lable at ScienceDirect

Developmental and Comparative Immunology 53 (2015) 105e111

Contents lists avai

Developmental and Comparative Immunology

journal homepage: www.elsevier .com/locate/dci

Nasal vaccination of young rainbow trout (Oncorhynchus mykiss)against infectious hematopoietic necrosis and enteric red mouthdisease

I. Salinas a, *, S.E. LaPatra c, E.B. Erhardt b

a Center for Evolutionary and Theoretical Immunology, Department of Biology, University of New Mexico, Albuquerque, NM, USAb Mathematics and Statistics, University of New Mexico, Albuquerque, NM, USAc Clear Springs Foods Inc., Buhl, ID, USA

a r t i c l e i n f o

Article history:Received 16 March 2015Received in revised form27 May 2015Accepted 27 May 2015Available online 23 June 2015

Keywords:NALTRainbow troutDevelopmentNasal vaccines

* Corresponding author. Center for Evolutionary a(CETI), Department of Biology, MSC03 2020, 1 Univquerque, NM 97131, USA.

E-mail address: isalinas@unm.edu (I. Salinas).

http://dx.doi.org/10.1016/j.dci.2015.05.0150145-305X/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Determining the earliest age at which farmed fish can be successfully vaccinated is a very importantquestion for fish farmers. Nasal vaccines are novel mucosal vaccines that prevent aquatic infectiousdiseases of finfish. The present study investigates the ontogeny of the olfactory organ of rainbow trout byhistology and aims to establish the earliest age for vaccination against infectious hematopoietic necrosis(IHN) and enteric red mouth (ERM) disease using the nasal route. Rainbow trout (Oncorhynchus mykiss)were vaccinated intranasally (I.N) at three different ages: 1050� days (DD) (group A); 450 DD (group B);and 360 DD (group C), or 70, 30 and 24 days post-hatch (dph), respectively. The mean weights of groupsA, B and C were 4.69 g, 2.9 g and 2.37 g, respectively. Fish received either a live attenuated IHN virusvaccine, ERM formalin killed bacterin or saline (mock vaccinated). Fish were challenged to the corre-sponding live pathogen 28 days post-vaccination. IHN vaccine delivery at 360 DD resulted in 40%mortality likely due to residual virulence of the vaccine. No mortality was observed in the ERM nasaldelivery groups. Following challenge, very high protection rates against IHN virus were recorded in allthree age groups with survivals of 95%, 100% and 97.5% in groups A, B and C, respectively. Survival againstERM was 82.5%, 87.5% and 77.5% in groups A, B and C, respectively. Survival rates did not differ amongages for either vaccine. Our results indicate the feasibility and effectiveness of nasal vaccination as earlyas 360 DD and vaccination-related mortalities when a live attenuated viral vaccine was used in theyoungest fish.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Many diseases affect early life stages of fish so it is important todetermine the earliest age when fish can be successfully vaccinated(Ellis, 1988). The earliest time to vaccinate salmonids has beendetermined in a number of disease models such as vibriosis, entericred mouth disease (ERM) or viral hemorrhagic septicemia (VHS). Inrainbow trout (Oncorhynchus mykiss), cellular immunity is func-tional and main lymphoid organ development is completed at~200� days (DD) (Tatner and Manning, 1983). However, mucosal

nd Theoretical Immunologyersity of New Mexico, Albu-

immunity develops later (Salinas et al., 2011) than systemic im-munity in teleosts. As a consequence, and generally speaking, 0.5 g(10 weeks post-hatch) at 10 �C (¼700 DD) is considered the earliesttime to vaccinate rainbow trout using immersion vaccination(Tatner and Manning, 1983; Obach, 1991; Ellis, 1988). Currently,immersion vaccination of young fingerlings is the industry goldstandard for ERM vaccination. On the other hand, injecting vaccinesinto small fish is impractical and often leads to high mortalities dueto the handling. As a result, 1 g is considered the smallest size forinjection vaccination of salmonids whereas 2.5 g is considered theoptimal size to achieve maximum protection for all species(Johnson et al., 1982; Ellis, 1988).

In fish, traditional mucosal vaccination strategies include im-mersion and oral vaccination. Recently, nasal vaccines were addedto this list. Nasal vaccination can be a very effective way to control

Table 1p-Values obtained from the statistical analyses performed in the present. Study.“Side” indicates mortalities recorded prior to challenge to the live pathogen.

Description p-Value

Age_ERM_Side 1Age_IHNV_Side 6.15E-10Age_Control_Side 0.775538147TreatmentPairs_C_ERM_A_B 1TreatmentPairs_C_ERM_A_C 1TreatmentPairs_C_ERM_B_C 1TreatmentPairs_C_IHNV_A_B 0.001415719TreatmentPairs_C_IHNV_A_C 2.89E-10TreatmentPairs_C_IHNV_B_C 0.001796509TreatmentPairs_C_Control_A_B 0.497487437TreatmentPairs_C_Control_A_C 1TreatmentPairs_C_Control_B_C 1TreatmentPairs_A_ERM_Control_Vaccinated 1.41E-09TreatmentPairs_A_IHNV_Control_Vaccinated 1.48E-16TreatmentPairs_B_ERM_Control_Vaccinated 8.21E-12TreatmentPairs_B_IHNV_Control_Vaccinated 2.53E-18TreatmentPairs_C_ERM_Control_Vaccinated 5.17E-09TreatmentPairs_C_IHNV_Control_Vaccinated 1.94E-13Age_ERM_Control 1Age_ERM_Vaccinated 0.549087619Age_IHNV_Control 0.302214595Age_IHNV_Vaccinated 0.772112235

I. Salinas et al. / Developmental and Comparative Immunology 53 (2015) 105e111106

aquatic infectious diseases in finfish (Tacchi et al., 2014; LaPatraet al., 2015). The use of this delivery route has been tested insmall trout but age was not controlled in those studies. Nasalvaccination of young non-aquatic vertebrates such as piglets,newborn mice, calves and rabbits (Guzman-Bautista et al., 2014;Sandbulte et al., 2014; Sabirov and Metzger, 2008; Ellis et al.,2010; Lancaster et al., 1960) is feasible, effective and does notinduce tolerance. In fact, the human nasal vaccine FluMist (LAIV) isrecommended for use in young infants (6e59 months) since itprevents about 50%more cases than the injected flu vaccine (TIV) atthis age (Carter and Curran, 2011).

We currently lack information regarding the age at whichrainbow trout fry can be vaccinated nasally. The aim of the presentstudy is to determine the earliest age at which rainbow trout caneffectively be vaccinated using the nasal route. Two different vac-cinemodels are used, a live attenuated viral vaccine and a formalin-killed bacterial vaccine. Our results are coupled with histologicalobservations of the ontogeny of the trout olfactory organ fromhatch to 1050 DD. Altogether, the present study provides novelinsights into fish nasal immunity and contributes towards theimplementation of nasal vaccines in rainbow trout aquaculturefacilities.

2. Materials and methods

2.1. Fish

Three hundred specific-pathogen-free (spf) rainbow trout ofthree different ages (1050� days (DD) (group A); 450 DD (Group B);and 360 DD (group C)), or 70, 30 and 24 days post-hatch (dph),were obtained from Clear Springs Foods Inc. (Buhl, Idaho). Themean weight of fish in group A was 4.69 g; the mean weight of fishin group B was 2.9 g whereas in group C was 2.37 g. Fish weremaintained in 378 L tanks that received single-pass spf springwater at a constant temperature of 14.5 �C and a dissolved oxygencontent of 9.2 ppm. Fish were fed twice daily a commercial rainbowtrout diet (Clear Springs Foods, Inc.).

2.2. Vaccination trials and challenge experiments

Vaccination experiments were conducted using two differentvaccine models: an IHN live attenuated viral vaccine (LaPatra et al.,2004) and an ERM bacterin (Nelson et al., 2015). One hundred fishfrom each age group were vaccinated with each vaccine or salineusing a 10 or 25 ml volume dependent on the size of the fish.Duplicate 25-fish groups from each treatment were challengedwith either IHNV or Yersinia ruckeri at 28 (400 DD) days post-immunization (dpi) and followed for an additional 28 days aspreviously described (Tacchi et al., 2014). Kidney, spleen and liverwere collected from rainbow trout that died prior to challenge.Tissues were weighed and homogenized. The presence of IHNV inhomogenized samples was measured as explained elsewhere(LaPatra et al., 1989).

2.3. Histology

Whole rainbow trout (N ¼ 5) were sampled every day fromhatching (15 DD) to 600 DD. The heads from these series of sampleswere dissected. Additionally, olfactory organs from adult in-dividuals (N ¼ 4) were also collected. All samples were fixed in 10%neutral buffered formalin overnight and embedded in paraffinfollowing standard histological procedures. Five mm-thick sectionswere stained with hematoxylin-eosin and observed under a ZeissAxioscope microscope coupled with a digital camera using theAxioVision software.

2.4. Statistical analysis

To estimate the expected mortality profile of vaccination trialsand challenge experiments in practice, KaplaneMeier survivalcurves with pointwise 95% confidence bands were computed andplotted. To test for differences in the proportion of mortality bytreatment and age for each pathogen in both the vaccination trialsand challenge experiments, the 28-day survival and mortality fre-quencies were used in Fisher's exact test of conditional indepen-dence for two-dimensional contingency tables as implemented in Rversion 3.1.2 stats package (R Core Team, 2014) fisher.test() func-tion. Familywise error rate was controlled by the most conservativeBonferroni correction by multiplying all p-values by the totalnumber of hypothesis tests performed (16 tests). All test results arepresented in Table 1.

All p-values are shown in Table 1.

3. Results

3.1. Vaccination-related mortalities prior to challenge

The KaplaneMeier survival profiles shown in Fig. 1a indicateincreased and earlier mortality only for younger fish vaccinatedwith live attenuated IHN vaccine; total mortalities are shown inFig. 1b. No mortalities associated with I.N delivery of ERM vaccineor saline were observed in any of the age groups that were vacci-nated (p-value ¼ 1). Vaccination with the live attenuated IHNvaccine resulted inmortalities of 4%,19% and 40% in groups A, B andC, respectively (Fig. 1b). Furthermore, all age group pairs weresignificantly different (A vs B p-value ¼ 0.0227, A vs C p-value ¼ 4.62 E�9, and B vs C p-value ¼ 0.0287). This means thatearly life stages of rainbow trout are more susceptible to residualvirulence present in the IHNV vaccine than older life stages testedin this study (p-value¼ 9.87 E�9). Some rainbow trout that died as aresult of nasal IHNV delivery showed typical gross signs of IHNalthough no attempts to characterize the histopathology of mori-bund fish were performed. IHNV levels were titrated in kidney,spleen and liver homogenates of 43% (27/63) of the fish that died.IHNV was detected in 70% (19/27) of the animals tested. The meantiters were 6.0 � 103 plaque forming units (pfu)/g for group A;1.4 � 103 pfu/g for group B and 4.9 � 105 pfu/g for group C.

I. Salinas et al. / Developmental and Comparative Immunology 53 (2015) 105e111 107

3.2. Challenge-related mortalities

The KaplaneMeier survival profiles shown in Fig. 2a and bindicate in challenge experiments, replicate tanks had similarmortality profiles, therefore final mortality frequencies werecombined from replicate tanks; total mortalities are shown inFig. 2c.

Fig. 1. a) Vaccination trials KaplaneMeier survival curves of the three experimental ageattenuated IHN vaccine or ERM vaccine. b) Vaccination trials total mortality at 28 days po(control) or I.N vaccination with either live attenuated IHN vaccine or ERM vaccine.

3.3. Protection against ERM challenge

All three vaccinated groups were significantly more protectedthan their age-matched mock-vaccinated controls with mean sur-vival of 82.5%; 87.5% and 77.5% in groups A, B and C, respectively(Fig. 2a). No significant effect of age on survival was found in thevaccinated groups (p-value ¼ 1) (Table 1).

groups (A, B and C) following I.N saline (control) or I.N vaccination with either livest-vaccination of the three experimental age groups (A, B and C) following I.N saline

I. Salinas et al. / Developmental and Comparative Immunology 53 (2015) 105e111108

3.4. Protection against IHNV challenge

All three vaccinated groups were significantly more protectedthan their age-matched mock-vaccinated controls with mean sur-vival of 95%, 100% and 97.5% in groups A, B and C, respectively(Fig. 2b). No significant effect of age on the percentage survival wasfound for the vaccinated groups (p-value ¼ 1) (Table 1).

Fig. 2. a and b) Challenge experiments KaplaneMeier survival curves of the three experivaccination. c) Challenge experiments total mortality at 28 days post-challenge of the three

3.5. Histological studies of NALT development in rainbow trout

In an effort to determine when rainbow trout can first bevaccinated via the nasal route we have conducted a developmentalseries histological study from newly hatched to 40 days post-hatch(600� days, DD) and compared them to adult specimens. Newlyhatched larvae (15 DD) already showed open olfactory pits where

mental age groups (A, B and C) challenged with live IHNV or Y. ruckeri 28 days postexperimental age groups (A, B and C) following I.N saline (control) or I.N vaccination.

I. Salinas et al. / Developmental and Comparative Immunology 53 (2015) 105e111 109

water is able to circulate. The olfactory organ at this point consistedof a pair of flat olfactory epithelia with a homogenous morphology(not shown). At 45 DD the primary invagination is evident forminga flat olfactory pit (Fig. 3A). Ciliated undifferentiated cells form theprimordial olfactory epithelium. No epithelial folds can be observedyet. Mucosal goblet cells are present in the epithelium surroundingthe olfactory pit but not within the neuro-olfactory epithelium.Capillaries are absent in the lamina propria (LP). By 90 DD cellproliferation is evident with the basal region showing mitotic fig-ures (Fig. 3B). Flat apical nuclei can be seen and cilia are moreabundant. Neither epithelium folding nor capillary formation isobserved yet. At 195 DD the first primordial fold (primary lamella)start to form (Fig. 3C). The LP is still not very vascularized. The sizeand height of the primary fold increases at 240 DD (Fig. 3D). At thispoint the sensory and mucosal portions are not distinguishable butthe number of capillaries augments in the LP. At 315 DD (Fig. 3E),the folds are still primordial but some small invaginations indica-tive of a second primary fold forming can be observed. Three dayslater, at 360 DD (Fig. 3F), the olfactory organ presents two primaryfolds, one clearly much larger than the other. At this point myeloidcells were observed in the LP. Importantly, at 450 DD (Fig. 3G) the

Fig. 3. Histological study (hematoxylin and eosin stains) of the development of the olfactororgan of a 90 DD old rainbow trout. C) Olfactory organ of a 195 DD old rainbow trout. D) Olftrout. F) Olfactory organ of a 360 DD old rainbow trout. G) Olfactory organ of a 450 DD old raadult rainbow trout. NC: nasal cavity; OE: olfactory epithelium; LP: lamina propria; SF: secGreen arrowheads indicate mucosal epithelium regions. Scale bar: 50 mm. (For interpretativersion of this article.)

first mucosal regions within the olfactory epithelium appear. Mostof the goblet cells are located at the valleys of the primary fold. By555 DD (Fig. 3H) three primary folds are visible and the number ofgoblet cells in the valley of the folds has increased. In adult fish(Fig. 3I), however, there is a greater number and complexity of theolfactory organ with clear secondary fold formation in each of theprimary folds including apical mucosal epithelial areas. Due to theimportant histological changes observed around 360 and 450 DD,we selected these time points for the vaccination trials.

4. Discussion

Fish vaccination is one of the most effective ways to controlinfectious aquatic diseases. Vaccines against viral and bacterialdiseases are commonly administered to farmed fish worldwide. Ofparticular interest is the vaccination of young life stages of fish dueto their high susceptibility to pathogens. The recommendedvaccination route at this early life stages depends on the type ofvaccine and disease. For instance, ERM vaccines are routinelydelivered by immersionwhereas IHNV vaccines are not effective viathe immersion route and therefore injection is preferred. Yet,

y organ of rainbow trout. A) Olfactory organ of a 44 DD old rainbow trout. B) Olfactoryactory organ of a 240 DD old rainbow trout. E) Olfactory organ of a 315 DD old rainbowinbow trout. H) Olfactory organ of a 555 DD old rainbow trout. I) Olfactory organ of anondary folds. Black arrows indicate primary folds. Red arrows indicate blood vessels.on of the references to colour in this figure legend, the reader is referred to the web

I. Salinas et al. / Developmental and Comparative Immunology 53 (2015) 105e111110

recommendation guidelines pertaining the earliest time to vacci-nate exist for a limited number of vaccines only and are usuallylimited to the immersion or injection routes.

Young rainbow trout become immunocompetent on the basis oflymphoid organ development and cellular immunity approxi-mately at 14 dph (196 DD) (Tatner and Manning, 1983). Youngrainbow trout fingerlings are usually vaccinated when they are 1 gin body weight for maximum protection (Ellis, 1988) but thenumber of available studies is scarce in models other than vibriosisand ERM. Additionally, guidelines for alternative routes of vacci-nation such as intranasal delivery are not available.

Nasal vaccination of young children as well as newborn mice,piglets, calves and chickens (Guzman-Bautista et al., 2014;Sandbulte et al., 2014; Sabirov and Metzger, 2008; Ellis et al.,2010; Lancaster et al., 1960) has been performed successfully.Interestingly, the power of diffuse NALT to induce effective im-munity following nasal vaccination in the absence of organizedNALT was unequivocally demonstrated in newborn mice whoseorganized NALT had been surgically removed (Sabirov andMetzger,2008). Recently, we characterized teleost NALT, which consists ofdiffuse NALT only lacking organized lymphoid structures such astonsils or adenoids (Tacchi et al., 2014).

The discovery of nasal immunity in teleosts prompted the firststudies concerning nasal vaccines for use in aquaculture (Tacchiet al., 2014; LaPatra et al., 2015). Both ERM and IHNV vaccines arevery effective when delivered intranasally to approximately 5 grainbow trout. In this study, we sought to determine the earliesttime that nasal vaccines can be delivered to young rainbow troutusing the intranasal route. It is worth highlighting that the presentstudy was not aimed to compare nasal vaccination with otherroutes of vaccination but rather establish the earliest time at whichnasal vaccines are both effective and feasible for delivery to youngrainbow trout. We selected three different ages for our study: 1050DD, 450 DD and 360 DD; which included fish of 4.69 g; 2.9 g and2.37 gmeanweight, respectively. The largest fish groupwas used asa tentative positive control based on our previous studies (LaPatra eal., 2015; Tacchi et al., 2014). The other two earlier life stages wereselected based on the findings from our histological results.

The olfactory organ is the first chemosensory system to developin fish, preceding the appearance of solitary chemosensory cells(Kotrschal et al., 1997) and taste (Hansen et al., 2002). Our histo-logical study is in agreement with previous ontogenic descriptionsof teleost olfactory organs (Evans et al., 1982; Hansen and Zeiske,1998; Hansen and Zielinski, 2005; Zielinski and Hara, 1988;Zielinski and Toshiaki, 1988). Generally speaking, differentiationof mucosal versus sensory areas, increasing numbers of primaryfolds and appearance of secondary folds take place as rainbow troutincrease in size/age (Pfeiffer, 1963; Olsen, 1993). We found that themucosal and sensory portions of the olfactory epithelium formed atapproximately 450 DD. This is in agreement with previous studiesthat combined light and electron microscopy to describe thedevelopment of the olfactory organ of salmonids (Kudo et al.,2009). However, younger trout (360 DD ¼ 24 dph), whose olfac-tory epithelium did not show mucosal portions yet, were alsosuccessfully vaccinated via the nose using both vaccine models(albeit the losses recorded for the IHNV vaccine). Interestingly,rainbow trout vaccinated by intraperitoneal injection or immersionagainst coldwater disease using a heat-inactivated vaccinewere notprotected if the vaccine was delivered at 30 or 40 dph (Obach andLaurencin, 1991). Future studies should address the expression ofimmune markers in NALT in order to better resolve the onset ofnasal immunity in trout larvae.

Vaccination of early developmental stages can result in the in-duction of tolerance if the antigen is delivered prior to the onset of afully functional immune system. For instance, in teleosts, early

vaccination of seabream with a vaccine against vibriosis can resultin tolerance issues (Mulero et al., 2008). Other examples includeearly studies in carp (Mughal et al., 1986). Based on our results,delivery of nasal vaccines to early life stages of rainbow trout doesnot affect the efficacy of the vaccine. Even if the youngest aged fishtested in this study suffered from some mortality when vaccinatedwith IHNV, vaccinated fish that lived and were challenged 28 dayslater were all protected against IHN.

Live attenuated vaccines elicit the strongest immune responsesand are amongst the most efficacious vaccines available. Liveattenuated vaccines rely on the effective clearance of the livemicroorganism by the host's immune system. Thus, there are somerisks associated with this type of vaccines, which are not recom-mended for use in immunocompromised hosts or hosts that lack afully functional immune system (Medical Advisory Committee ofthe Immune Deficiency Foundation et al., 2014). Our resultsstrongly support the idea that the immune system of 360 DD-oldrainbow trout failed to clear IHNV at a fast enough rate leading togreater IHNV titers in tissues and mortality. The fact that no losseswere observed in the same age fish following ERM delivery furthersupports the idea that live attenuated vaccines are not safe inimmunocompromised hosts.

5. Conclusions

In conclusion, the present study shows that ERM nasal vaccinesare very effective in early rainbow trout life stages as young as 360DD (24 dph). Whereas live attenuated IHNV vaccine caused sig-nificant mortalities, formalin killed bacterins such as ERM appear tobe completely safe. Importantly, no tolerance was induced sug-gesting the presence of a functional autologous NALT in trout asearly as 360 DD. Our current nasal delivery protocol is still time-consuming and not practical if millions of fingerlings need to bevaccinated. Future efforts should be directed towards the compar-ison of nasal and immersion routes in small fingerlings in order toassess the cost-benefits of each strategy. Finally, the design of massvaccination methods that can deliver vaccines nasally to salmonidfingerlings would significantly increase the commercial applicationof nasal vaccines in aquaculture.

Acknowledgements

This work was funded by USDA AFRI Grant# 2DN70-2RDN7 to ISand the National Institutes of Health COBRE Grant# P30GM110907.Authors thank the technical staff at Clear Springs Foods ResearchDivision for their help with fish maintenance and sampling and K.Crossey for help with histological sample preparation.

References

Carter, N.J., Curran, M.P., 2011. Live attenuated influenza vaccine (FluMist®; Flu-enz™): a review of its use in the prevention of seasonal influenza in childrenand adults. Drugs 71, 1591e1622.

Ellis, A.E., 1988. Fish Vaccination. Academic Press, London.Ellis, J.A., Gow, S.P., Goji, N., 2010. Response to experimentally induced infection

with bovine respiratory syncytial virus following intranasal vaccination ofseropositive and seronegative calves. J. Am. Vet. Med. Assoc. 236, 991e999.

Evans, R.E., Zielinki, B., Hara, T.J., 1982. Development and regeneration of the ol-factory organ in rainbow trout. In: Hara, T.J. (Ed.), Chemoreception in Fishes.Elsevier Scientific Publishing Co, Amsterdam, Oxford, New York, pp. 15e37.

Guzman-Bautista, E.R., Garcia-Ruiz, C.E., Gama-Espinosa, Al., Ramirez-Estudillo, C.,Rojas-Gomez, O.I., Vega-Lopez, M.A., 2014. Effect of age and maternal antibodieson the systemic and mucosal immune response after neonatal immunization ina porcine model. Immunology 141, 609e616.

Hansen, A., Reutter, K., Zeiske, E., 2002. Taste bud development in the zebrafish,Danio rerio. Dev. Dyn. 223, 483e496.

Hansen, A., Zeiske, E., 1998. The peripheral olfactory organ of the zebrafish, Daniorerio: an ultrastructural study. Chem. Senses 23, 39e48.

Hansen, A., Zielinski, B.S., 2005. Diversity in the olfactory epithelium of bony fishes:

I. Salinas et al. / Developmental and Comparative Immunology 53 (2015) 105e111 111

development, lamellar arrangement, sensory neuron cell types and trans-duction components. J. Neurocytol. 34, 183e208.

Johnson, K.A., Flynn, J.K., Amend, D.F., 1982. Onset of immunity in salmonid fryvaccinated by direct immersion with Vibrio anguillarum and Yersinia ruckeribacterins. J. Fish. Dis. 5, 197e205.

Kotrschal, K., Krautgartner, W.D., Hansen, A., 1997. Ontogeny of the solitary che-mosensory cells in the zebrafish Danio rerio. Chem. Senses 22, 111e118.

Kudo, H., Shinto, M., Sakurai, Y., Kaeriyama, M., 2009. Morphometry of olfactorylamellae and olfactory receptor neurons during the life history of chum salmon(Oncorhynchus keta). Chem. Senses 34, 617e624.

Lancaster, J.E., Merriman, M., Rienzi, A.A., 1960. The intranasal Newcastle diseasevaccination of chicks from immune parents. Can. J. Comp. Med. Vet. Sci. 24,52e56.

LaPatra, S., Kao, S., Erhardt, E.B., Salinas, I., 2015. Evaluation of dual nasal delivery ofinfectious hematopoietic necrosis virus and enteric red mouth vaccines inrainbow trout (Oncorhynchus mykiss). Vaccine 33, 771e776.

LaPatra, S.E., Clouthier, S., Anderson, E., 2004. Current trends in immunotherapy andvaccine development for viral diseases of fish. In: Leung, K.Y. (Ed.), CurrentTrends in the Study of Bacterial and Viral Fish Diseases. World Scientific Pub-lishing Co. Pte. Ltd, pp. 363e389.

LaPatra, S.E., Roberti, K.A., Rohovec, J.S., Fryer, J.L., 1989. Fluorescent antibody testfor the rapid diagnosis of infectious hematopoietic necrosis. J. Aquat. Anim.Health 1, 29e35.

Medical Advisory Committee of the Immune Deficiency Foundation, Shearer, W.T.,Fleisher, T.A., Buckley, R.H., Ballas, Z., Ballow, M., Blaese, R.M., Bonilla, F.A.,Conley, M.E., Cunningham-Rundles, C., Filipovich, A.H., Fuleihan, R.,Gelfand, E.W., Hernandez-Trujillo, V., Holland, S.M., Hong, R., Lederman, H.M.,Malech, H.L., Miles, S., Notarangelo, L.D., Ochs, H.D., Orange, J.S., Puck, J.M.,Routes, J.M., Stiehm, E.R., Sullivan, K., Torgerson, T., Winkelstein, J., 2014. Rec-ommendations for live viral and bacterial vaccines in immunodeficient patientsand their close contacts. J. Allergy Clin. Immunol. 133, 961e966.

Mughal, M.S., Farley-Ewens, E.K., Manning, M.J., 1986. Effects of direct immersion inantigen on immunological memory in young carp, Cyprinus carpio. Vet.Immunol. Immunopathol. 12, 181e192.

Mulero, I., Sepulcre, M.P., Fuentes, I., García-Alc�azar, A., Meseguer, J., García-Ayala, A., Mulero, V., 2008. Vaccination of larvae of the bony fish gilthead

seabream reveals a lack of correlation between lymphocyte development andadaptive immunocompetence. Mol. Immunol. 45, 2981e2989.

Nelson, M.C., LaPatra, S.E., Welch, T.J., Graf, J., 2015. Complete genome sequence ofYersinia ruckeri str. CS007e82, etiologic agent of red mouth disease in salmonidfish. Genome Announc. 3 (1) e01491e14.

Obach, K., Laurencin, F.B., 1991. Vaccination of rainbow trout Oncorhynchus mykissagainst the visceral form of cold-water disease. Dis. Aquat. Org. 12, 13e15.

Ols�en, K.H., 1993. Development of the olfactory organ of the Arctic charr, Salvelinusalpinus (L.) (Teleostei, Salmonidae). Can. J. Zool. 71, 1973e1984.

Pfeiffer, W., 1963. The morphology of the olfactory organ of the pacific salmon(Oncorhynchus). Can. J. Zool. 41, 1233e1236.

R Core Team, 2014. R: a Language and Environment for Statistical Computing. RFoundation for Statistical Computing, Vienna, Austria. URL: http://www.R-project.org/.

Sabirov, A., Metzger, D.W., 2008. Intranasal vaccination of infant mice inducesprotective immunity in the absence of nasal-associated lymphoid tissue. Vac-cine 26, 1566e1576.

Salinas, I., Zhang, Y.-A., Sunyer, J.O., 2011. Mucosal immunoglobulins and B cells ofteleost fish. Dev. Comp. Immunol. 35, 1346e1365.

Sandbulte, M.R., Platt, R., Roth, J.A., Henningson, J.N., Gibson, K.A., Raj~ao, D.S.,Loving, C.L., Vincent, A.L., 2014. Divergent immune responses and diseaseoutcomes in piglets immunized with inactivated and attenuated H3N2 swineinfluenza vaccines in the presence of maternally-derived antibodies. Virology464e465, 45e54.

Tacchi, L., Musharrafieh, R., Larragoite, E.T., Crossey, K., Erhardt, E.B., Martin, S.A.,LaPatra, S.E., Salinas, I., 2014. Nasal immunity is an ancient arm of the mucosalimmune system of vertebrates. Nat. Commun. 5, 5205.

Tatner, M.F., Manning, M.J., 1983. The ontogeny of cellular immunity in the rainbowtrout, Salmo gairdneri Richardson, in relation to the stage of development of thelymphoid organs. Dev. Comp. Immunol. 7, 69e75.

Zielinski, B., Hara, T.J., 1988. Morphological and physiological development of ol-factory receptor cells in rainbow trout (Salmo gairdneri) embryos. J. Comp.Neurol. 271, 300e311.

Zielinski, B., Toshiaki, J.H., 1988. Morphological and physiological development ofolfactory receptor cells in rainbow trout (Salmo gairdneri) embryos. J. Comp.Neurocytol. 271, 300e311.