Refinement of a colostrum-deprived pig model for infectious disease research

Graphical abstract


Method details
Pig infectious disease researchers can chose to use conventional pigs, colostrum-deprived (CD) pigs [1][2][3], caesarian-derived colostrum deprived (CDCD pigs) or gnotobiotic/germfree pigs, each with their own advantages and disadvantages (Table 1; Fig. 1). Moderate to high mortality at an early age as the direct result of colostrum deprivation requires refinement of procedures [4] in sourcing and rearing pigs for research. The customized model for CD pigs described herein uses elevated pens split into quarters which can house 1-2 pigs per quarter (Fig. 2). This prevents direct contact between piglets and reduces movement of feces and waste between pens minimizing risk of transmission of diseases. The initial set-up of the research facility, on-farm derivation of piglets, preventive medical treatment protocols and a simplified feeding schedule, compared to previous protocols [1,5], are described in here. This refined model has been used at Iowa State University several times over the last few years resulting in survival rates of over 90% of the CD pigs derived for various research trials lasting from a few days to several weeks.

Initial room set-up at the research facility
Cleaning procedure The rooms and equipment are washed and disinfected ahead of time using a degreaser followed by a disinfectant [6]. Rooms, walls and equipment are thoroughly cleaned with 70 C water using a highpressure (180 psi) nozzle and then sprayed with a degreaser (PRL-Grease Free; Pharmacal Research Laboratory Inc, Naugatuck, Connecticut), which is allowed at least 10-15 min contact with surfaces. Rooms and equipment are then fumigated with Virkon S (Dupont, Pharmacal Research Laboratories, Inc, Naugatuck, Connecticut) at the manufacturer's recommended concentration and the room is allowed to completely dry (1-2 days).

Room stocking
After the rooms are dry, it is stocked with personal protective equipment, supplies, and broad spectrum antibiotics, as described in Tables 2 and 3. Animal rooms are set up so that personnel enters through anterooms, where room specific boots, disposable N95 respirators, and disposable coveralls are stored.

Pig housing tubs
In each animal room there is one tub made from heavy-duty, UV-stabilized polyethylene (Fig. 2) which is supported by a heavy-duty stand manufactured with a 22.2 mm solid rod steel, raising the unit 30.5 cm off the floor. The overall dimension of the tub is 91.5 cm (height) Â 135 cm (width) Â 170 cm (length). Below the floor is a 136 L capacity catch pan with a 7.6 cm slide gate for easy draining. Each tub is subdivided into four smaller compartments or pens equipped with clear plastic divisions which allow eye contact between pigs. Newborn pigs without teat access have a tendency to suckle on their littermates which can lead to inflammation and abscesses, often involving the umbilical cord stump. The presence of another pig, even if separated through a plexi-glass, may help to improve the welfare and comfort of the pigs (Fig. 3). For this reason, commonly only a single pig is housed in a compartment. However, it is possible to house up to 2-4 pigs per compartment which requires much closer monitoring of pigs for evidence of injuries and associated skin infections and less ability to accurately monitor milk and feed consumption per pig. Each compartment is equipped with a height adjustable heat lamp, a nipple drinker, a plastic feeder and environmental enrichment such as rubber or plastic dog toys. In addition, in the center of the tub (away from the heat lamp) is a sturdy ceramic cup for milk replacer. Rooms are heated and equipped well in advance of the arrival of the pigs.

Temperature, humidity and photoperiod schedule
If possible, the environmental temperature and ventilation should be regulated by a ventilation system to approximately 30-40% to avoid skin infections. The temperature in the utilized rooms is set to 30 C from day 1 to day 10 and is reduced by 1 C per week thereafter. The heat lamp is turned off on day 28 or sooner based on pig behavior. The photoperiod is set to 12 h light and 12 h dark.

Personal movement
To minimize biosecurity risks, any personnel that enter the animal room is required to shower and wear disinfected boots and clean coveralls to walk from the shower room to the animal room. Boots are removed prior to the entrance into the animal ante-room. Disposable coveralls, N95 respirators, and designated room-specific boots are put on in the ante-room prior to entrance into the animal rooms and disposable gloves are put on after entering the animal room.

Farm selection
Depending on the requirement of the research being conducted, dams are sourced from highhealth status farms that routinely monitor for common vertically transmitted viruses such as porcine reproductive and respiratory syndrome virus, porcine parvovirus and porcine circovirus type 2. Serum or other appropriate sample types from a representative number of sows is collected in advance for screening against the pathogens of interest.

Selection and preparation of pregnant dams
Depending on the number of piglets needed, several pregnant dams close to farrowing (presence of colostrum in mammary glands; behavioral changes), housed individually in farrowing crates on a   commercial sow farm geographically close to the research facility are pre-selected. For the selected dams, the vulva is cleaned with a povidone scrub and a clean cotton towel is placed below the vulva to decrease contamination (Fig. 4A). If the towel is contaminated by fecal material or saturated with fluid, it is replaced. The selected dams are closely monitored in order to avoid contact between the newly born piglets and the floor or dam. Having multiple people is advantageous.

Piglet management during farrowing
As the piglets are coming out of the birth canal, they are picked up using clean gloves without touching the farrowing crate or the floor, dried off with a clean towel, their body is wiped with a povidone scrub, and they are put in a new large plastic tote with clean towels positioned under a heat lamp. Alternatively, rags slightly moistened with 70% ethanol may also be used to clean the pigs. The strongest and healthiest pigs are selected for the project. The tote is placed on a large plastic bag in a

ml intramuscular) At arrival and upon onset of clinical signs
Clostridium perfringens types C and D antitoxin (Colorado Serum Company, 3 ml orally and 3 ml subcutaneously, 25 ml of procaine penicillin are added to a 250 ml bottle) Days 3 and 7 Injectable fortified iron (VetOne1; 100 mg intramuscular) clean area of the barn away from the farrowing area (Fig. 4B). The umbilical cord of any pig bleeding excessively is tied with a clean zip tie. Once the required number of pigs has been collected, the tote with the pigs is transported immediately to the research facility.

Arrival at the research facility
Upon arrival, a zip tie is placed on the umbilical cord, approximately 2.5 cm from the body wall, and the umbilical cord stump is sprayed with iodine. Each pig is treated with antimicrobials as specified in Table 3, receives a commercial colostrum replacer of bovine origin (Tables 2 and 4), and 1-2 pigs are placed in each compartment of the plastic tubs (Fig. 3). Using a bovine colostrum replacer will provide the piglets with antibodies against some common pathogens. On the day of arrival, appropriate sample types such as serum or feces can be collected from the piglets and tested to assure freedom of pathogens of interest prior to initiation of the experiment.

Colostrum
Colostrum replacer is only given once upon arrival of the pigs to the research facility. The colostrum replacer is prepared according to the instructions of the manufacturer using hot water (approximately 60 C) and a clean plastic dish, and the piglets are syringe-fed when the mixture has cooled down to body temperature. The amount of colostrum fed depends on the product and the willingness of the pig to eat. Commercially available spray-dried bovine colostrum products are not sterile. A disposable syringe is filled with the colostrum replacer and the piglet is held carefully with the index finger at the angle of the jaw, forcing the mouth open while the palm of the hand is around the back of the head. The colostrum is squirted into the back of the piglet's mouth at a rate that allows the pig to suckle and swallow the milk with a maximum amount of 25-30 ml colostrum or milk replacer during the first 2-3 days. Alternatively, piglets can be fed with disposable bottles. However, usage of syringes allows a more precise monitoring of the volume ingested by each pig. Alternating the piglets after 5-10 ml can be helpful when getting them started with syringe feeding. As a last resort, if syringe feeding is not possible, a rubber stomach feeding tube (Table 2) is used; however, this involves a higher risk of injury to the piglet and should be avoided if possible. If necessary, the feeding tube is slowly inserted over the back of the tongue, and the pig is observed carefully for a swallowing reflex before continuing with the procedure. If accidental introduction of the tube into the trachea occurs, the pig will start coughing and the tube needs to be immediately withdrawn. Once the tube is in the pig's stomach a syringe is used to pump the food into the pig. A list in which feeding times and amount of food being consumed are tracked is helpful, especially during the first days of life.

Milk replacer
After the first feeding, all subsequent feedings consist of commercially available sterile liquid milk replacer (Table 4) as described [7] providing a portion orally by a disposable syringe (20-40 ml) or a disposable bottle and the remainder in ceramic bowls. Syringe or bottle feeding is only done for the first 2-3 days at which time the pigs readily take milk from the bowl. Any milk leftover between feedings is discarded, and the bowl is washed with water and cleaned with a disinfectant wipe prior to re-fill. Milk disappearance and body condition are monitored closely. Any reduction in milk consumption could indicate that a pig may require additional care such as assisted feeding (syringe or tube feeding) or medication. If all milk is gone upon arrival for the next feeding, the amount of feed needs to be increased as appropriate (typically 10%) and recorded on the feeding sheet. The timing of the feedings is important; however, 1-2 h of variation between feedings is acceptable. Table 4 Feeding schedule. Milk replacer feeding schedule adapted from protocols obtained through the National Animal Disease Center, USDA, Ames, Iowa [7]. Abbreviations used: C = Colostrum (including the amount given); M = Milk replacer (including the amount given); Y = One tablespoon of plain natural yogurt; F = One tablespoon of pre-starter feed.

Feed supplementation after 3 days of age
If piglets are being held longer than 3-5 days, one tablespoon of plain yogurt is added to the milk twice a day. If piglets are being held longer than 7-10 days one tablespoon of pelleted pre-starter feed is added to the milk at each feeding time to create a gruel.

Personnel required
Feeding of the pigs and cleaning of the equipment and pens can be easily done by a single person. A shower and clothing change is required between rooms. During the first two days after the pigs arrive when individual feeding is required every few hours, having additional people is helpful.
Pig maintenance during the trial period

Medication schedule
The pigs receive intramuscular injections of iron at 3 and 7 days of age (Table 3) and they are prophylactically treated with antimicrobials 6 h ahead of viral challenge. Once the pigs have been inoculated, disposable OB sleeves ( Table 2) that are changed between each pig are put on over the disposable coveralls and latex gloves. Clinical signs including inappetence, lethargy, diarrhea, labored respiration, fever, loss of body condition or dehydration can appear quickly and almost always require immediate intervention. In addition to antibiotics, dehydrated animals can be given balanced electrolyte solutions.

Cleaning of the pig housing
The cleaning of the pens during the trials is typically limited to washing the ceramic bowls with hot water followed by cleaning them with disinfectant wipes. On rare occasions, visibly contaminated areas, including feeders, are wiped by hand using a disinfectant towel. The fecal material and urine produced by the pigs is collected underneath the raised tubs and removed at the termination of the trial.

Additional information
Infectious disease research is often hindered by the presence of the pathogen of interest or due to presence of antibodies against that pathogen. Sows have an epitheliochorial placenta [8], which prevents the transfer of maternal antibodies to the offspring through the placenta and piglets are born free of antibodies. Antibody transfer from the dam to the piglet occurs via colostrum to protect pigs at a young age [9]. Colostrum supplies a pig with energy and protein and depending on the exposure and vaccination history of the dam, immunoglobulins to agents other than the pathogen of interest for the research project. This makes CD and CDCD pigs extremely susceptible to develop project-unrelated diseases that often cause high mortality [5] and model refinement is needed.
Sourcing and raising protocols for CD piglets should and can be adapted based on the purpose of the research. Protocols used for rearing of CD pigs as a newborn infant model for studying immune responses [10,11] or neurological development [12] have been recently described. Although several manuscripts report the use of CD pigs for study of infectious pathogens, few detail the rearing conditions [1,5,13,14]. Rearing protocols for CD piglets with limited or no use of antibiotics and more intensive feeding schedules have been previously described [1,5] and would be more appropriate for bacterial challenge studies. Raising and housing CD piglets under the conditions described in this manuscript typically results in high survival rates and these pigs can be used at an early age to study non-vertically transmitted pig viruses.

Conflict of interest
None declared.