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.

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
<ol>
	<li>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 ar...

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&#39;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...

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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Intranasal Immunization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Machine/Vaporizer</td>
			<td>Vet Equip</td>
			<td>901807</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle (25 g)</td>
			<td>Terumo</td>
			<td>07-806-7584</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (250 mL Bottle)</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td>2-4%</td>
		</tr>
		<tr>
			<td>Luer Lock Syringe (1 mL)</td>
			<td>Air-Tite</td>
			<td>07-892-7410</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucosal Vaccine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Experimental Vaccine</td>
		</tr>
		<tr>
			<td>Nose Cone</td>
			<td>McCulloch Medical</td>
			<td>07891-1097</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>VWR</td>
			<td>53503-290</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>VWR</td>
			<td>89079-962</td>
			<td></td>
		</tr>
		<tr>
			<td>PromAce (Acepromazine maleate)</td>
			<td>Boehringer Ingelheim</td>
			<td>07-893-5734</td>
			<td>1mg/kg IM</td>
		</tr>
		<tr>
			<td>Puralube Sterile Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>07-888-2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Milk Collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep 2-ply</td>
			<td>Covidien</td>
			<td>07-839-8871</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Machine/Vaporizer</td>
			<td>Vet Equip</td>
			<td>901807</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needles (25 g)</td>
			<td>Terumo</td>
			<td>07-806-7584</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (250 mL Bottle)</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td>2-4%</td>
		</tr>
		<tr>
			<td>Luer Lock Syringe (1 mL)</td>
			<td>Air-Tite</td>
			<td>07-892-7410</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Woven Sponge (4x4)</td>
			<td>Covidien</td>
			<td>07-891-5815</td>
			<td></td>
		</tr>
		<tr>
			<td>Nose Cone</td>
			<td>McCulloch Medical</td>
			<td>07891-1097</td>
			<td></td>
		</tr>
		<tr>
			<td>PromAce (Acepromazine Maleate)</td>
			<td>Boehringer Ingelheim</td>
			<td>07-893-5734</td>
			<td>1mg/kg IM</td>
		</tr>
		<tr>
			<td>Puralube Sterile Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>07-888-2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Conical Vial (15 mL)</td>
			<td>Falcon</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
	</tbody>
</table>

intranasal immunization and milk collection in studies of maternal immunization in new zealand white rabbits (oryctolagus cuniculus)

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.

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...

Watch this Scientific Journal Video about Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus at JoVE.com

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

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.

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

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...