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Tuesday, January 26, 2010

Differences in Listeria monocytogenes contamination of rural Ohioresidences with and without livestock.(Report). USA, LLCAppliances at

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To determine the contribution of on-site livestock to the environmental contamination of rural households with Listeria monocytogenes, a total of 1779 environmental and food samples were collected from 26 ruminant-farm households and 26 rural households in Ohio. L. monocytogenes isolates were identified and differentiated using sequence comparisons of the intragenic regions of inlB and inlC. L. monocytogenes was isolated from shoes, 9.6% (20/208); utility gloves, 5.4% (6/111); kitchen sinks, 1.5% (3/204); washing machines, 0.96% (2/204); food, 1.11% (7/631); and animal feces, 8.7% (9/104), over the course of four household visits at monthly intervals. Notably, L. monocytogenes-contaminated shoes were identified more frequently from ruminant farmhouses than from rural households that did not raise ruminants on site (odds ratio= 4.8). L. monocytogenes isolated from animal feces was indistinguishable from strains recovered from shoes and gloves stored in several homes. Our results highlight the potential of the rural household environment as source of L. monocytogenes exposure.

Full Text :COPYRIGHT 2010 Mary Ann Liebert, Inc.

ARM RESIDENCY HAS BEEN IDENTIFIED as a risk factor for several zoonotic infections such as campylobacteriosis, salmonellosis, and Escherichia coli 0157:H7. Notwithstanding, the specific on-farm behaviors that result in increased exposure to zoonotic pathogens among farm residents have not been clearly identified. Exposure could occur during direct animal contact, feeding, or cleaning practices. Moreover, many zoonotic pathogens have prolonged environmental survival and may be considered environmentally acquired infections (Stanford et al., 1990; Lengerich et al., 1993; Aureli et al., 2000; Blancou et al., 2005; Jayarao et al., 2006). If farm household contamination occurs, farm residents may be exposed to an under-recognized source of infection. This is particularly important because even those residents who do not have direct animal care responsibilities, such as infants and young children, may also be exposed to these microorganisms.

Listeria monocytogenes is an important foodborne pathogen because infections in high-risk individuals--the fetuses of pregnant women, neonates, elderly, and other immunocompromised--can lead to bacteremia and meningoencephalitis with associated mortality rates approaching 30%. More recently, L. monocytogenes was recognized as a causative agent for acute febrile gastroenteritis in healthy individuals (Ryser and Marth, 2007). This organism is readily isolated from many natural sources such as soil, water, and organic materials (plant and animal origin), and it infects birds, ticks, fish, crustaceans, and over 50 mammals, including domestic livestock (Borucki et al., 2004; Nightingale et al., 2004). Ruminant animals play an important role in the amplification and dispersal of this pathogen. Rich in organic matter, the agricultural environment is a niche suitable for the survival of Listeria spp. (Jemmi and Stephan, 2006).

The specific objectives of this study were to ascertain the magnitude of household environmental contamination with zoonotic pathogens, using L. monocytogenes as an indicator organism (Nam et al., 2004). Specifically, we determined the prevalence of L. monocytogenes in rural household--both where ruminants were housed on the same site (ruminant-farm households) and in rural households where ruminants were not housed on the same premises (rural households). We also explored potential sources and routes of L. monocytogenes contamination in the home.

Materials and Methods

Study population

A convenience sample of 26 rural households (defined as not raising ruminants on site) and 26 ruminant-farm households (defined as a holding of five or more ruminant animals on the same premise) were recruited in Ohio over a 2-year period beginning in February 2006 and ending in August 2007. Selection criteria included planning on living in the same residence for 3 years and willingness to participate in a larger, ongoing microbiological and educational study. A compensation of $35 was offered to all enrolled study participants. The study was approved by the Institutional Review Board for human subject research at The Ohio State University.

Sample collection

Each house was visited four times during the course of the study at approximately monthly intervals. Food items, environmental samples, and human and ruminant feces were collected by the homeowner under the direction of investigators. All specimens were tested for the presence of L. monocytogenes. Food items were selected based on their potential to be contaminated with L. monocytogenes and being eaten without further cooking. Foods were categorized into three categories: dairy items (yogurt, sour cream, cheeses, and any raw dairy products), leftovers, and ready-to-eat deli products. If one of these items was unavailable, fresh produce was collected. Food samples (approximately 25 g) were collected with a clean kitchen implement owned by the homeowner and placed into a sterile 532 mL bag (Whirl-Pak, Nasco, and Weber Scientific, Fort Atkinson, WI).

Five environmental samples were collected, including sponge swab (Hydrasponges[TM]; Biotrace Intl., Muncie, IN) samples of various surfaces, including the following: (1) refrigerators (door handle, and a drawer or shelf); (2) kitchen sinks (faucet and drain); (3) washing machines (under the lid or drum area); (4) kitchen counter tops (rural households [without ruminants] only) or a work glove (ruminant-farm households); and (5) the bottom of a shoe/boot. Human feces were provided by some participants in Commode Specimen Collection Systems (Fisherbrand, Florence, KY), or as soiled disposable diapers. Samples of approximately 10 g each were collected from three recently voided ruminant feces and pooled. Samples were kept on ice during transport and stored at 4[degrees]C until processing, usually within 24 hours of collection. All sampling was done wearing latex medical gloves (Dot Scientific, Burton, MI), researchers wore disposable booties in houses, and single-use plastic boots in animal pens.

Microbiological analysis

For food and fecal samples, Universal Pre-enrichment Broth (Acumedia, Lansing, MI) was added to the specimen to achieve a 1:10 dilution. Twenty milliliters of Universal Pre-enrichment Broth was added to the pre-moistened Hydrasponges TM used for environmental samples to achieve a 1:10 dilution. Solid samples were homogenized, 5 strokes/sec for 2 minutes using a laboratory stomacher (Masticator IUL Instruments, Barcelona, Spain). All specimens were pre-enriched at 35[degrees]C for 24 hours (Zhang and Knabel, 2005), following which 1 mL of the pre-enrichment culture was transferred to 9 mL of Fraser Broth (Acumedia) and incubated at 35[degrees]C. A loopful (10 [micro]L) of darkened Fraser Broth was streaked on PALCAM (Acumedia) after 24 or 48 hours of incubation. PALCAM plates were incubated for 48 hours at 35[degrees]C. Subsequently, up to four black colonies surrounded by blackened media were transferred to blood agar and incubated at 37[degrees]C for 24 hours for detection of beta-hemolysis. The colonies exhibiting beta-hemolysis were further screened biochemically on RAPID Umono (Bio-Rad, Hercules, CA). A blue colony without a yellow halo was considered consistent with L. monocytogenes and preserved at -80[degrees]C in Brain Heart Infusion broth with 30% vol/vol glycerol. Up to four presumptive positive colonies per sample, if present, were banked.

A multiplex PCR assay (Zhang et al., 2004) was used for genotypic characterization of presumptive L. monocytogenes isolates. This assay allowed for confirmation of suspect isolates as L. monocytogenes species. Moreover, serotypes 4b and 1/2a generate unique amplification patterns that permitted these two serotypes to be differentiated from all other L. monocytogenes serotypes. To assess the relationship among isolates recovered within each household, molecular sequence analysis was performed on inlB and inlC amplicons generated from L. monocytogenes isolates obtained from each source from households where L. monocytogenes was recovered more than once. If more than one serotype was recovered from a single sample, the additional isolates were sequenced as well.

Statistical analysis

Neighbor joining analysis was conducted using inlB and inlC intragenic sequences (Bionumerics; Applied Maths, Austin, TX). Fisher's exact test, and odds ratio were used to test for an association between L. monocytogenes contamination and the type of residence (i.e., ruminant-farm household versus rural household). Comparisons were run on a household level. A household was considered positive if L. monocytogenes was isolated from any source (food, environmental, or ruminant feces), regardless of whether it was isolated only once from a single source or on multiple occasions from more than one source.


A total of 1779 samples were collected during the course of the study from 26 ruminant-farm households, which included 7 beef operations, 9 sheep farms, and 10 dairy farms, and 26 rural households. A total of 109 L. monocytogenes isolates were identified from 47 samples, including the following: food items, 1.1% (7/631); shoes, 9.6% (20/208); work gloves, 5.4% (6/111); kitchen sinks, 1.5% (3/204); washing machines, 1.0% (2/204); and animal fecal samples, 8.7% (9/104). All samples from human feces, refrigerators, or counter tops (collected only from rural households) were negative for L. monocytogenes (Table 1). Isolates were categorized into three serogroups based on PCR serotyping: 4b, 1/2a or 1/2c, "other" (those isolates confirmed as L. monocytogenes, but not exhibiting the multiplex PCR profile of the two aforementioned serotypes). Thirty-three of 109 (30.3%) isolates were serotype 4b, 14/109 (12.8%) were serotype 1/2a, and 62/109 (56.9%) were other. Four specimens yielded more than one serotype.

Examining the data on a household basis, L. monocytogenes was isolated from a total of 6/26 (23.1%) rural households and from a total of 14/26 (53.8%) ruminant-farm households, including composite ruminant fecal samples (p = 0.05). Excluding farms (ID 14, 25, and 43) that were only positive for ruminant fecal samples, there was no significant difference in L. monocytogenes isolation frequency between the two household types. However, when both ruminant fecal samples (not present on rural households) and food samples (which may have been contaminated from off farm sources) were excluded, the odd of detecting L. monocytogenes in household environmental samples was 5.6 times higher (95% confidence interval: 1.4-21.8) among household where ruminants were keep nearby.

Contaminated foods were found on one farm (ID 13) (1/26, 3.8%) and in three rural households (3/26,11.5%) for a total of 4 out 52 (7.7%) sampled households. On one dairy farm (ID 13), three food samples were contaminated with L. monocytogenes: raw milk on two separate visits, and a cookie made with raw milk intended to be consumed without further cooking. Both the cookie and milk samples were contaminated by L. monocytogenes serotype 4b. Two rural households (ID 28 and 46) had one positive food sample each: a strawberry (serotype other) and raw poultry (serotype 4b). The third rural home (ID 49) had L. monocytogenes-positive samples on the second and third visit, bologna and grapes, respectively, both serotype other (Table 2). There was no statistically significant association between the presence of Listeria in food and household status (p = 0.61).

Two rural households had work gloves available for sampling, and neither home had L. monocytogenes identified on the gloves. All ruminant-farm households had gloves available. There were 104 pairs of work gloves sampled from ruminant-farm households, and 5 out of the 26 ruminant-farm households (19.2%) had L. monocytogenes-positive gloves on at least one visit (Table 2). We sampled 208 shoes, 104 from rural households and 104 from ruminant-farm households. Only one shoe from a pair was sampled each time. Three out of 26 rural households (11.5%) had an L. monocytogenes-positive shoe on one visit each, and 10 out of 26 ruminant-farm households (38.5%) had a positive shoe on at least one visit (Table 2). Shoes from ruminant-farm households were more frequently contaminated than shoes from rural households (odds ratio = 4.8; 95% confidence interval: 1.2-18.7). One rural house out of 26 (3.8%) had a positive sink, and 2 ruminant-farm households out of 26 (7.7%) had positive sinks. This difference was not significant (p =1.0). Contaminated washing machines were found in 2 out of 26 (7.69%) ruminant-farm households, but not in any rural households (Table 2) (p = 0.49). Eight out of 26 ruminant-farm households (30.8%) had positive ruminant fecal samples on one or more occasions (Table 2). Bovine fecal serotypes included 1/2a and other L. monocytogenes serotypes, but not 4b.


Isolates from households where L. monocytogenes was isolated only once were not sequenced. DNA sequence analysis differentiated the 43 L. monocytogenes isolates tested into 25 unique sequence types (Fig. 1). Results from multiplex PCR and sequence typing detected L. monocytogenes in ruminant fecal samples that were indistinguishable from environmental samples (washer, gloves, and gloves) collected from the same household (ID 3, 13, and 30, respectively). The isolate obtained from the cookie made with raw milk was indistinguishable from an isolated recovered from raw milk used by the homeowner. Indistinguishable subtypes were recovered from different ruminant farms on several occasions. For example, an indistinguishable 4b subtype was isolated on ruminant farms ID 6, 18, and 17, on the fourth visit. Indistinguishable subtypes were also identified on a dairy farm (ID 25) and two rural households (ID 31 and 49). Some subtypes were repeatedly identified on the same farm. For example, dairy farm ID 13 had the same subtype isolated on visits 3 and 4.


Serotypes of L. monocytogenes commonly associated with human disease (4b and 1/2a) were isolated from rural homes. These data show that work gloves and shoes may be vehicles for the introduction of L. monocytogenes into the home. On ruminant farms, feces from the animals held on the premises may be the source of contamination. Symptomatic and asymptomatic livestock excrete L. monocytogenes in feces (Nightingale et al., 2004). As a result, the ruminant farm environment including manure, soil, feed, and water can become heavily contaminated with a mixture of L. monocytogenes strains, including those that are pathogenic to humans, such as serotypes 1/2a and 4b (Sauders et al., 2006). A study conducted by Nightingale et al. (2004) found a prevalence of L. monocytogenes of 20.2% for healthy bovine feces and 5.9% for healthy sheep feces. On farms, soil, manure, animal feedstuffs, and wildlife and plant materials can readily serve as reservoirs for Listeria spp., and there are many opportunities for rural residents to come into contact with these sources, resulting in contamination of shoes, gloves, clothing, and hands. L. monocytogenes has also been identified in soil, water, and other environmental samples taken from urban and pristine environments, although at considerably lower frequency than for samples taken from farms (Marcus, 2008).

Several farms had shoes testing positive on more than one occasion, indicating either contamination reoccurred or that the organism was not completely removed by cleaning. We also identified two Listeria-positive washing machines on farms. The contamination may have been a result of clothing worn during farm chores. Regardless of the source, contaminated washers could contribute to cross-contamination within the house, if not properly sanitized after use, especially with soiled farm clothing.

The prevalence of L. monocytogenes-positive foods in this study was comparable to that reported by others: Wagner et al. (2007) reported that 1.7% of 640 foodstuffs were positive for L. monocytogenes. In this study, L. monocytogenes was isolated from fresh fruit that is not typically associated with foodborne outbreaks. This could be of particular concern depending on how the fruits are handled before consumption, with exposure occurring through consumption of contaminated fruit or through cross-contamination of the kitchen. We also identified L. monocytogenes serotype 4b from raw milk samples on two separate sampling dates as well as food made from raw milk, highlighting the potential health risks associated with consuming raw milk (Lejeune and Rajala-Schultz, 2009). Jayarao et al. (2006) reported that 42% of dairy producers in Pennsylvania, the state immediately east of Ohio, consumed raw milk and that L. monocytogenes was present in 2.8% bulk tank milk samples.


We observed indistinguishable subtypes on repeat visits to the same farm as well as among farms with no reported epidemiological connection. Precautions were taken by researchers to avoid cross-contamination, and some of these farms were visited by different teams of researchers. Although pulsed-field gel electrophoresis currently is considered the gold standard for subtyping L. monocytogenes, its utility in detecting whether isolates that are part of the same chain of transmission are descendants of a common source strain is uncertain (Maiden et al., 1998). In contrast, sequence typing provides a highly accurate and epidemiologically relevant method for evaluating genetic relatedness (Hyytia-Trees et al., 2007). The sequence-typing methodology used herein is reported a discriminatory power similar to that of Apal pulsed-field gel electrophoresis (Davis et al., 2003; Zhang and Knabel, 2005).

Cattle and, to a lesser extent, sheep play a key role in amplifying Listeria on the farm (Borucki et al., 2004b); more importantly, the farm environment can serve as a reservoir for human epidemic clones (Chen et al., 2007). Thus, certain strains may be geographically widespread in rural environments. From a public health perspective it is important to characterize and determine the extent of overlapping of indistinguishable subtypes in the rural environment as not to cluster isolates from outbreaks solely on molecular similarity. Continued efforts to characterize subtypes endemic to the rural environment will shed light on the ecology and transmission of Listeria in the farm to fork continuum and assist in determining if clonally related strains isolated in the face of human outbreaks are attributed to a single, or multiple epidemiologically unrelated, sources.

In conclusion, using Listeria as an indicator organism of hygiene, we have shown that zoonotic pathogens can be introduced from livestock into the home via shoes and gloves. Our study was conducted in households where the farm animals and the farm household were adjacent. This type of situation is common among family-owned dairy operations in Ohio and other places. We did not test households of dairy farm workers whose residences were off-site. These dairy farm workers may also be taking pathogens with them on their hands, footwear, and clothing when they leave the farm. A potential for cross-contamination exists when proper attention to home hygiene is not observed. Simple modifications in daily routines like not wearing shoes into the home, leaving work gloves in the barn, and taking special precautions in the kitchen with raw food items could help reduce household contamination with L. monocytogenes and potentially other zoonoses common to the farm environment.


This work was supported in part by Grant #2005-5111002347, National Integrated Food Safety Initiative, Cooperative State Research, Education and Extension Service (CSREES), United States Department of Agriculture (USDA), and by state and federal funds allocated to the Ohio Agricultural Research and Development Center.

Disclosure Statement

No competing financial interests exist.

DOI: 10.1089/fpd.2009.0318


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Ann L. Kersting, (1) Lydia C. Medeiros, (2) and Jeffrey T. LeJeune (1)

(1) Food Animal Health Research Program and (2) Human Ecology, The Ohio State University, Wooster, Ohio.

Address correspondence to:

Jeffrey T. Lejeune, D.V.M., Ph.D.

Food Animal Health Research Program

Ohio Agricultural Research and Development Center

The Ohio State University

1680 Madison Ave.

Wooster, OH 44691-4096



No. of
Source n Positive (%) isolates 4b

Animal feces 104 9 (8.70%) 11 0
Gloves 111 6 (5.4%) 17 3
Shoes 208 20 (9.6%) 47 17
Sink 204 3 (1.47%) 8 1
Washing machine 204 2 (0.98%) 5 0
Refrigerator 204 0 0 0
Kitchen counter 99 0 0 0
Human stool 14 0 0 0
Food 631 7 (1.11%) 21 12
Totals 1779 47 109 33

Source % Total 1/2a % Total

Animal feces 0 3 27.30
Gloves 17.64 4 23.50
Shoes 36.17 6 12.80
Sink 12.50 1 12.50
Washing machine 0.00 0 0.00
Refrigerator 0 0
Kitchen counter 0 0 0
Human stool 0 0 0
Food 57.10 0 0.00
Totals 14

Samples with
Source Other % Total serotypes

Animal feces 8 72.70 0
Gloves 10 58.80 0
Shoes 24 51.10 3
Sink 6 75.00 1
Washing machine 5 100.00 0
Refrigerator 0 0
Kitchen counter 0 0 0
Human stool 0 0 0
Food 9 42.90 0
Totals 62 4

Table 2. Distribution of Listeria monocytogenes
Serotypes Among Households Testing Positive on One
or More Occasions

Visit 1 Visit 2

ID Status Source Serotype Source Serotype

2 NF
3 DF Feces (a) Other
6 DF
13 DF Shoe 4b
Glove Other
14 BF
17 DF Shoe 4b
18 SF Shoe 4b
19 SF Shoe 1/2a,
Shoe (b) Other
24 BF Shoe Other
25 DF
26 SF Shoe 1/2a, Shoe Other
Shoe (b) Other
Glove Other
28 NF
30 DF
31 NF
38 NF
39 DF Glove 1/2a
43 DF
44 DF Shoe Other
Shoe Other
46 NF Poultry 4b
49 NF Bologna Other

Visit 3 Visit 4

ID Status Source Serotype Source Serotype

2 NF Shoe Other
3 DF Washer Other
6 DF Shoe 4b
13 DF Raw milk 4b Raw milk 4b
Cookie 4b
Shoe Other
Feces (a) Other
14 BF Feces (a) Other
17 DF Sink 1/2a, Shoe 4b
Sink (b) Other
18 SF Sink 4b Glove 4b
19 SF Shoe Other
Feces (a) Other
24 BF
25 DF Feces (a) Other
26 SF Shoe Other
28 NF Strawberry Other
30 DF Shoe Other
Glove Other
Feces (a) Other
31 NF Sink Other
Shoe Other
38 NF Shoe 1/2a
39 DF Shoe 1/2a
Shoe (b) Other
Glove 1/2a
43 DF Feces (a) 1/2a Feces (a) 1/2a
44 DF Washer Other Feces (a) 1/2a
Shoe Other
46 NF
49 NF Grape Other

(a) Ruminant source.

(b) Sample yielded isolates with more than one serotype.
NF, rural home; DF, dairy farm; BF, beef farm; SF, sheep farm.Source Citation
Kersting, Ann L., Lydia C. Medeiros, and Jeffrey T. LeJeune. "Differences in Listeria monocytogenes contamination of rural Ohio residences with and without livestock." Foodborne Pathogens and Disease 7.1 (2010): 57+. Academic OneFile. Web. 26 Jan. 2010. .

Gale Document Number:A216896478

Disclaimer:This information is not a tool for self-diagnosis or a substitute for professional care.

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