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                    John Gay, DVM PhD DACVPM               AAHP                  FDIU             VCS

Version 1.1 uploaded 12/4/03 modified 12/19/05

Introduction

Enteric infectious disease is a complex, multifactorial condition with numerous factors including pathogen exposure, strain variation, environmental conditions, management conditions, nutritional state and immune status all interacting to cause loss. Most, if not all, of these factors are related to biosecurity. Many are under management control and most are not specific to a single infectious agent. Biosecurity is not a new concept in animal agriculture but rather is largely a redefinition of earlier ideas and practices historically considered to be good animal husbandry. This is evident when one observes in early veterinary texts the calls for cleanliness, disinfection and isolation of herd replacements and sick animals (3).

Most enteric agents transmit predominately by the fecal-oral route from the feces of infected animals to the mouths of susceptible animals and do so very efficiently. Immediate transmission occurs when infected animals are housed with susceptible animals under conditions that allow nose-to-nose contact or inhalation of aerosols produced by coughing, urinating or defecating. Indirect contact transmission requires that the infectious agent survive in the environment. Most all agents survive well under most environmental conditions, remaining in the environment where they can be transmitted indirectly by contact with contaminated feces, fomites such as equipment or mechanical vectors such as flies. For enteric agents transmitted by indirect contact, key factors include the number of organisms shed in the feces and their survival characteristics in the environment compared with the infectious dose required to initiate infection in susceptible hosts. Information on the environmental survival characteristics of an indirectly transmitted agent is needed to determine how long that agent is likely to remain at an infectious dose once the area is contaminated with it. All of this is critical information for determining how to manage livestock flow through an existing set of facilities and to otherwise minimize disease transmission through management practices. The relationship between infecting agents and the environment is complex, involving factors such as the physical characteristics of the substrate material (e.g., feces, water, milk, manure slurry, dust), temperature, pH, water activity, and competing microorganisms. As a consequence, these relationships are not well defined for many combinations encountered in the farm environment.

With the rare exception, it is likely that most infectious enteric agents of cattle co-evolved with their bovine hosts long before their domestication thousands of years ago (25, 26). Therefore, if an agent was able to survive under the free-range conditions of the wild bovine, it is likely that transmission will occur relatively easily in the environment of the intensively managed domesticated bovine of today. Indeed, these agents are shed by infected animals in numbers several logs higher per gram of feces than the total number required to infect the typical susceptible animal. Additionally, these agents have been shown to be extraordinarily flexible with regard to their genetic make-up and through survival of the fittest can rapidly take advantage of new environments and management systems. Consequently, intervention strategies devoted to single control point may be successful in the short run but are likely to prove unsuccessful over the long run.

Appropriate cleaning and disinfection procedures are critical to breaking fecal-oral transmission cycles of enteric agents that contaminate housing, feeding or treatment equipment or other vectors and fomites. However, cleaning and disinfection procedures are not without pitfalls and adherence to a sound protocol covering all of the infectious agents of concern is critical for long term success. Procedures that do not impact a resistant agent, such as Cryptosporidia oocysts or rotavirus, may spread it from areas of high concentration across previously uncontaminated surfaces where it can then contaminate materials such as water and feed at sufficient levels to provide an infectious dose to the more susceptible animals.

The most important first step is thorough cleaning to remove organic material (e.g., feces, milk film) prior to applying disinfectant (22). Vigorous cleaning (scraping, scrubbing, flushing) cannot be replaced by applying disinfectants in larger quantities or under higher pressure. For any protocol or in nature, destruction of microorganisms initially follows a first order logarithmic decay process and then slows (35). In relation to the amount of time required to destroy one-half of the initial population, approximately three time periods are required for a one log (90%) reduction, six for a two log (99%) reduction, nine for a three log (99.9%) reduction, and so on. In addition to contact time, the concentration, temperature, pH, water content, water hardness, and amount of organic material present are critical variables determining the success of chemical disinfection. The relationships between these factors are not straightforward (27). For example, halving the concentration of formaldehyde requires a 2-fold increase in contact time to obtain similar microbial destruction, whereas halving the concentration of phenolics requires a 64-fold increase in contact time. A 10^oC rise in temperature increases the activity of alcohols 30-fold, yet only increases the activity of formaldehyde 1.5-fold. Iodophors are highly active at low pH but are inactive at an alkaline pH. In general, live steam effectively applied inactivates the broadest range of microorganisms. Antec Virkon S is an example of a widely used, broad spectrum disinfectant.

Sodium hypochlorite (bleach, NaOCl) at a sufficient concentration, contact time and temperature combination is effective against the bacterial and viral agents of neonatal enteric disease (39), but at practical levels not Cryptosporidium oocysts. It is readily available as 5.25% (household bleach) and 12.75% solutions, is cost effective and is environmentally safe. However, as it begins dissipating upon dilution, the Centers for Disease Control recommends that diluted solutions should be used within 24 hours and that they be stored in opaque containers. It is rapidly inactivated by the presence of any appreciable organic material. For example 1% albumin reduces its effectiveness by 6 logs and increasing concentration or contact time does not recover this loss. Bacteria in biofilms are 150 to 3000 times more resistant. In solution, hypochlorus acid is the active form of the free chlorine. It is most available at a pH 6, dropping to 80% of the free chlorine at pH 7 and to 25% at pH 8, suggesting that the pH of disinfectant solutions should be monitored regularly as part of disinfection protocols. Below pH 6 it is more corrosive to metals and more chlorine gas is released. Testing kits can be used to monitor free chlorine as part of disinfection protocols. However, as these kits measure both hypochlorus acid and hypochlorite ion (non-active form), pH must also be considered. Recommended concentrations for use in human environments range from 500 ppm (1:100 dilution of 5.25% household bleach) and 10 minute contact time at room temperature to 5000 ppm (1:10 dilution of 5.25% bleach) and 1 minute contact time at room temperature, the higher concentrations being used in more critical areas. For viruses in veterinary hospitals and kennels, a recommended dilution of household bleach is 1:32, which results in a 0.175% sodium hypochlorite solution, and a 10 minute contact time at room temperature (42) at pH 6 to 7.

See Chlorox Company's "Bleach Solutions for Surfaces" for further information on using bleach.

The characteristics of environmental surfaces targeted for disinfection in the farm environment also influences the success or failure of various procedures (31). For example, unfinished plywood retains 15-fold more microorganisms than varnished plywood, which supports 15-fold more microorganisms than plastic surfaces. On smooth, ideal surfaces physical removal of visible contamination by thorough washing with soap and water removes 99% of the microbial load (2 logs). However, on typical housing surfaces washing only removes 90% (1 log). Proper disinfection removes an additional 6 to 7% and terminal fumigation will remove 1 to 2%. Disinfection after washing is an important step, particularly if the surface remains damp because remaining bacteria can proliferate in the minimal nutrients leaching from wet wood and because washing can disperse an infectious agent from limited areas of high concentration broadly across other surfaces. The application of high pressure sprays can aerosolize organisms allowing dissemination to distant sites and posing a risk to operators if zoonotic organisms are present.

Sufficient ventilation is important to the health of housed animals (4, 46). Besides being critical for removal of transpired water vapor and reducing humidity, sufficient ventilation in enclosed housing also removes infectious aerosols. Reducing humidity can also reduce the survival time of aerosolized and surface-borne infectious agents. Although more important for respiratory disease, these factors in turn have an effect on the risk of enteric disease. As many infectious agents can be transmitted by aerosols, procedures that produce aerosols (pressure washing, housing flush systems) considerably increase the risk of transmission (6). For example, Mohammed et al. found that dairy calves raised outside or in mechanically ventilated buildings were five-fold less likely to shed Cryptosporidia parvum oocysts than those raised in non-ventilated barns (30).

As personal hygiene is crucial to stopping the transmission of these agents in the human hospital environment, it is also likely a critical component in the intense livestock production environment as well. This personal hygiene includes frequent, effective hand washing of sufficient duration with soap followed by an alcohol-based hand disinfectant, (24) cleaning and disinfecting boots and washing work clothes with bleach followed by hot air drying. Handwashing should be include vigorous rubbing for at least 15 seconds. The newer alcohol-based hand rubs contain emollients that prevent hand cracking, provide a greater amount of bacterial reduction for longer time than antibacterial soaps. If an adequate amount (several mL) of an alcohol-based hand rub is used, the gel will not be dry before at least 15 seconds of hand rubbing has occurred.

Hand Hygiene in Healthcare Settings (CDC webpage)

Guideline for Hand Hygiene in Health-Care Settings (MMWR (10/25/02) 51:RR-16) (pdf)

Infectious Agent Examples

Salmonella spp. are very hardy organisms that are very well adapted to surviving in the environment (16). They are able to proliferate rapidly at high ambient temperatures in waste milk, colostrum and moist feeds. In the absence of direct sunlight or predation by other microorganisms, Salmonella spp. can survive in wet or dry substrates or on surfaces for years, particularly if they are protected by biological films such as dried saliva, milk or fat. Biological films also protect organisms from the action of chemical disinfectants. In an experiment that simulated a barn floor under defecating cows, Salmonella spp. were shown to survive for 5 ½ years (34). These researchers also found S. Typhimurium in an empty slurry pit that had not been used for 4 years.

Because of significant health risks, indirect as well as direct contact between susceptible individuals and livestock potentially infected with these agents should be minimized. Hands should be washed well, using soap and warm water and scrubbing for 15 seconds followed by an alcohol-based antiseptic hand rub, (24) before eating or returning to the household. Inhalation of potentially contaminated dusts or aerosols, particularly those generated by cleaning procedures such as high pressure washing, should be minimized. To reduce the likelihood of introducing these agents into the household and their transmission to susceptible humans or domestic pets, equipment, outer garments and footwear exposed to potentially infected animals and their discharges should not be brought into the household.

As a major bacterial component of feces from warm-blooded animals, E. coli are ubiquitous in the environment. Although not as hardy as Salmonella spp., E. coli survive well on typical farm environmental surfaces and in feces and dust protected from moisture and direct sunlight (5, 28). In experimentally inoculated cow manure or fresh slurry under common farm environmental conditions both organisms decrease by one log in one to three weeks (20). Depending on the surface characteristics, the numbers of organisms decline at about 0.25 log per day. Generally the rate of decline is slower at lower humidity but proliferation can occur on surfaces under saturated conditions with minimal organic nutrients (0.5 mg/L). Ultraviolet in direct sunlight rapidly kills the organism (13).

Rotavirus is a double-stranded, non-enveloped RNA virus. Being non-enveloped, the virus is relatively stable in the environment, being infectious in feces for up to 6 months at 25^oC. In smears of human feces, human rotavirus was more stable at lower temperatures and at humidity extremes (29). Infectious particles declined by 1 log in 29 days at 4^oC and 93% relative humidity, in 16 days at 4^oC and 13% relative humidity, in 2.2 days at 20^oC and 55% relative humidity and in 1.5 days at 37^oC and 13% relative humidity. Some research suggests that bovine rotavirus may be more resistant than human rotavirus. Virus stability in water varies with water quality and temperature, ranging from being very stable in clean water at 4^oC to falling 2 logs in 10 days in typical river water at 20^oC (84). As temperatures above 60^oC are lethal to the virus (33), standard milk pasteurization procedures are effective against it. Rotavirus is susceptible to sufficient concentrations of sodium hypochlorite (1750 ppm) but is relatively resistant to many common disinfectants, such as chlorhexidine, under the same exposure conditions. Because as a non-enveloped virus it is not affected by soaps, washing with soap alone may actually spread the virus around on the washed surface (12).

Coronavirus is an enveloped single-stranded RNA virus and is not as stable in the environment as rotavirus. Because of their envelope, these viruses retain infectiousness better at lower rather than higher relative humidity (13) and are considerably more sensitive to soaps and common disinfectants than are non-enveloped viruses. This virus is more active in the colder climates (9).

Unlike most other enteric protozoa, Cryptosporidium are immediately infectious when passed and can infect other susceptible hosts through direct contact. Because Cryptosporidium can auto-infect the original host, the infectious dose can be exceedingly small. For example, the median infectious dose for humans is only 87 oocysts (15). In the environment, cryptosporidia are extremely resistant to most veterinary disinfectants except 5% ammonia, 6% hydrogen peroxide or 10% formalin (7, 38, 47). They survive very well in water, requiring 4 to 11 weeks to decline by one log (8). As the oocysts adhere in large numbers to the plastic and rubber surfaces of common calf feeding and treatment equipment such as nipples, bottles and buckets (E.R. Atwill, personal communication), common sanitation procedures likely don't prevent fomite transmission by these items. A portion of the oocysts still retain their infectivity after mild freezing (14). On the other hand, complete drying in thin, naturally infected fecal smears on wood kills the oocysts within one to 4 days (2). Finally, as moist heating at 45^oC for 20 minutes kills the oocysts (1), standard pasteurization procedures (e.g., 63^oC for 30 minutes, 72^oC for 15 seconds) are effective.

One of the most overlooked vectors that presents a significant disease transmission risk are the nuisance flies, particularly the house fly, Musca domestica (18). During summer months prior to severe frosts, fly populations typically increase to very high numbers around concentrated livestock operations such as dairies and calf raising operations. Liquids such as diarrhea and milk or materials containing soluble components such as dried molasses and solid feces are very attractive to nuisance flies. Because the larvae require >90% humidity to develop, dampened organic calf bedding materials such as straw and sawdust provide are an ideal substrate (40, 41). The ability of these insects to transmit enteric pathogens from feces is well documented (10, 23). Specific physical characteristics of flies including mouth parts, body hairs and spines, and sticky foot pads can carry infectious agents in large numbers. Some pathogens pass through the fly digestive tract and remain viable in their feces. When feeding, the fly frequently moistens surfaces by regurgitating a "vomit drop" from their crop that contains residue, including infectious agents, from their previous meal. "Fly spots" are either such vomit drops or feces, both of which can contain high numbers of infectious agents. Studies have determined that flies are attracted to diarrheic feces, that they can transmit Cryptosporidia in numbers above the minimal infectious dose for healthy humans, and that they can harbor this agent for 3 weeks after exposure (17). Methods for controlling fly populations at different points in their life cycle have been reviewed (44). However, it is important to point out that control methods based on chemical means alone are usually inadequate because flies readily develop resistance to such chemicals.

Rodents are also a frequently overlooked source of enteric pathogens in the farm environment. They have been implicated in the transmission of salmonellosis in dairy (43) and beef herds (21) and in poultry flocks (11, 19). As the feces from infected mice typically contain up to 1 x 10⁴ salmonella per pellet (21), a single pellet may exceed the infectious dose for a susceptible animal. Current work suggests that rodents are a significant non-livestock reservoir of Cryptosporidium as approximately one-third of rodents of any age, even in non-livestock ecosystems, shed C. parvum at an average of 1 x 10³ oocysts per fecal pellet (36, 45). Importantly, significant rodent populations can be present long before their signs (e.g., rodent droppings and runways) are obvious or noticeable. Raccoons have also been reported to harbor S. Typhimurium (32).

Selection and Use of Disinfectants (Nebraska NebGuide G00-1410-A, 2000)

Cleaning and Sanitizing Fresh Produce and Fresh Produce Handling Equipment, Utensils and Sales Areas (D Schlimme, Maryland Cooperative Extension Fact Sheet 715, pdf)

Chlorine - Lenntech Disinfectants

Evaluation of the efficacy of a peroxygen compound, Virkon®S, as a boot bath disinfectant (pdf)

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