Prevalence of Clostridium difficle (C. diff) in Connecticut Swine farms

Final Report for GNE10-010

Project Type: Graduate Student
Funds awarded in 2010: $12,520.00
Projected End Date: 12/31/2010
Grant Recipient: Yale University School of Public Health
Region: Northeast
State: Connecticut
Graduate Student:
Faculty Advisor:
Dr. Robert Heimer
Yale University School of Public Health
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Project Information

Summary:

There is a growing concern over the antibiotic-use associated emergence of bacteria such as Clostridium difficle in livestock and its implication for public health. C. difficle (C. diff) is the main cause of antibiotic induced diarrhea acquired in hospital settings. Recently, infection has not only been seen in individuals with no hospital contact – referred to as Community acquired Clostridium difficle associated diarrhea (CA-CDAD) – but infections have also increased in severity. This study determined if the farm environment is a potential source of infection by looking at colonization rates for C. diff in CT swine industry. This study searched for the presence of C.diff in a convenience sample of 15 swine farms in the southern New England region (MA and CT), 80% of which reported the non-routine use of antimicrobials, a major risk factor for C. diff colonization and infection.

From Nov 2010 to July 2011, 90 freshly voided fecal samples were collected from eleven breeding (farrow-finish), three 4H, and one finishing farm in the southern New England region. Using a rapid membrane enzyme immunoassay (EIA) test, all samples tested negative presence of C. diff antigen and toxins, A (tcdA) and B (tcdB). Twenty samples were also analyzed using CDC`s culture and isolation protocol for C.diff. All of these were also negative for bacterium growth.

This result mirrors that from testing the meat (pork, chicken turkey and beef) supply from local grocery stores, and together suggests that the recent increase in incidence rates of community associated C.diff infection in humans may be due to other reasons.

Introduction:

Swine flu has highlighted the importance of research at the animal-human interface, and how infectious agents can adapt to different hosts and settings. Over the years there has been a push to include swine workers in pandemic preparedness(1), and recent events have justified such push. Apart from the flu, swine workers are constantly exposed to different pathogens due to their direct contact with swine waste such as manure, urine and tissues. These wastes contain organisms such as Streptococcus, Salmonella, leptospira and Clostridium Spp which can infect humans and may be virulent(2). Thus, the purpose of this project was to determine the prevalence, and characteristics of Clostridium difficle (C. diff) on CT swine farm. C. diff is a spore forming and toxin producing bacteria that is widely spread in the environment (3). It is present in about 2% of healthy adults but may be seen in 10-20% of the elderly population (3). In North America and Europe it accounts for approximately 25% of diarrheas associated with antibiotic use (4). Historically C. difficile-associated disease (CDAD) has been associated with the hospital environment, with major risk factor being antibiotic use, but since 2005 CDAD has not only increased in severity it is being seen in otherwise healthy individuals with no hospital contact, thus signaling the emergence of a strain that may be community acquired. This condition is referred to as community acquired CDAD (CA-CDAD).

In 2006, CA-CDAD was added to the list of conditions reportable by Connecticut health-care providers after CDC published a study conducted in CT, in which data were collected on patients who presented with gastrointestinal symptoms with no history of overnight hospitalization (5). According to these data a quarter of the cases had no established risk factors for CDAD including advanced age, hospital contact or antibiotic use, thus indicating other factors.

Although C. difficle infection is a known cause of neonatal diarrhea in swine, with mortality as high as 30%, there are little data on the prevalence rate in animals especially in the North-East. As far as we know only one study has been done looking for C.diff amongst swine, on a Texas farm-an intensive breeding operation with the highest prevalence (50%) amongst suckling piglets (6). Other studies from Europe and Canada have reported C. difficle isolates in pork, veal, and beef (7). Molecular analysis has shown similarities between the human and animal isolates, highlighting the possibility of interspecies transmission. Thus this pilot study looked at the C. difficle colonization rate of CT breeding farms, highlighting the possibility of the farm environment as a potential source of infection for both farmers and pigs.

Relevance to sustainable agriculture

Human community-acquired C. difficle has been gaining attention in recent years, and studies, albeit few, in livestock and animal products have given rise to questions regarding the role of animals in the epidemiology of this pathogen, and its zoonotic potential. As earlier mentioned there has been an increase in human CA-CDAD in CT with no known risk factor, thus it has become necessary to investigate possible sources of infection. In addition C. difficle is an established cause of neonatal diarrhea in piglets, and even agalactiae in breeding sows(8), thus this disease may be a potential threat to not only the farmer and swine health but may also affect the sustainability of the already dwindling swine industry in this region. Furthermore, since C. difficle spores are hardy and are resistant to most environmental conditions, this study will determine the importance of the farm environment as a possible source for both human and animal infection. It will not only help close the existing knowledge gap but also aid in making policy decisions about husbandry practices, such as better use of personnel protective equipment (PPE) amongst swine workers. It will also contribute to a better understanding of the role of C. difficle in animal and human gastroenteritis, thereby informing clinical decision-making, for example about including C. difficle testing in otherwise healthy individuals and pigs. Finally, CDAD is a major cause of enteritis in neonatal pigs in major swine producing regions, and even otherwise healthy pigs are colonized with the bacteria. However, nothing is known about the burden of this disease in small swine producing areas such as CT, where farms are directly linked to community due to increased demand for local animal products and a renewed interest in recreational animal rearing.

Project Objectives:
  1. This study had two objectives:
    1) To determine the prevalence and the characteristics of Clostridium difficle in Connecticut farms.
    2) To conduct knowledge, attitude and belief (KAB) survey regarding bio-safety practices.

Cooperators

Click linked name(s) to expand
  • Dr. Robert Heimer

Research

Materials and methods:

This pilot was incorporated into a longitudinal study investigating methicillin resistant Staphylococcus aureus (MRSA) in CT swine and swine workers. Fifteen farms were sampled over an eight month period from Nov 2010 to July 2011, one farm; a farrow-finish operation was located in neighboring Massachusetts (MA). The CT farms were sampled as an adjunct to the mandatory annual sampling of breeding farms for brucellosis and pseudorabies conducted by the CT Department of Agriculture, with prior approval of farm owners. We selected breeding farms for this sampling study because, apart from the convenience of incorporating the sampling into the state annual testing, available literature had shown that suckling swine along with sows had a higher rate of C.diff colonization than other types of production and may be the source of infection for growers/finishers and subsequently the food chain.

Based on USDA classifications (9) about a quarter (26%) of the farms sampled were either large (50-99 pigs) or medium (25-49 pigs) while the remaining were small (</=25 pigs). All farm owners resided on or within a mile of farm property and about half raised other livestock. All farms sourced swine from within the United States; mainly from Pennsylvania.

Prior to sample collection, information about the animals (age, type, presence of groupings) and management practices such as antimicrobial use and housing were collected from the farm owner/manager. Sampling was based on size of farm, number of pens and availability of freshly voided feces on pen/barn floor. Farms with farrowing herds (sows and piglets) had such herds housed separately and two farms had a separate nursery for weanling pigs. Such herds were over-sampled given higher colonization rates in piglets and sows. Using a sterile pouch, a composite sample consisting of 2-3 fecal pats was collected from the floor of pens, barns and even the bare ground in outdoor raised swine. The pouch was labeled, placed on ice, and transported to laboratory. On the day of sampling, a farm assessment form was used to evaluate bio-safety practices such as availability of hand wash facilities; established visitor policy and isolation of new or sick animals. Collection date, temperature, animal type and number of swine per pen were recorded on a spreadsheet. Farmers on participating farms were monetarily compensated for their contribution.

Laboratory Analysis

In the laboratory, fecal samples were manually mixed for two minutes using sterile applicator stick as described by Norman et al. 2009 (6). Using C.diff Quick Check complete (Wampole™ C.Diff Quik Chek Complete®, New Jersey), samples were screened for the glutamate dehydrogenatase antigen and toxins A and B of C.diff. Testing was done according to manufacturer’s protocol which included negative and positive controls. This test is highly specific at almost 100% (range 91.2-99.7%) thereby an excellent screening test. In addition the first 20 samples were cultured for the presence of C.diff according to CDC protocol with preliminary steps adopted from Rodriquez-Palacios et al 2006 (10). Since both the culturing and the rapid test kit yielded same results for these 20 samples a decision was made to first screen tool samples with the kit; then confirm the presence of toxin B in any toxin positive samples by real time PCR according to Belenger et al 2003 (11) and Peterson et al (12) and lastly culture and isolate C.diff in both antigen and/ or toxin positive samples as outlined in Figure 1 (lab sampling scheme).

Objective 2: Knowledge, Attitude and Belief (KAB) survey regarding bio-safety practices.

As part of the MRSA study, we worked with a swine veterinarian at the University of Connecticut (UCONN) and an occupational health group at Yale with an interest in farmers` health. In consultation with these experts and a statistician a questionnaire was developed looking at bio-safety practices on farm. This questionnaire was piloted on 15 swine workers present on the 7 farms sampled in this study. It was administered in person and took a maximum of 10mins to administer.

Research results and discussion:

As seen in table 1 about half of the farms had less than 50 pigs present on day of sampling, six farms (40%) had young pigs (suckling and weanling pigs) present. About a third of all farms sampled were free range, one of which had pasture-fed swine. None of these farms slaughtered swine on farm and half of them raised other livestock (goats, sheep, poultry and horses). Only three (20%) used antibiotics, one during the winter months when swine were indoors and the other two in drinking water for piglets 8-10 weeks. All farms sampled were negative for either C.diff antigen or its toxins. This is not surprising given the low use of antibiotics on these farms and swine were healthy with no history of neonatal diarrhea in the past one year or a diagnosis of C.diff infection. Also the number of suckling and weanling pigs present was small compared to the other age group; only 6 of the 15 farms sampled had young swine present.

Human characteristics

Fifteen humans agreed to participate in the study. About 60% (10/15) were male and mean age was 45 years old. Nearly half of the respondents (7/15) were farm owners, 40% (6/15) had jobs in non-agricultural sectors (pharmaceutical, education and retail).

Looking at the C.diff risk factors, only one participant had at least 3 episodes of diarrhea within 24 hours in the past 3 months. It was resolved within one day. None consumed antibiotics in the previous 3 months. Three of the respondents had children less than 5 years old, one of these had a child less than a year old.
As for personal protective equipment use (PPE), all farmers had designated farm clothing and wore cloth gloves while handling pigs. About ninety percent (13/14) took work clothes home that were laundered separately. None of them washed their hands after animal related tasks. Also none of them wore any form of face mask, eye goggles, or hair cover (not baseball caps).

Regarding biosafety guidelines, about a third (5/15) were aware of guidelines, two of these were specifically aware of USDA guidelines. Only one worker was concerned about infection from swine. Four (26%) of the respondents had flu shots in 2009-2010, three of these had both seasonal and HIN1 flu shots and two of these got flu shots the following year (2011).

Research conclusions:

Although C.diff was not found, this study demonstrates the feasibility of on-farm screening for emerging zoonoses and the need to conduct separate studies in small swine enterprise since there are obvious husbandry differences from the well known confined animal feeding operations (CAFOs). It also confirms what has been seen in other studies that for healthy animals C.diff colonization/infection is not a problem as compared to other bacteria such as MRSA. Another notable point is that this result was mirrored in a parallel study conducted by the CT dept of public health in raw fresh meat (pork, beef and ground turkeys) from randomly sampled monthly from Sept 2010 to Apr 2011. Although there has been an increase in the incidence rate of human CA-CDAD, a recent report by the Yale/CT Emerging Infections Program (13) showed a link with increased antimicrobial use and underlying medical conditions in such cases.

In addition, this is the first study investigating bio-safety awareness amongst swine workers in the region, thereby providing insights about knowledge gaps and how to tailor existing guidelines to this subpopulation of small farms that have a closer tie to the community with an increasing demand for organic animal products coupled with emphasis on patronizing local farmers. Studies such as these are great compliments to existing surveillance systems that are mostly focused on the CAFOs.

Limitation

Due to the small sample size of both farms and humans, results from this study cannot be generalized beyond Connecticut farms. Although study highlights the need to investigate other possible risk factors for human C.diff infection such as antibiotic use.

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

Two manuscripts based on Objective 2 are in preparation. One entitled “Bio-safety issues in Swine workers” has been submitted for peer-review and the other is currently being drafted. In conjunction with Yale occupational group and UCONN a bio-safety guideline tailored to the needs of small swine enterprise is being formulated with input from farmers. A prototype was sent to participants in this study for their comments on feasibility of such measures.

Project Outcomes

Project outcomes:

No economic analysis was conducted for this study. However; it provides basic information for cost analysis and subsequent cost-benefits of on-farm screening as a compliment to existing surveillance system.

Farmer Adoption

Farm results were sent to farmers with a bio-safety prototype mentioned above suggesting appropriate personal protective equipments, farm bio-safety practices and sources of available guidelines.

Assessment of Project Approach and Areas of Further Study:

Areas needing additional study

This study needs to be expanded beyond Connecticut to enable full insights about C.diff prevalence in the area. It may also be necessary to include screening of other bacteria such as Clostridium perfringes which is the third cause of food-borne infection in the United States. With regards to bio-safety awareness, more workers in the New England region need to be recruited to accurately identify knowledge gaps.

References

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Programs. Nature 2007;82(6):3.
2. Cole D, Todd L, Wing S. Concentrated Swine Feeding Operations and Public Health: A Review of
Occupational and Community Health Effects. Environmental Health Perspectives 2000;108(8):12.
3. Olsen KEP, and Hansen F. Clostridium difficle- A potentially foodborne zoonosis? Significance in
humans, animals and food. NMKL Technical Report No.3 2009.
4. Songer JG and Anderson MA. Clostridium difficle: An important pathogen of food animals.
Anaerobes 2006:12; 1-4.
5. CDC. Surveillance for Community –Associated Clostridium difficle. Connecticut, 2006. MMWR 2008:
57 (13); 340-343.
6. Norman KN, Harvey RB, Scott HM et al. Varied prevalence of Clostridium difficle in an integrated
swine system.
7. Keel K, Braizer KW, Post S, et al. Prevalence of PCR ribotypes among Clostridium difficle isolates
from pigs, calves and other species. J. Clin. Microbiol. 45: 1963-1964.
8. Post KW, Songer JG, Jost BH et al: The emergence of Clostridium difficle as a cause of porcine
neonatal enteritis. In; Proceedings of the 32nd annual meeting of the AASV, Nashville, TN, 2001.
9. USDA. Small enterprise swine: Reference of management practices on small enterprise swine
operations in the United States.
10. Rodriguez-Palacios, HR Staempfli, T Duffield, A Peregrine, LA Trotz-Williams, LG Arroyo, JS
Weese, J Brazier. Clostridium difficile PCR ribotypes in calves, Canada. Emerg Infect Dis 2006;
12:1730-1736.
11. Belanger SD, Boissinot M, Clairoux N et al. Rapid detection of Clostridium difficle in feces by Real-time PCR. J of Clin. Micro 2003; 41(2):730-734.
12. Peterson L R. Detection of toxigenic Clostridium difficile in stool samples by real-time polymerase chain reaction for the diagnosis of C. difficile-associated diarrhea. Clin Infect Dis 2007; 45:1152–60.
13. Lyons C, Fowler H, Meek J et al. Community-associated Clostridium difficle infection in selected towns; Laboratory-based active surveillance Connecticut-2010.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.