Jacobs Journal of Veterinary Science and Research

Cryptosporidium parvum Co-Infection on the Magnitude of Escherichia coli O157:H7 Shedding in Experimentally Inoculated Pigs

*Edward R. Atwill
Western Institute For Food Safety And Security, University Of California-Davis, California-Davis, United States

*Corresponding Author:
Edward R. Atwill
Western Institute For Food Safety And Security, University Of California-Davis, California-Davis, United States
Email:ratwill@ucdavis.edu

Published on: 2019-03-30

Abstract

Escherichia coli O157:H7 [E. coli O157:H7] and Cryptosporidium parvum [C. parvum] are zoonotic pathogens that have potential for economic impact worldwide. Neonatal animals are especially vulnerable to these types of pathogens. The significance of single vs. concurrent [multiple] infections are unclear. Our objective was to determine differences in the magnitude of daily shedding and colonization by E. coli O157:H7 between pigs co-infected with C. parvum and pigs inoculated only with E. coli O157:H7. Fifteen out of 27 weaned 2-month-old pigs were inoculated with a C. parvum isolate from naturally-infected calves. On the fourth day of infection, 23 pigs were inoculated with approximately 102, 103, 104, 105, or 107 colony forming units or CFU of an E. coli O157:H7 strain. On days 3 and 4 post-inoculation [PI] with E. coli O157:H7, one pig co-infected with C. parvum and 107 CFU E. coli was euthanized and necropsied to determine the enumeration and distribution of colonized E. coli O157:H7. On days 7 and 14 PI, one co-infected and non co-infected pig from each inoculum group was also euthanized for enumeration and distribution of colonized E. coli O157:H7. The total environmental shedding load of E. coli O157:H7 suggested that the amount of time PI and the dose of E. coli O157:H7 are important factors in relation to the magnitude of E. coli O157:H7 shedding. Trends between co-infected and non co-infected groups were seen, but the relationship was non-significant in this model. The infectious dose for 50% [ID50] of the pigs inoculated using the outbreak E. coli O157:H7 strain was determined to be in the range of 103-104 CFU.

Keywords

Escherichia coli O157:H7; Cryptosporidium parvum; Co-Infection; Pigs; Fecal Shedding

Introduction

E coli O157:H7 and other serotypes of Shiga toxigenic E. coli [STEC] cause an estimated 110,000 cases of human illness yearly in the United States. Most of these cases are thought to occur as a result of the ingestion of contaminated food or water, although cases of direct contact with animal feces and person-to-person transmission have been reported. The infection is characterized by diarrhea but it can progress to hemorrhagic colitis in humans and in some cases cause hemolytic uremic syndrome [HUS], leading to acute renal failure and mortality.  This syndrome occurs in approximately 6% of people infected with the O157:H7 serotype and is most commonly seen in children.  Cattle are considered to be a major reservoir of STEC, but evidence has been collected to suggest that domestic pigs sometimes serve as reservoirs of E. coli O157:H7 as well.  Cornick and Helgerson determined that E. coli O157:H7 is transmitted from donor to naïve pigs, and infected pigs shed the bacteria in their feces for up to two months.  In 2006, a field investigation suggested that feral swine were also possible carriers of the E. coli O157:H7 serotype that was associated with a nationwide outbreak involving the consumption of fresh spinach grown at a ranch in San Benito County, California. This investigation identified a relatively high percentage of positive fecal specimens [~15%] collected among the feral swine population. Since E. coli O157:H7 has a diverse range of reservoir hosts, a better understanding of its pathogenic mechanisms is needed. There has been extensive work using animal models to elucidate host-pathogen interactions. It has been shown that neonatal animals, especially calves, are the most susceptible age class for E. coli O157:H7 related diseases and symptoms [5]. The attachment of E. coli O157:H7 to the host’s microvillous border causes malabsorption and maldigestion due to the loss of villous enterocytes, leading tothe clinical presentation of diarrhea [5, 6].

In addition to E. coli O157:H7, young animals are also vulnerable to co-infections with other bacterial pathotypes, protozoal, and viral pathogens. One major pathogen that causes diarrhea in young animals and can co-infect with E. coli O157:H7 is C. parvum. Similar to E. coli O157:H7, C. parvum infects multiple mammalian species and has a significant impact on both human and domestic farm animal health. During the incubation period and clinical course of the infection, the parasite proliferates mainly inside the epithelial cells of the jejunum and the ileum, causing secretory diarrhea by interference with enterocyte function. However, after the start of oocyst shedding, the lesions can spread to other parts of the small and the large intestine [7

E. coli O157:H7 and C. parvum initially colonize the distal small intestine, with the potential extension into the large intestine, particularly in regard to E. coli O157:H7. Unfortunately, it’s still uncertain how these two pathogens interact if co-infected into the same host. Enemark et al [8] noticed that the unintended presence of rotavirus in experimentally infected pigs with C. parvum resulted in an additive or synergistic effect between the two pathogens resulting in prolonged diarrhea, increased oocyst shedding, and decreased weight gain. The same synergistic effect was also described by Newsome and Coney [9] with rotavirus and enterotoxigenic E. coli inoculated in mice, as well as with turkey coronavirus and enteropathogenic E. coli coinfection in turkeys [10]. On the other hand, La Ragione et al [7] noticed changes in E. coli O157:H7 shedding trends when inoculated into young lambs that were predisposed to C. parvum, but could not conclude that concurrent C. parvum infection altered the shedding of the E. coli pathogen

Here, we examine the infection pattern of E. coli O157:H7 experimentally inoculated into weaned pigs that had been previously infected with C. parvum. Our objective was to determine differences in the magnitude of daily shedding of and colonization by E. coli O157:H7 between pigs co-infected with C. parvum and pigs only inoculated with E. coli O157:H7.We also examined the dose-dependent characteristics of an E. coli O157:H7 strain between co-infected and non-co-infected groups. In conclusion, we found that time post-inoculation [PI] and dose of E. coli O157:H7 were important factors in relation to the magnitude of E.coli O157:H7 shedding, not the state of being co-infected with C. parvum.

 

Material and Methods

Escherichia coli O157:H7 and Cryptosporidium oocysts

The E. coli O157:H7 strain used in the current study was isolated during the 2006 spinach outbreak in San Benito County, CA. The strain was also evaluated using endpoint polymerase chain reaction [PCR] for Shiga toxins [Stx 1 and Stx 2], intimin [eaeA], and hemolysin A [HlyA] virulence genes [Eppendorf, Hauppauge, New York] protocol [11]. Three replicate growth curves were made by incubating a bead of stock inoculum, in 150 mL of brain heart infusion, BHI [Sigma-Aldrich, St. Louis, Missouri] broth. Each broth was shaken [at 100 rpm] for six hours at 37° C, while checking optical density every 30 minutes with a spectrophotometer [Shimadzu, Torrance, California] at 610 nm. Serial dilutions from 10-1 to 10-10 were made in 9 mL of phosphate buffered saline [PBS; Sigma-Aldrich, St. Louis, Missouri]. Dilutions were plated on Luria Base [LB] agar [Sigma-Aldrich, St. Louis, Missouri] and incubated overnight at 37° C. Using the number of colonies that grew from each dilution, the overall concentration at each time point was calculated. On the day of inoculation, the same method of growth was used to reach the observed turbidity, but then the flasks were placed on ice for 15 minutes. The optical density was then measured at 610 nm and the concentration of E. coli in the stock solution was calculated by using the generated growth curve from the O157:H7 strain. The inocula was then prepared in PBS from the stock solution.

The C. parvum oocysts used in this study were isolated in fecal samples collected from 10-15 day old calves at a local dairy in Davis, CA. Oocysts were purified and then quantified with immunofluorescent microscopy [12]. To confirm the oocysts were C. parvum, DNA was extracted with Qiagen QIAamp DNA stool extraction kit [Qiagen Inc, Valencia, California] and a nested PCR was performed using primers to detect the 18 s small-subunit rRNA gene [13, 14]. Confirmed C. parvum oocysts solutions were used to prepare inoculums for the animal experiments.

 

Ethics Statement

All animal experiments were conducted under the approval by Institutional Animal Care and Use Committee [IACUC] of the University of California [UC] Davis Animal Care and Use program [Protocol #15450]. Experimental procedures followed the federal guidelines outlined in the “Animal Welfare Act” and “Health Research Extension Act,” where personal protective equipment for each pathogen, standard operating protocols for pathogens used, daily cleaning/observation/animal enrichment, and sedation/ euthanasia/necropsy methods were described in detail for the review and approval by the committee

Animal Experiments

Twenty-seven 2-month-old Yorkshire/Hampshire piglets were purchased from UC Davis Swine Teaching and Research Center. Each pig was housed individually in a biosafety level-II animal facility at the Teaching and Research Animal Care Services [TRACS]. The pigs were fed maintenance pellets [Purina, St. Louis, Missouri] twice a day and provided free access to water. Misters and fans were also provided during warm ambient temperatures. Bedding material was removed while pens were bleached and rinsed daily, to ensure fresh fecal collection. Upon arrival, each pig was examined by UC Davis Campus Veterinary Service and only pigs deemed healthy were used for the experiment. Additionally, each pig was tested for the presence of Cryptosporidium sp. with an acid-fast stain and E. coli O157:H7 with an enrichment procedure on fresh feces collected on three consecutive days. Ten grams of each fecal sample was measured into 100 mL of Tryptic Soy Broth [TSB]. The samples were incubated at 25° C for 2 hours, 42° C for 8 hours followed by immunomagnetic separation [IMS] using anti-O157 antibodies [Dynal Inc, Camarillo, California]. Beads with attached bacteria were cultured on MacConkey agar with sorbitol, cefixime, and potassium tellurite [Becton, Dickinson, Co, Sparks, Maryland], and Rainbow agar [Biolog, Hayward, California] to detect the presence of E. coli O157:H7. If any colonies were considered O157:H7 suspects, end-point PCR] was performed using a set of specific primers to detect O-antigen-encoding rfb regions of E. coli O157:H7 [11]. Once the pigs were confirmed negative forCryptosporidium and E. coli O157:H7 prior infections, they were assigned an E. coli O157:H7 inoculum dose and either grouped for co-infection with C. parvum or non-co-infection with only E. coli O157:H7 or assigned to a control group. 

Inoculation Of Animals

Primary Inoculation with C. parvum

One week after arriving at the TRACS facility, fifteen pigs were individually inoculated with C. parvum by adding approximately an aliquot of 500 µl that contains 105 oocysts to the center of a feed ball constructed using crushed feed pellets and peanut butter. The feed ball was sticky and dense enough to absorb and retain the inoculum inside, as well as small enough so that the pigs could consume it in a short amount of time. The animals were observed until all inoculated feed mixture was consumed [approximately 15- 20 minutes]. Primary infection was allowed to develop for four days PI with C. parvum. Fecal samples were collected and processed from each pig on days 3 and 4 PI. Acid-fast stain was used to confirm primary Cryptosporidium infection.

Secondary inoculation withE. coli O157:H7

On the fourth day of primary infection with C. parvum, 23 pigs were inoculated with approximately 102 , 103 , 104 , 105 , or 107 colony forming units [CFU] of the E. coli O157:H7 the same way as inoculation with the Cryptosporidium oocysts. Each inoculum dose was administered to 2 co-infected pigs and 2 non co-infected pigs, except for the 107 CFU inoculum groups which had 5 co-infected pigs and 2 E. coli O157:H7 only pigs. Two pigs served as negative controls and two pigs were inoculated with C. parvum only, which served as Cryptosporidium controls. Serial dilutions of the inoculum from 10-1 to 10-10 were made in 9mL of PBS and plated in triplicate on LB agar for each inoculum and stock solution. After an overnight incubation at 37° C, numbers of colonies on LB plates were counted in order to calculate the true concentrations of each inoculum.

Daily Sampling and Detection of E. coli O157:H7

Fecal samples were collected late morning from each pig daily until the 14th day PI. Coveralls, gloves, bootcovers and face masks were used during sample collection with gloves and boot covers changed between pens to prevent cross-contamination. Fresh fecals were collected from the floor of the pen, where no water or disinfectant pooled. At each collection, the negative control was always sampled first leading to the pigs inoculated with the lower doses of the E. coli O157:H7 inoculums and ending with the highest dose. Approximately 50 g of fecal material was collected from each pen, transported to the laboratory on ice and stored at 4° C upon arrival to be processed within 24 hours of sampling. A 10 g sample was placed in whirl pac bags [eNasco, Fort Atkinson, Wisconsin] containing 90 mL TSB and then incubated for 2 hours at 25° C, 8 hours at 42° C, and held overnight at 6° C. E. coli O157:H7 was recovered using IMS and the Dynal Bead Retriever, with 50 μL of washed beads streaked for isolation on Rainbow agar and another 50 μL streaked for isolation on Sorbitol MacConkey Agar. As performed in our laboratory, this IMS method has been shown to detect as few as 1 CFU/10g of pig feces. Two suspect colonies per positive plate were confirmed with end-point PCR [15]. Confirmed colonies were stored at -80° C in Microbank vials for further analysis

Quantification of E. coli O157:H7 in Feces

If the fecal sample was positive for E. coli O157:H7 on IMS, then another 10 g of feces was placed in a 50 mL conical vial containing 30 mL of PBS and homogenized for 15 minutes using a wrist-action shaker [Burrell Scientific, Pittsburgh, Pennsylvania]. The fecal mixture was then centrifuged at 500 g for 5 minutes. Ten-fold serial dilutions of the supernatant were made in PBS and spread onto two Rainbow agar plates. Plates were incubated at 37° C for 24 hours and colonies counted to determine E. coli O157:H7 concentrations CFU/g in feces. Two colonies per plate were confirmed E. coli O157:H7 positive with end-point PCR [15].

Determination of the Detection Limit in Feces Using the Serial Dilution Assay

To determine the detection limit in fecal samples, 50 g of feces were collected from four isolated pigs at the UC Davis Swine Teaching and Research Center. Ten grams of feces from each pig were then tested for the presence of E. coli O157:H7 with the method described above. If confirmed negative, three original fecal samples were divided into four 10 g amounts and spiked with approximately 50, 100, 500, or 1000 CFU of the E. coli O157:H7 inoculum. The fourth original sample served as the negative control for each spiked dose. Fecal samples were then processed in the same manner as described above for quantifying the spiked E. coli O157:H7. 

Verification of Shed Bacteria

In order to confirm the E. coli O157:H7 shed in feces PI were the same strain as the inoculated bacteria, PulsedField Gel Electrophoresis [PFGE] was performed. Two isolates, one early and one late in the trial, were chosen from each positive pig. Each isolate was digested using the Centers for Disease Control and Prevention PulseNet PFGE with XbaI restriction enzyme [16]. The isolates were run alongside the inoculum strain on the CHEF Mapper [Bio-Rad Laboratories, Hercules, California] to determine whether the E. coli O157:H7 that was collected during the experiment in the feces was the original inoculated strain. In addition, each chosen late isolate and the original outbreak E. coli O157:H7 was tested for both Shiga toxins, hemolysin A [hlyA], and attaching/effacing [eaeA] genes [11].

Detection and Quantification of C. parvum

Daily fecal samples were processed for C. parvum detection and quantification in the same manner as described above for the oocyst inoculum preparation. Immunofluorescent microscopy determined the number of oocysts present for each co-infected pig on every other day of collection. One hundred microliters of fecal suspension was used for nested PCR confirmation. On day 7 and 14 PI, all fecal samples, including the negative controls and non-co-infected pigs, were tested using immunoflourescent microscopy or PCR for the presence of C. parvum as described above.

Quantifying E. coli O157:H7 Concentrations in Tissues

On days 3 and 4 PI, one pig co-infected with C. parvum and 107 CFU E. coli O157:H7 was sedated with a mixture of ketamine and xylazine intramuscularly, and theneuthanized intravenously with Euthasol solution [1 mL/10 lbs of body weight, Virbac Animal Health, Fort Worth, Texas]. During necropsy, sections of the pars esophagea, pylorus, duodenum, proximal jejunum, middle jejunum, distal jejunum, proximal ileum, middle ileum, distal ileum, cecum, spiral colon, proximal colon, distal colon, and rectum were collected for bacterial culture, as well as the tonsils, pancreas, liver, mesenteric lymph node, gall bladder, lung, and kidney. Each intestinal segment was tied with string at both ends and maintained on ice for transport to the laboratory so that the contents and mucosa could be cultured for the presence of E. coli O157:H7. One end of the tissue segment was cut and the contents were collected in a whirl-pac bag containing 90 mL of TSB. The intestinal tissue was then cut longitudinally to expose the mucosa and placed in a separate whirl bag with 90 mL of TSB. The non-intestinal tissues were placed in a bag containing 50 mL of TSB. All TSB bags were then incubated for 2 h at 37° C, 8 h at 42° C, and then held overnight at 6° C. E. coli O157:H7 was recovered using the same method as described above

If an intestinal tissue segment was positive for E. coli O157:H7 with IMS, then 5 g of that saved segment stored at -20° C, for no more than 48 hours, was placed in a whirl-pac bag containing 20 mL of PBS and homogenized for one minute at 230 rpm in a stomacher 400 circulator [Seward, Port Saint Lucie, Florida]. Fifty and 5 µL of stomach contents were spread onto one of two Rainbow agar plates and incubated at 37° C for 24 hours. Colonies were counted to determine CFU/g of tissue and two colonies per plate were confirmed as E. coli O157:H7 with end-point PCR [17]. On days 7 and 14 PI, two pigs from each inoculum dose, one co-infected and one infected with only E. coli O157:H7, were euthanized and necropsied in the same manner for culture.

Determination of the Detection Limit on Intestinal Tissue using the Serial Dilution Assay

One pig from the UC Davis Swine Teaching and Research Center, roughly the same age and weight as the experimental pigs, was tested for the presence of E. coli O157:H7 as described above one day prior to slaughter. The pig was confirmed negative for E. coli O157:H7 and the entire intestinal tract was collected during slaughter atthe UC Davis Meat Laboratory and transported on ice for processing. Intestinal sections ranging from the stomach to the rectum was then trimmed into ten 5 g pieces and cut longitudinally to expose the mucosa. Nine of the 5 g pieces for each intestinal section were then spiked in triplications with approximately 1000, 10000, or 25000 CFU of the E. coli O157:H7 inoculum strain. The last 5 g piece served as a negative control for each section. All tissue pieces were processed in the same manner as described above to quantify the spiked E. coli O157:H7. 

Data Analysis

Bacterial counts from daily fecal collections were converted to total environmental shedding for each pig by multiplying daily bacterial counts with the starting weight of the pig and the standard amount of feces produced by weaned production pigs [18]. The total environmental shedding load was then standardized by adding 1 and converted to log10. Colonized bacterial counts in each tissue segment [CFU/gram of tissue] were also standardized by adding 1 and converted to log10. The effects of dose, time, and co-infection with C. parvum on the magnitude of daily shedding and colonization of E. coli O157:H7 was analyzed with Poisson regression performed in StataIC 12 computer software [7, 9], with the random-effect being the pig. The infectious dose at 50% [ID50] for the 2006 outbreak E. coli O157:H7 strain was calculated by using binary logistic regression with StataIC 12 [StataCorp LP, College Station, Texas] computer software.

Results

An infected pig was defined as one that shed the E. coli O157:H7 inoculum strain for more than 48 hours PI. This was to account for passage of the inoculum through the gastrointestinal tract. The E. coli O157:H7 outbreak strain used for this study was PCR-confirmed positive for Shiga toxin 2, attaching/effacing [eaeA], and hemolysin A [hlyA] genes, which was the same virulence pattern as the strain isolated from an infected human specimen. All E. coli O157:H7 isolates had the same virulence genes as the inoculated E. coli O157:H7 strain. In addition, Pulsed-Field Gel Electrophoresis [PFGE] confirmed that all E. coli O157:H7 isolates shed by positive pigs were identical to the inoculated strain. For each pig, fecal and selected intestinal tissues were confirmed positive for the E. coli O157:H7 inoculum. 

During this study, pigs showed no clinical signs of infection, even though E. coli O157:H7 and/or C. parvum were detected in each daily sample. No E. coli O157:H7 was detected in feces or tissue from either negative or C. parvum controls. In 6 of the 12 pigs not experimentally inoculated with C. parvum, an unanticipated C. parvum infection developed despite the careful cleaning and sampling regimen. Four pigs inoculated only with the E. coli O157:H7 strain and both negative controls were discovered to be infected with C. parvum during the processing of fecals on days 7 and 14 PI. This is taken into consideration in the statistical analysis by counting them as infected with C. parvum.

On days 3 and 4 PI, one pig co-infected with C. parvum and 107 CFU E. coli O157:H7 was euthanized and necropsied for the enumeration and distribution of E. coli O157:H7. The pig euthanized on day 3 PI had greater concentrations [CFU/g of tissue] of E. coli O157:H7 in the intestinal tissues when compared to the pig euthanized 4 days PI [Figure 1]. The pig euthanized 3 days PI also had a greater distribution of colonized bacteria in its intestine. For both pigs, C. parvum was observed in the ileum and daily fecal samples were positive for the presence of E. coli O157:H

Figure 1: Concentration of Colonized E. coli O157:H7 from Co-Infected Pigs days 3 and 4 PI

Concentration (Log10 (CFU/g of tissue)) of Colonized E. coli O157:H7 from Pigs inoculated with C. parvum and 107 CFU outbreak E. coli O157:H7 strain then euthanized on days 3 and 4 Post-Inoculation with E. coli O157:H7

One co-infected and one non co-infected pig from each inoculum group was euthanized on days 7 and 14 PI with E. coli O157:H7 to determine the distribution and concentration of E. coli O157:H7 colonized in the selected intestinal tissues. Five of the nine pigs euthanized 7 days PI wereconsidered infected with E. coli O157:H7. One C. parvum control pig was also euthanized at this time point, but fecal samples and tissue were not positive for the presence of E. coli O157:H7. Pigs inoculated with 105 and 107 CFU of E. coli O157:H7 co-infected and not, had a longer period of total environmental shedding load of E. coli O157:H7 in their feces during the 7 days compared to the pigs inoculated with the lower CFU doses that either shed for 2-3 days PI or not at all 

Table 1: Daily Total Environmental Shedding load of fecal E. coli O157:H7 (Log10 (CFU/g of feces)) from pigs euthanized 7 days PI. (A) Pigs inoculated with E. coli O157:H7 only (B) Pigs inoculated with E. coli O157:H7 and C. parvum with amount of oocysts shed on day of euthanasia (7 days PI).

(+) Indicates IMS positive results, but concentration of E. coli O157:H7 in the fecal sample was too low to detect with serial dilutions, so considered positive by culture and PCR confirmation; E. coli O157:H7 negative pigs in each group were excluded from the tables (4 experimental pigs in total); The C. parvum control pig euthanized at 7 days PI was negative for the presence of E. coli O157:H7

The co-infected pigs with higher E. coli O157:H7 inoculum doses also shed higher concentrations of C. parvum oocysts in their feces on day 7 PI compared to the lower co-infection inoculum doses of E. coli O157:H7. The concentration [Log10 (CFU/g of tissue)] of E. coli O157:H7 present in the intestinal tissues of these pigs are shown in Figure 2

Figure 2: Concentration of Colonized E. coli O157:H7 from Pigs euthanized 7 days PI

Concentration (Log10 (CFU/g of tissue)) of Colonized E. coli O157:H7 from IMS positive pigs euthanized 7 days PI with E. coli O157:H7, (A) E. coli O157:H7 only pigs, (B) Co-infected pigs with E. coli O157:H7 and C. parvum. The pig only inoculated with 105 CFU E. coli O157:H7 had a wider distribution of colonized bacteria in its large intestine with the highest concentration detected in the spiral colon compared to the pig inoculated with 103 CFU E. coli O157:H7 only. The co-infected group inoculated at 107 CFU had the highest concentration and widest distribution of E. coli O157:H7 in its tissues compared to the other co-infected pigs. No E. coli O157:H7 was detected in feces from the group co-infected with C. parvum and 104 CFU E. coli O157:H7, but E. coli O157:H7 was recovered in the cecumand IMS was positive in the proximal colon at necropsy. This means that the proximal colon was culture positive, but numbers were too low to determine quantitatively. Eight out of 12 pigs euthanized 14 days PI were considered infected with E. coli O157:H7. Pigs of both negative controls and the C. parvum control euthanized at this time point were negative for the presence of E. coli O157:H7 in daily fecal samples and tissues. For this group euthanized 14 days PI, fecal E. coli O157:H7 was recovered from pigs co-infected and non-co-infected that had been inoculated with 104 , 105 , and 107 CFU E. coli O157:H7 doses [Table 2]. Both negative controls and the C. parvum control were negative for the presence of E. coli O157:H7. The pig inoculated with E. coli O157:H7 at 104 CFU

Table 2: Daily total environmental shedding load of fecal E. coli O157:H7 (Log10 (CFU/g of feces)) from pigs euthanized 14 days PI. (A) Pigs inoculated with E. coli O157:H7 (B) Pigs inoculated with E. coli O157:H7 and C. parvum with amount of oocysts shed on the day of euthanasia (14 days PI)

had the longest total environmental shedding period, 14 days PI, compared to the other pigs only inoculated with E. coli O157:H7. In contrast, the co-infected pigs followed more of a dose-dependent trend. The pig co-infected and inoculated with E. coli O157:H7 at 107 CFU had the longest period of environmental shedding as well as the highest concentrations of C. parvum oocysts in feces detected on the 14th day PI. Even though E. coli O157:H7 was detected in daily feces from 8 pigs, only 3 of these pigs had detectable

E. coli O157:H7 in their intestinal tissue. Serial dilutions detected the concentration [Log10 (CFU/g of tissue)] of colonized E. coli O157:H7 in both co-infected and non-co-infected pigs inoculated at 104 CFU [(Figure 3], where the non-co-infected had a wider distribution of colonized tissues compared to the co-infected group. The non-co-infected 105 CFU E. coli O157:H7 group was culture positive with IMS and PCR confirmation, but concentrations were too low to quantify with serial dilu

Figure 3: Concentration of Colonized E. coli O157:H7 from pigs euthanized 14 days PI.

Concentration (Log10 (CFU/g of tissue)) of colonized E. coli O157:H7 from IMS positive pigs euthanized 14 days post-inoculation with E. coli O157:H7, Jejunum and Ileum Tissues were negative for the presence of E. coli O157:H7 for both pigs

Statistically, the time PI and the dose of inoculated E. coli O157:H7 had a significant effect on the total environmental load of shed E. coli O157:H7 in all groups [p=0.001 and p=0.000], while the initial inoculation of C. parvum and the amounts of daily C. parvum oocysts were non-significant [p=0.701 and p=0.495]. This interaction can be graphically seen in Figure 4, which demonstrates that at lowerinoculum doses, the predicted total environmental load of fecal E. coli O157:H7 increases slightly over time [p=0.00]. At the inoculum dose of 104 CFU, the environmental load of E. coli O157:H7 starts out at a higher value and gradually decreases. The slope of the line becomes greater as the inoculum dose increases. In contrast, the amount of time PI, the inoculated dose of E. coli O157:H7, and the initial inoculation of C. parvum had no significant effect on the concentration of colonized E. coli O157:H7 [Log10 (CFU/g of tissue)] in the different intestinal segments, despite seeing trends between doses and co-infected groups[p>0.05].

Figure 4: Predicted values of fecal E. coli O157:H7. Predicted total environmental shedding load of fecal E. coli O157:H7 over time while dose is held constant.

The infectious dose at 50% [ID50] was calculated to show that the approximate range of 103-104 CFU is the ID50 value for this outbreak E. coli O157:H7 strain inoculated in 2 month old domestic pigs [Figure 5] 

Figure 5: Infectious dose curve. The infectious dose at 50% (ID50) curve for the outbreak E. coli O157:H7 strain in two month old domestic pigs

Table 3: Detection level of the outbreak E. coli O157:H7 strain with the serial dilution assay

(A). Fecal detection limit with four different spiked doses of E. coli O157:H7 and three replications each;

(B) Tissudetection limit with three different spiked doses of E. coli O157:H7 and three replications each, Negative controls for fecal and tissue were negative for the presence of spiked E. coli O157:H7. 

 

Discussion

ussion The ID50 for the outbreak E. coli O157:H7 strain in 2 month old domestic swine was approximately 103 -104 CFU. This was similar to what has been reported by Cornick and Helgerson [1] in which the infectious dose of E. coli O157:H7 for 3-to-4 month old pigs was approximately 6x103 CFU in vitro grown bacteria. This is also in agreement with what has been reported for young calves [5x103 to 9x103 CFU], although lower doses may be infectious to an occasional animal [19]. It was important to show that the outbreak E. coli O157:H7 inoculum had a low ID50 since the strain was a key isolate during the 2006 nationwide spinach outbreak, which infected humans and possibly other animals [3]. Due to it’s virulence genes, the E. coli O157:H7 strain also had the capability of causing infection once experimentally inoculated into a swine model.

No detectable clinical signs of disease were observed in this study even though both daily fecal samples and tissues collected at the time of necropsy were positive for the inoculated E. coli O157:H7. Due to the warm temperatures during the study, water misters and fans were placed towards the back of each pen. Neurological signs did not develop in any of the pigs, unlike what has been described for gnotobiotic, cesarean section-derived colostrum deprived, and naturally farrowed suckling pig models inoculated with E. coli O157:H7 [20, 21]. Despite the careful cleaning and sampling regimen, 6 of the 12 pigs not experimentally inoculated with C. parvum became accidentally infected with C. parvum. The source of the infection was thought to be the inter-connected drainage system that ran along each of the pens, where co-infected and non co-infected pigs would sometimes defecate. With the water from the misters pooling in the back of the pens and the drainage system, C. parvum was thought to have accidentally infected 4 E. coli O157:H7 only pigs and the 2 negative controls from neighboring co-infected pigs.

The previously reported synergistic effect of C. parvum/rotavirus infection in calves, rotavirus/E. coli infectionin mice, and rotavirus/enteropathogenic E. coli infection in weanling pigs [8, 9, 22], raised the question of whether C. parvum has an effect on E. coli O157:H7 colonization and shedding in pigs. Concurrent infections with two or more enteropathogens, including Cryptosporidium in association with E. coli, have previously been described in naturally infected diarrhoeic calves and goats [7, 23, 17]. In theory, the two pathogens should interact because C. parvum invades and develops inside the epithelial cell of the jejunum and ileum, although on the edge of the host cell cytoplasm. After one life cycle, the thin-walled oocysts can spread and infect other parts of the small and the large intestine [7, 24], possibly inhibiting the initial attachment and colonization of the E. coli O157:H7 in the distal ileum and large intestine if the host is co-infected. It has also been documented that both pathogens use actin polymerization to initiate infection, which could explain a possible co-localization [12, 25]. In this study, the differences of E. coli O157:H7 fecal concentration in pigs co-infected with C. parvum and E. coli O157:H7 versus pigs only inoculated with E. coli O157:H7 was not significant; meaning a primary infection with C. parvum had no effect on the total fecal shedding of E. coli O157:H7. It’s possible that our dose of 105 oocysts was not high enough for our pig model to show an effect. Even though co-infection with C. parvum and E. coli O157:H7 was not significant in our study, trends were seen between co-infected and non-co-infected groups suggesting that there is a possible protective interaction between the two pathogensas seen in La Ragione e

. The amount of time PI and the dose of inoculated E. coli O157:H7 had a significant effect on the concentration of fecal shedding of E. coli O157:H7 in all positive pigs. With low inoculum doses, there is a slight increase of predicted E. coli O157:H7 as time increases, fluctuating within a range or tapering off, compared to high inoculum doses that start out high and decrease over time. With each dose increase, the greater the slope of decline becomes for the predicted number of E. coli O157:H7. This can be supported by Cornick and Helgerson [1] where they determined that at least in the early days following inoculation, the inoculum dose has an important influence on the magnitude of E. coli O157:H7 fecal shedding. They concluded that both the inoculum dose and an individual’s susceptibility to colonization are important factors in the persistent shedding of E. coli O157:H7

With the methods of IMS and serial dilution, we were able to detect and quantify the colonization and daily shedding of E. coli O157:H7 from each positive pig. The fecal and tissue detection levels of the serial dilution method in the current study were not as low as 50 CFU/g stated by Cornick and Helgerson [1], but were still successful in quantifying the E. coli O157:H7 in feces [ ≥ 100 CFU/g] and intestinal tissue [ ≥ 1000 CFU/g]. The variations in protocol, such as centrifugation of the fecal suspension in our study, might account for the difference in the detection limit. When the fecal suspension was not centrifuged, it appeared that background bacteria inhibited the growth of the E. coli O157:H7 inoculum.

 

Conclusion

Collectively, the data in this study suggest that the dose of E. coli O157:H7 and the length of duration of infection are important factors in relation to the total environmental shedding of E. coli O157:H7. Initial exposure and colonization of C. parvum in 2-month-old domesticated pigs does not appear to enhance or abrogate shedding of E. coli O157:H7. Trends between co-infected and non-co-infected groups were seen suggesting a possible protective interaction, but the relationship was non-significant using this pig model.

Acknowledgements

The authors are grateful to the Univeristy of California, Davis, Teaching and Research Animal Care Services (TRACS) for their assistance on the purchasing, care, and transportation of the animals used for this experiment. The authors would also like to thank the California Animal Health and Food Safety Laboratory at the Univeristy of California, Davis, for their assistance during necropsies. This research was funded by the Center for Food Animal Health at the University of California, Davis, School of Veterinary Medicicne

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