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 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.</li>
	<li>Wait 10-15 minutes to allow the acepromazine to take effect.</li>
	<li>Anesthetize the rabbit with isoflurane by placing the connected nose cone over the animal&#39;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.</li>
	<li>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.</li>
	<li>Continually monitor reflexes and breathing during anesthesia, and reduce the isoflurane rate to 1-2% once an adequate plane of anesthesia has been reached.</li>
</ol>
2. Intranasal immunization
<ol>
	<li>Prepare immunization solution prior to animal handling.</li>
	<li>Sedate the rabbit as described above.</li>
	<li>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.</li>
	<li>Place the rabbit in dorsal recumbency, and prop the neck and head at an approximate 45&#176; angle that allows easy access and visualization of both nares by the lab member administering the vaccine.</li>
	<li>Load the pipette with no more than 100 &#181;L of the vaccine solution, and quickly administer the solution in each nostril. The pipette should be held at an approximate 45&#176; 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.</li>
	<li>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 &#181;L per nostril at a time. If a larger volume is to be administered, with a maximum total of 500 &#181;L, the vaccine can be given in 100 &#181;L 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.</li>
	<li>Following immunization, place the rabbit on the ventrum for recovery and closely monitor the animal until it can maintain sternal recumbency.</li>
</ol>
3. Milk collection
<ol>
	<li>Sedate the lactating rabbit as described above.</li>
	<li>Clean the skin over the marginal ear vein with the alcohol swab/wipe.</li>
	<li>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.</li>
	<li>Following oxytocin administration, apply pressure to the injection site with the piece of gauze.</li>
	<li>While maintaining the anesthesia mask over the rabbit&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

The authors have nothing to disclose.

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. 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 progesterone34,37,38,42,43. 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 rabbits37,44, several studies have assessed progesterone levels in pregnant rabbits and demonstrated elevated levels relative to non-pregnant rabbits, particularly during organogenesis around mid-gestation34,43,44. 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 daily45,46, 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, respectively45,47. 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 literature13,46,48. 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 &#176;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 mice50,51. 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 &#181;L per nostril with a maximal intranasal volume of 500 &#181;L.
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.

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><tbody><tr><td>&lt;strong&gt;Intranasal Immunization&lt;/strong&gt;</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>&lt;strong&gt;Milk Collection&lt;/strong&gt;</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 2.5% cream). The presence of antigen-specific IgG levels can be measured in these samples. Female rabbits are immunized via the intranasal route, as described in the protocol and demonstrated in the video. Depending on the study, the vaccine may require a boost or may need to be given through an additional route (e.g., intramuscular or subcutaneous). Following study initiation, rabbits are 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, and at approximately 30-32 days post-breeding, pregnant does will kindle. We recommend limiting handling of 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 (Figure 3). In addition to a wide variety of nutrients, kits receive IgA from the lactating doe while nursing. Kits are typically weaned at 4-8 weeks, but prior to weaning, milk can easily be collected from lactating does, as demonstrated in the video. The collected milk samples can then be processed for the detection of total and antigen-specific IgA levels (Figure 4). Depending on the study, vaccines (+/- boosts) 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.
For maternal studies, determining pregnancy as early as possible is helpful for the study design and for ensuring the doe does not need to be rebred. Progesterone measurements can be used as a means to detect pregnancy. As shown in Figure 2, 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. There are additional methods for pregnancy detection, including manual palpation, ultrasound, and radiographs; however, these require well-trained personal and proper equipment.
Antigen-specific IgG that was transferred transplacentally while in utero can be measured in the serum of kits. Blood can be collected from a small number of kits at or near the time of birth to assess early antigen-specific antibody levels, but serial blood collection is technically much easier as the kits age and increase is size. As depicted in Figure 3, serum levels of antigen-specific IgG in the kits can be measured by ELISA and compared to the maternal levels. Maternally transferred IgG levels tend to be higher at birth and decrease over time.
As a type of mucosal sample, milk can be collected and processed to measure total or antigen-specific antibody levels. As shown in Figure 4, IgA makes up a significant portion of the total antibody levels within the breast milk that is being transferred to the kits via lactation. Our results demonstrate that breast milk IgA produces a slightly higher ELISA signal (relative light units, RLU) when compared to IgG, and both IgA and IgG produce a signal that is much higher than the signal for IgM. These results are in agreement with results from others that suggest that rabbit milk contains around 4.5 mg/mL IgA, 2.4 mg/mL IgG, and 0.1 mg/mL IgM26,27.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62317/62317fig01.jpg" /> 
Figure 1. Sample timeline for a maternal intranasal immunization study design in a rabbit (Oryctolagus cuniculus) model. Female rabbits are immunized via the intranasal route, as described in the protocol and demonstrated in the video. Depending on the study, the vaccine may require a boost or may need to be given through an additional route (e.g. intramuscular or subcutaneous). Rabbits are then bred. Antigen-specific IgG is transferred transplacentally to the kits, and at approximately 30-32 days post-breeding, pregnant does will kindle. IgA is passed to the kits from the lactating doe while nursing. Prior to weaning, milk can easily be collected from the lactating does to assess total and antigen-specific IgA levels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62317/62317fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62317/62317fig02.jpg" /> 
Figure 2. Progesterone levels in pregnant and non-pregnant rabbits at 3 weeks post-breeding. Blood was collected from rabbits at 3 weeks post-breeding. Rabbits were confirmed either pregnant or non-pregnant based on ability to kindle a litter at 30-32 days post-breeding. Serum progesterone levels were measured through the Michigan State University Veterinary Diagnostic Laboratory using a chemiluminescent immunoassay (CLIA) with an immunoassay system (e.g., Siemens Healthineers IMMULITE 2000). Error bars represent standard error of the mean, and the sample size consisted of 4-6 rabbits per group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62317/62317fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62317/62317fig03.jpg" /> 
Figure 3. Antigen-specific IgG levels in kits (relative to maternal levels) at birth and at 3 weeks of age following a series of maternal immunizations. Blood was collected from the does and kits soon after kindling and at 3 weeks of age. Antigen-specific IgG levels within the serum were detected using a fluorescent ELISA as previously described28. Antigen-specific IgG is plotted as a ratio of levels detected in the kit serum and maternal serum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62317/62317fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62317/62317fig04.jpg" /> 
Figure 4. A comparison of IgA, IgM, and IgG levels in rabbit milk. Rabbit milk was collected as described and demonstrated in the video. Milk was processed by a long centrifugation (13,000 x g for 4.5 hours at 4 &#176;C), and the clear middle layer was isolated following processing. Total IgA, IgG, and IgM levels were measured in this clear layer by fluorescent ELISA as previously described28, except that plates were coated with polyclonal anti-IgA, anti-IgG, or anti-IgM to detect total rabbit IgA, IgG, or IgM, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62317/62317fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

intranasal-immunization-milk-collection-studies-maternal-immunization

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.5K Views.  Duke University Medical Center. 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. 

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

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

Medicine

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Bioengineering

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Biology

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and minimizing systemic drug exposure. The transcutaneous technique is simple and allows for the IT delivery of substances to the lung of prematurely born rabbits shortly after birth. Newborn pups are anesthetized with inhaled Isoflurane before being placed in a supine position with the neck extended. The larynx is identified and stabilized before transcutaneous placement of a 26-gauge (G) catheter into the trachea. Following catheterization of the trachea, a 30 G blunt needle attached to a Hamilton syringe is introduced into the IT catheter and is used for delivering a precise volume into the trachea during spontaneous respiration. After the IT injection is completed, the needle and catheter are withdrawn, and the pup is allowed to recover from anesthesia. Transcutaneous IT injection delivers a large proportion of the injected substance to the lung, with the majority remaining in the lung 3 hours after the intervention. The injections are well tolerated from the day of birth and can be repeated for multiple consecutive days without influencing survival. This technique can be used to investigate the effect of pharmaceutical agents on lung development and in the prevention of neonatal lung injury in preterm rabbits.

Transcutaneous intratracheal injection allows for effective intrapulmonary drug delivery during spontaneous respiration. Single and multiple injections are well tolerated with no effect on survival. The technique is simple to perform and can examine the effect of substances on lung development and the prevention of lung injury in newborn rabbits.

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and ...

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

Pregnancy and Nursing Management for Embryo-Transferred and Genetically Modified Rabbits

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding management systems for gene-edited rabbits, leading to low survival rates among gene-edited rabbits prepared by many inexperienced researchers. Therefore, proper guidance is essential. This article summarizes the pregnancy and feeding practices for genetically modified rabbits developed in the author's laboratory and outlines a set of fundamental processes. These include pregnancy diagnosis, antenatal care, midwifery, assisted breastfeeding, weaning, and other procedures, along with the rescue and care of weak newborn rabbits. Compared to the traditional natural childbirth and nurturing methods used in rabbit farms, this approach involves more refined management, requiring additional time and effort but significantly increasing the survival rate of suckling rabbits. The methods described in this article are suitable for most laboratory breeding scenarios involving gene-edited or embryo-transferred rabbits and provide a straightforward and effective reference for other researchers.

This protocol describes a pregnancy and feeding management technique for embryo-transferred and genetically modified rabbits, aimed at reducing newborn rabbit mortality and enhancing the preparation efficiency of gene-edited rabbits.

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding ...

Optimized Single&mdash;Person Method for Milk Collection in Lactation Studies of Mice

An Efficient Single-Person Technique for Milk Sampling from Laboratory Mice

Rodent models have been widely used in biology since the early 20th century to study basic physiology and biochemical mechanisms. In lactation and neonatal development research, there is a need to sample milk to determine the composition and how milk may be affected by experimental manipulations, thereby impacting newborn development. Collecting biofluids in sufficient amounts from small rodents can be difficult. Several studies have demonstrated different techniques to obtain milk from mice. However, these techniques often require at least two trained personnel, which may be challenging in some cases.&#160;
Here, we demonstrate a modified milk sampling technique based on a method published in 2009. With this affordable and easy-to-assemble method, one trained person can routinely obtain 0.1&#8211;0.5 mL of milk from a dam under anesthesia in less than 10 min. Using this method, we collected sufficient milk on post-delivery day 2 (PD2) and post-delivery day 10 (PD10). We measured milk macronutrient components and compared them with the existing literature to validate our collection method.&#160;
We recorded that on PD2, milk protein averaged at 70.1 &#177; 5.2 g/L, milk fat was 174.4 &#177; 7.1 g/L, and milk lactose was 12.3 &#177; 0.6 g/L. On PD10, while milk lactose and protein remained at similar concentrations as on PD2, milk fat was significantly higher (224.4 &#177; 17.1 g/L). We also observed that the relative abundance of individual milk proteins varied between PD2 and PD10. More specifically, &#945;S1-casein and whey acidic protein were higher, while &#946;-casein was lower at PD10 compared with PD2. Overall, we demonstrate an efficient single-person technique for milk sampling from mice using a device that can easily be assembled with commonly used laboratory equipment.

An Efficient Single&mdash;Person Technique for Milk Sampling from Laboratory Mice

This article describes how to assemble a device that can be used to collect milk from laboratory mice. This device is affordable, portable, and allows a collection of 0.1&#8211;0.5 mL of milk by one trained person. A detailed milking protocol is also included.&#160;

Rodent models have been widely used in biology since the early 20th century to study basic physiology and biochemical mechanisms. In lactation and ...

Intranasal Immunization with BCG in a Mouse Model

Source: Uranga, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54440">Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates intranasal BCG immunization in mice, activating both innate and adaptive immune responses, leading to the development of lung-resident memory T cells and effector B cells, thus enhancing protection against Mycobacterium infections.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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