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

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. There are several informative videos and tutorials on rabbit breeding that can be observed online²⁹, but after breeding, the male will typically fall over and may vocalize. Once this is observed, the doe can be returned to her cage. A buck may breed 2-3 times a day with no decrease in sperm count³⁰. In our IACUC-approved protocol, bucks are limited to breeding 10-12 does per week and are provided at least two rest days per week, as sources state that a single buck is usually sufficient to service 10-15 does³¹. Our group suggests purchasing proven breeders from the vendor to improve the breeding success rate. As rabbits are induced ovulators and ovulation typically occurs 10-13 hours post-copulation³¹, we have experienced a higher rate of pregnancy in does that are bred in the morning and then again in the afternoon, or twice within that 10-13 hour window (observed increase from 75% to 95% success rate, unpublished). Based on the literature, typical breeding success rates vary from 57-100%^{32,33,34,35,36,37,38,39,40}, and litter sizes average approximately 7-9 kits³¹.

Determining pregnancy as early as possible is helpful in maternal studies to confirm that the doe does not need to be rebred or removed from the study. Options for pregnancy detection include palpation (as early as 14 days)³¹, ultrasound (as early as 5-9 days)⁴⁰, radiographs (as early as day 11)³¹, weight gain, and molecular techniques, such as measurements of insulin-growth factors⁴¹ (IGF) and progesterone^{34,37,38,42,43}. Previous work has indicated significant elevations of IGF-II levels in pregnant rabbits compared to levels in non-pregnant rabbits⁴¹. However, in our hands, we were unable to detect a difference in IGF-II levels between pregnant and non-pregnant rabbits (unpublished). As adequate progesterone levels are necessary for the maintenance of pregnancy in rabbits^37,44, several studies have assessed progesterone levels in pregnant rabbits and demonstrated elevated levels relative to non-pregnant rabbits, particularly during organogenesis around mid-gestation^34,43,44. Our group was unable to detect differences in progesterone levels using an ELISA between pregnant and non-pregnant rabbits, but preliminary results using an automated chemiluminescent assay at the Michigan State University Veterinary Diagnostic Laboratory indicate elevated progesterone levels in pregnant rabbits compared to non-pregnant rabbits even after matings by a buck were confirmed for all does assessed (Figure 2).

Lactating does can produce approximately 250 mL, or 60 mL/kg, of milk daily^45,46, allowing for large volumes for experimental assays to assess total and antigen-specific antibody responses/concentrations. Rabbit milk contains high levels of fat and protein, containing 2 and 3 times more concentrated levels of fat and protein, as compared to cow and sow milk, respectively^45,47. Due to the high fat content in the milk, the samples require significant processing, depending on the assays to be conducted. Following centrifugation of the milk sample, three distinct layers are separated out, including the cells within the bottom layer, clear intermediate layer containing the immunoglobulins, and fat within the top layer⁴⁸. Immunoglobulins, within the clear intermediate layer, are present at high concentrations within the colostrum and milk and are primarily made up of IgA, IgG, and IgM (Figure 4). Though milk samples are easily collected by hand, and this is our preferred technique within the lab, vacuum systems have also been reported in the literature^13,46,48. Yoshiyama et al.⁴⁸ collected milk samples using negative pressure and described a long centrifugation (15,000 x g for 4 hours) for separation of the milk layers prior to passing the clear intermediate layer through a Sepharose 4B column for immunoglobulin removal. Using this method, authors were able to detect cholera toxin-specific antibodies within the rabbit milk of orally immunized rabbits at sufficient levels for protection against Vibrio cholerae-induced secretion in the intestine⁴⁸. Peri et al.¹³ processed milk samples collected by aspiration with a water vacuum system by centrifugation at 4 °C for 2 hours at 24,000 x g. In this study, authors were able to detect anti-RSV IgA in the colostrum, milk, and bronchial and intestinal secretions following mucosal immunization (oral or intratracheal), but not intravenous immunization, whereas anti-RSV IgG was detected in colostrum, milk and serum regardless of immunization route¹³.

Care should be taken during the intranasal immunization procedure to confirm the rabbit is in an adequate plane of anesthesia and to avoid administering large volumes of vaccine at once. Previous work from our group has shown that the efficacy of the intranasal immunization was influenced by the use of anesthesia⁴⁹. Specifically, deep anesthesia induced by the combination of ketamine and xylazine correlated with increased immunogenicity of the nasally administered vaccine and improved nasal retention of the solution. These levels were significantly higher than retention and immunogenicity in rabbits intranasally immunized following sedation with acepromazine and butorphanol⁴⁹. Similar findings have also been demonstrated following intranasal immunization in mice^50,51. Additionally, our work demonstrated increased immunogenicity following intranasal immunization under the deep anesthesia over animals that were fully awake and over animals undergoing shorter duration anesthesia with a combination of acepromazine and isoflurane. This difference in IgG immunogenicity between deep versus shorter duration anesthesia was significant at the 42 day time point but not at the 56 day time point. Although increased immunogenicity and retention may result from the deeper anesthesia, rabbits required at least 30 minutes to recover; whereas, rabbits were upright and mobile within 5 minutes of receiving the nasal vaccine following the shorter duration isoflurane-induced anesthesia. Although anesthesia may not be ideal for mimicking the clinical setting for intranasal immunization in humans, a shorter duration anesthesia (e.g., isoflurane) may be preferred to a longer, deep anesthesia with injectable agents (e.g., ketamine + xylazine). The use of an animal model, as demonstrated in Gwinn et al.⁴⁹, will often require the use of sedation or anesthesia to allow for safe animal handling and effective and consistent vaccine delivery. A potential limitation for anesthesia use with vaccine administration in animal models is its effect on the ability of the animal to induce an appropriate immune response to the vaccine. Interestingly, reports in the literature suggest that isoflurane can attenuate systemic inflammatory responses, providing protection against challenges⁵² and reducing oxidative stress and inflammation⁵³.

With respect to minimization of vaccine loss and adequate intranasal delivery, our group recommends a rest period of 30 seconds after administering each aliquot of the intranasal vaccine to allow for retention of the solution within the nasal cavity and to prevent the solution from seeping out from the nostrils if the rabbit is being immediately placed in sternal recumbency and returned to the cage. Additionally, it is important to limit the volume of the aliquot administered to ensure intranasal immunization, as mucosal absorption is limited by the surface area of the nasal passage. If a large volume is administered, the solution may bypass the nasal mucosa and result in inhalational or gastric immunization. Our group recommends aliquots of 100 µL per nostril with a maximal intranasal volume of 500 µL.

This article focuses on mucosal vaccine delivery, specifically via the intranasal route, but there are multiple methods of immunization, both mucosal and parenteral that have been demonstrated in rabbits. These additional methods include, but are not limited to, oral, subcutaneous, intramuscular, and intradermal. As such, samples to be collected and assessed vary based on the experimental goals and route of immunization.