Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Studies of maternal immunization and antibody transfer are invaluable for numerous reasons as this is the initial route of immunity transfer and subsequent protection from pathogens and disease in newborns and infants. Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. In this video article we demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White Rabbits, including intranasal immunization and milk collection.

We will first describe an example timeline and series of events for a mucosal maternal immunization study in New Zealand White Rabbits. Female rabbits are immunized via the intranasal route, as demonstrated in this video. Depending on the study, the vaccine may require a boost or may need to be given through an additional route, such as intramuscular immunization.

Though not depicted, blood should be collected prior to the initial immunization for baseline measurements, and throughout the remainder of the study at regular intervals. Blood is easily obtained by the central ear artery with mild sedation and a topical analgesic agent. The presence of antigen-specific IgG levels can be measured in these samples.

Rabbits are then bred. We prefer to purchase proven breeders from vendors to use to ensure a higher pregnancy rate for these studies. Depending on the immunization timeline, rabbits may receive additional immunizations throughout pregnancy.

Antigen-specific IgG is transferred transplacentally to the kits. At approximately 30 to 32 days post-breeding pregnant does will kindle. We recommend limiting handling of the kits for the first several days to minimize rejection from the does.

Blood samples can be collected from the kits to assess antigen specific IgG levels that were transferred transplacentally. Blood can be collected from a small number of kits at or near the time of birth to assess early antigen specific antibody levels. In addition to a wide variety of nutrients, kits receive antigen specific IgA from the lactating doe while nursing.

Kits are typically weaned at four to eight weeks of age. Prior to weaning milk can easily be collected from lactating does as demonstrated in this video. The collected milk samples are then processed for the detection of antigen specific IgA.

Depending on the study, vaccines and a boost can be administered to the kits and serial blood samples can be collected from the kits at a very early age using the lateral saphenous vein. We will now demonstrate intranasal immunization and milk collection in New Zealand White Rabbits.Protocol. All procedures were approved and performed in accordance with the Duke University Institutional Animal Care And Use Committee policies.

Intranasal Immunization. Materials Needed:Acepromazine, syringe and needle, ophthalmic lubricant, Isoflurane, nose cone, pipette, and pipette tips, mucosal vaccine. Prepare the immunization solution prior to animal handling.

Rabbits are sedated with Acepromazine one milligram per kilogram intramuscularly, and anesthetized with Isoflurane using a nose cone. Once fully anesthetized as assessed by eyelid reflex, an ophthalmic lubricant is added to each eye to prevent drying of the eyes and subsequent corneal ulceration. Once the lab member is ready to administer the vaccine and the rabbit is in an adequate plane of anesthesia, turn off the isoflurane and oxygen and remove the nose cone.

Place the rabbit in dorsal recumbency and prop the neck and head at an approximate 45 degree angle that allows easy access and visualization of both alars by the lab member administering the vaccine. Load the pipette with no more than 100 microliters of the vaccine solution and quickly administer the solution to one nostril and repeat the procedure for the second nostril. The pipette should be held in an approximate 45 degree angle.

Angle towards the medial aspect of the nasal passage. The goal of immunization is for the solution to contact the mucosal membranes of the nares. So the tip should not be placed within the nares as this may result in abrasion or irritation of the mucosal tissues and potentially influence the immunogenicity of the nasally administered vaccine.

The vaccine should be administered quickly and done in the same manner in the other nostril. Following administration in both nasal passages, the rabbit is maintained in dorsal recumbency for 30 seconds to minimize leakage of the vaccine solution. The lab will typically administer no more than 100 microliters per nostril at a time.

If a larger volume is to be administered with a maximum total volume of 500 microliters, the vaccine can be given in 100 microliter aliquots with a 30 second rest period between immunizations. And additional administrations of vaccine repeated with 30 seconds of rest between each administration until the total vaccine volume is delivered. Following immunization, place the rabbit on the ventrum for recovery and closely monitor the animal until it is able to maintain sternal recumbency.

In some instances, the vaccine formulation may not be completely delivered to the nasal cavity and a portion of the vaccine formulation may remain on the exterior portion of the nares. In this instance, because it is not clear of the exact volume of the vaccine that was properly delivered to the nasal cavity, do not recommend reapplication of additional vaccine material as this would add an undefined variable to the experiment. It is important to use a vaccine delivery technique that provides complete and consistent delivery of the vaccine formulation to the nares.

Milk collection. Materials needed:Acepromazine, syringes and needles, alcohol swab, gauze, ophthalmic lubricant, Isoflurane, nose cone, oxytocin, and a collection vial. Rabbits are sedated with Acepromazine one milligram per kilogram intramuscularly, and anesthetized with Isoflurane using a nose cone.

Once fully anesthetized as assessed by eyelid reflex, an ophthalmic lubricant is applied to each eye to prevent drying of the eyes and subsequent corneal ulceration. Clean the skin over the marginal ear vein with the alcohol swab. Using a one mil syringe and 25 gauge needle, administer approximately one to two international units of oxytocin intravenously by the marginal ear vein to induce milk let down.

Due to the smooth muscle relaxation, it is common for the rabbit to urinate or defecate following administration of oxytocin. Following oxytocin administration, apply pressure to the injection site with a piece of gauze to provide hemostasis. While maintaining the anesthesia mask or nose cone over the rabbit's nose, prop the rabbit up on its hind quarters.

As a side note, milk collection can also be performed with the animal in lateral recumbency, but the lab finds that collection is easier when the rabbit is propped up on it's rump with an assistant holding the rabbit upright with the anesthesia mask. Open the sterile tube to prepare for milk collection and locate the mammary tissue and associated teats. Rabbits typically have four to five pairs of mammary glands.

The teats are typically surrounded by wet fur from recent nursing and the mammary tissue is easily palpable when full of milk. Grasp at the mammary tissue associated with the teat between the thumb and forefinger and apply a gentle massaging pressure to the glandular tissue in the direction of the teat. Place the collection tube over the teat to collect the expressed milk.

It can sometimes take several minutes for the oxytocin to be effective, and milk production appears to vary among mammary glands. If milk expression is not successful, wait several minutes or rotate around to an additional mammary gland. Milk from all teats can be collected in the same vile.

Typically several milliliters of milk can be easily collected from a lactating doe. After the desired volume of milk has been collected, turn the Isoflurane and oxygen off and remove the nose cone from the rabbit, and allow the rabbit to recover while being closely monitored until the animal is able to maintain sternal recumbency. Representative Results.

Figure one. For maternal studies, determining pregnancy as early as possible is helpful to confirm that the doe does not need to be re-bred. Options for pregnancy detection include palpation, ultrasound, radiographs, weight gain, and molecular techniques such as measurements of insulin growth factors and progesterone.

Adequate progesterone levels are necessary for the maintenance of pregnancy in rabbits. Therefore several studies have also assessed progesterone levels in pregnant rabbits and demonstrated elevated levels relative to non-pregnant rabbits, particularly during organogenesis around mid gestation. As shown in figure one, elevated progesterone levels can be detected in pregnant rabbits compared to non-pregnant rabbits even after matings by a buck were confirmed for all does.

Serum progesterone levels were measured using an automated chemiluminescent assay. Figure Two. Antigen specific IgG that was transferred transplacentally during gestation can be measured in the serum of the kits by Elisa.

Blood can be collected from a small number of kits at or near the time of birth to assess early antigen specific antibody levels. Serial blood collection is technically much easier as the kits age and increase in size. As depicted in Figure Two, serum levels of antigen specific IgG in the kits can be measured and compared to the maternal levels.

Maternally transferred IgG levels are higher at birth and decrease over time. Figure Three. Rabbit milk can be collected as shown in this video, and processed to measure total and antigen specific antibody levels by Elisa.

As shown in this figure, IgA makes up a significant portion of the total antibodies within the breast milk that are available for passive transfer to the kits. Both IgA and IgG levels are considerably higher than IgM.Summary. This video article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

Depicted is a summary image for a typical maternal intranasal immunization study design incorporating the immunizations, breeding, kindling, lactation, and antibody transfer.