeon strain showed severe clinical symptoms fromday 5 p.i.: rugged appearance, apathia, and anorexia. Four ofthese five mice inoculated with the pigeon strain were moribundat day 7 p.i. and were euthanized. Significantly higher numbersof Salmonella bacteria were found in the ceca, livers, and spleensof mice inoculated with the pigeon strain than in those of miceinoculated with the human strain (Fig. 4) (P < 0.05, Mann-WhitneyU test).

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FIG. 3. Average number of bacteria ± standard error of the mean recovered from fecal pellets of BALB/c mice inoculated with either a human-derived (diamonds) or pigeon-derived (triangles) strain of variant Copenhagen.

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FIG. 4. Average number of bacteria ± standard error of the mean recovered from internal organs of BALB/c mice at 7 days p.i. inoculated with either a human-derived (black bars) or pigeon-derived (white bars) strain of variant Copenhagen.

DISCUSSION

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Discussion

The prevalence of variant Copenhagen in the city pigeon populationappears to be fairly low. However, taking into considerationthe huge numbers of these birds in the Belgian cities and theirrather close contact with humans, the risk of transfer of zoonoticagents to humans is not unthinkable. It would, therefore, beuseful to be able to estimate the public health risk of pigeonsas carriers of Salmonella. The results of the in vitro and invivo assessment of the virulence of pigeon-derived Salmonellastrains, however, were rather ambiguous. The in vitro assayssuggested that, whereas invasiveness was equal for both humanand pigeon strains, the human-derived strains were more capableof multiplying inside human macrophages. The association ofthe ability of intracellular multiplication with host adaptationhas been proven previously (2, 9, 12). In contrast, a clearassociation of invasiveness into intestinal epithelial cellsand host adaptation has not been found (4, 11). Hence, our invitro results suggest that the pigeon-derived variant Copenhagenstrains are less adapted to and, therefore, probably less virulentto humans than the human strains. This hypothesis is supportedby the lack of human isolates of PT 99 in the 2002 period inBelgium and its estimated low prevalence in the human population,despite the relatively high prevalence in the pigeon population.

The in vivo virulence assay in BALB/c mice demonstrated thatthe pigeon strain used was more virulent to mice than the humanstrain. Mice have been used previously as a model to assessvirulence of serovar Typhimurium for humans (1). Highly virulentmurine strains, however, do not necessarily have to be virulentin humans and vice versa (e.g., S. enterica serovar Typhi).Possibly, in the case of pigeon-derived Salmonella strains,rodents act as reservoirs for the bacterium to reside in, helpingto maintain and spread the infection in the pigeon population,for example, by contaminating food sources. Although supposedlyhost adapted, at least some of the pigeon-derived strains ofvariant Copenhagen are able to cause severe clinical salmonellosisin the murine host. This finding resembles the situation encounteredwith S. enterica serovar Dublin, which occasionally causes severeinfections in hosts other than cattle (10).

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology, Bacteriology, and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. Phone: 32 9 264 74 44. Fax: 32 9 264 74 94. E-mail: frank.pasmans{at}UGent.be.

REFERENCES

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