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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

Abstract

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Introduction

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 diseases in newborns and infants. Maternal immunization has the potential to positively impact both maternal and infant/child health at the global level by reducing morbidity and mortality associated with certain pathogens during this vulnerable period1. The main goal of this strategy is to increase the levels of specific maternal antibodies throughout pregnancy. These antibodies can then be transferred to the newborn and infant at levels sufficient enough to protect against infections until their immune system is mature enough to adequately respond to challenges1,2,3. Previous work has demonstrated that higher antibody titers at birth are associated with either complete protection or a delayed onset and reduced severity of numerous different infectious diseases in the newborn, including tetanus, pertussis, respiratory syncytial virus (RSV), influenza, and group B streptococcal infections1,2,3.

In humans, maternal antibodies are transferred passively across the placenta and are also transferred through the breast milk via nursing. Previous work has demonstrated that HIV-specific IgA levels in human breast milk from mothers infected with the virus were associated with reduced postnatal transmission of the virus, suggesting a protective role for breast milk anti-HIV IgA4. Studies in nonhuman primates have demonstrated that immunization against HIV can induce a significant antibody response in the breast milk, and although similar serum IgG responses were induced following systemic versus mucosal immunization, mucosal immunization induced a significantly higher IgA response within the milk5,6.

Identifying a translationally appropriate animal model for these studies should take into account the placentation type and mechanisms of passive antibody transfer, as well as the transfer of antibodies through breast milk. There are three main types of placentation in mammals based on the tissue types and layers at the materno-fetal interface, including hemochorial (primates, rodents and rabbits), endotheliochorial (carnivores), and epitheliochorial (horses, pigs, and ruminants). The hemochorial placenta is the most invasive type, allowing for direct communication between the maternal blood supply and the chorion, or the outermost fetal membrane. Based on the number of trophoblast layers, there are several variations of hemochorial placentation, including the hemomonochorial placenta found in primates, the hemodichorial placenta in rabbits, and the hemotrichorial placenta observed in rats and mice7. This direct contact between maternal blood supply and chorion allows for the passive transfer of antibodies across the placenta during gestation. IgG is the only antibody class that significantly crosses the human placenta8, whereas IgA is the predominant class of Ig found in human breast milk9. Of the scientifically relevant models, only primates (including humans), rabbits, and guinea pigs transfer IgG in utero and IgA in the milk10,11. Therefore, the rabbit model incorporates factors comparable to those in humans that control transplacental transfer of IgG and lactational transfer of IgA.

In addition to serving as an exceptional model for maternal immunity and vaccine development, similarities between the rabbit and human nasal cavities make them an appropriate model for intranasal immunization. The volume of the rabbit nasal cavity is more similar to humans than rodent models based on relative body mass12. Additionally, Casteleyn et al. 12 demonstrated that the nasal associated lymphoid tissue (NALT) is more voluminous in the rabbit compared to rodents. The NALT is located primarily at the ventral and ventromedial aspect of the ventral nasal meatus and at the lateral and dorsolateral aspect of the nasopharyngeal meatus in rabbits, whereas in rodents, the lymphoid tissue is only present along the ventral aspect of the nasopharyngeal meatus12. In rabbits, the structure and location of the intraepithelial and lamina propria lymphocytes, as well as the isolated lymphoid follicles, are similar to humans12.

Additional advantages of using the rabbit as a model for maternal and mucosal immunity include their high fecundity and relatively short gestation period. Large auricular blood vessels allow for relatively easy access to large volumes of blood for serial collections. A variety of mucosal samples can be collected for antigen-specific antibody response assays, including breast milk13 (when lactating), mucosal secretions or washes (e.g., oral14,15,16, bronchoalveolar lavage13,17,18,19, vaginal20,21,22), and feces20,23,24,25. Milk samples can be easily collected during lactation to assess the presence of antigen-specific antibody responses. Though not as abundant as for humans and mice, a wide variety of experimental reagents are available for rabbit-specific studies and assays. In this article, we will describe and demonstrate intranasal immunization and milk collection in New Zealand White rabbits (Oryctolagus cuniculus).

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Protocol

All procedures were approved and performed in accordance with the Duke University IACUC policies.

NOTE: Materials needed are provided in the Table of Materials.

1. Rabbit Sedation and Anesthesia

  1. Sedate the female rabbit (sexually mature; approximately 5-30 months old) by administering acepromazine intramuscularly (IM) at a dose of 1 mg/kg. Depending on the size of the animal, use a 1 or 3 mL syringe with a 25 G needle. Epaxial muscles are the preferred site of the intramuscular injection.
    NOTE: Acepromazine can also be administered subcutaneously, but IM is preferred by the lab, as it acts more rapidly and reduces the incidence of skin lesions.
  2. Wait 10-15 minutes to allow the acepromazine to take effect.
  3. Anesthetize the rabbit with isoflurane by placing the connected nose cone over the animal's nose. Adjust the vaporizer to up to 4% isoflurane combined with up to 4 liters/minute oxygen. Rabbits have a high aversion to isoflurane, so adequate restraint is necessary when masking the animal.
  4. Once fully anesthetized, as assessed by the pinna, pedal, and/or palpebral reflex, apply ophthalmic lubricant to each eye to prevent drying of the eyes and subsequent corneal ulceration.
  5. Continually monitor reflexes and breathing during anesthesia, and reduce the isoflurane rate to 1-2% once an adequate plane of anesthesia has been reached.

2. Intranasal immunization

  1. Prepare immunization solution prior to animal handling.
  2. Sedate the rabbit as described above.
  3. 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.
  4. Place the rabbit in dorsal recumbency, and prop the neck and head at an approximate 45° angle that allows easy access and visualization of both nares by the lab member administering the vaccine.
  5. Load the pipette with no more than 100 µL of the vaccine solution, and quickly administer the solution in each nostril. The pipette should be held at an approximate 45° angle, angled towards the medial aspect of the nasal passage.
    NOTE: The goal of immunization is for the solution to contact the mucosal membrane 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 nare.
  6. Following administration in both nasal passages, maintain the rabbit in dorsal recumbency for 30 seconds to minimize leakage of the vaccine solution.
    NOTE: The lab will typically administer no more than 100 µL per nostril at a time. If a larger volume is to be administered, with a maximum total of 500 µL, the vaccine can be given in 100 µL 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.
  7. Following immunization, place the rabbit on the ventrum for recovery and closely monitor the animal until it can maintain sternal recumbency.

3. Milk collection

  1. Sedate the lactating rabbit as described above.
  2. Clean the skin over the marginal ear vein with the alcohol swab/wipe.
  3. Using a 1 mL syringe and 25 g needle, administer approximately 1-2 IU of oxytocin intravenously via the marginal ear vein to induce milk letdown.
    NOTE: Due to the smooth muscle relaxation, it is common for the rabbit to urinate or defecate following administration of oxytocin.
  4. Following oxytocin administration, apply pressure to the injection site with the piece of gauze.
  5. While maintaining the anesthesia mask over the rabbit's nose, prop the rabbit on its hindquarters.
    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 its rump with an assistant holding the rabbit upright with the anesthesia mask.
  6. Open the sterile tube to prepare for milk collection and locate the mammary tissue and associated teats. The teats are typically surrounded by wet fur from recent nursing, and the mammary tissue is easily palpable when full of milk.
  7. Grasp the mammary tissue associated with a 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.
    NOTE: 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 the additional mammary glands. Milk from all teats can be collected in the same vial. Typically, several milliliters of milk can be easily collected from a lactating doe.
  8. Following milk collection, turn the isoflurane and oxygen off, and allow the rabbit to recover while being closely monitored until the animal is able to maintain sternal recumbency.

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Results

An overview of a typical maternal intranasal immunization study design is depicted in Figure 1, incorporating the immunizations, breeding, kindling, lactation, and antibody transfer. Though not illustrated, 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 via the central ear artery with mild sedation and a topical analgesic agent (e.g., lidocaine 2.5% and prilocaine 2...

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Discussion

Although not described in the above protocol, successful breeding of the rabbits is necessary for this maternal model and to allow for milk collection. Rabbits are easily bred by live cover in a research setting. It is recommended that does be transferred to the buck's cage for breeding, as does can be territorial and aggressive if kept in their own cage with the buck. If females are non-receptive after 15 minutes (as indicated by running away biting, or vocalizing), the doe should be placed back into her own cage. T...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to acknowledge the Division of Laboratory Animal Resources at Duke University and their husbandry team for their assistance and great care provided to the animals. Additionally, the authors would like to recognize the PhotoPath team within the Department of Pathology for their assistance with the audio and video portions of the manuscript. This work was supported by discretionary research funds from the Staats laboratory.

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Materials

NameCompanyCatalog NumberComments
Intranasal Immunization
Anesthesia Machine/VaporizerVet Equip901807
Hypodermic Needle (25 g)Terumo07-806-7584
Isoflurane (250 mL Bottle)Patterson Veterinary07-893-13892-4%
Luer Lock Syringe (1 mL)Air-Tite07-892-7410
Mucosal VaccineN/AN/AExperimental Vaccine
Nose ConeMcCulloch Medical07891-1097
Pipette TipsVWR53503-290
PipettorVWR89079-962
PromAce (Acepromazine maleate)Boehringer Ingelheim07-893-57341mg/kg IM
Puralube Sterile Ophthalmic OintmentDechra07-888-2572
Milk Collection
Alcohol Prep 2-plyCovidien07-839-8871
Anesthesia Machine/VaporizerVet Equip901807
Hypodermic Needles (25 g)Terumo07-806-7584
Isoflurane (250 mL Bottle)Patterson Veterinary07-893-13892-4%
Luer Lock Syringe (1 mL)Air-Tite07-892-7410
Non-Woven Sponge (4x4)Covidien07-891-5815
Nose ConeMcCulloch Medical07891-1097
PromAce (Acepromazine Maleate)Boehringer Ingelheim07-893-57341mg/kg IM
Puralube Sterile Ophthalmic OintmentDechra07-888-2572
Sterile Conical Vial (15 mL)Falcon14-959-49B

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