Use of the PACAP as a molecular adjuvant for vaccines

Abstract

The present invention relates to the use of the pituitary adenylate cyclase activating peptide (PACAP) as a molecular adjuvant for vaccines. Among other applications, these vaccines may be used in the protection against infectious agents such as viruses, bacteria and ectoparasites affecting mammals, birds and aquatic organisms. The PACAP, combined with a particular antigen, demonstrates its effectiveness as adjuvant increasing the host immune response against that antigen. This type of response can be observed when the vaccine compositions or combinations that include PACAP are administered orally, by injection, or by immersion baths, in case of aquatic organisms.

Claims

1. A vaccine composition comprising a pituitary adenylate cyclase activating peptide (PACAP), at least one vaccine antigen specific for an infectious agent, and a pharmaceutically acceptable vehicle or diluent, wherein said PACAP and said vaccine antigen are present in an amount sufficient for producing protection to an infectious disease caused by the infectious agent in fish.

2. The composition of claim 1 which is formulated to be administered orally, by injection, or by immersion baths.

3. The composition of claim 2 wherein the PACAP (1) is in a concentration of 50 to 750 g/Kg of feed if the composition is applied as a formulated feed, (2) produces a concentration of 0.1-10 g per gram of body weight if it is applied by injection, or (3) produces a concentration of 50-1000 g per liter of water if the composition is applied by immersion baths.

4. The composition of claim 1 wherein the PACAP is obtained by a) isolation from its natural source, b) by chemical synthesis, or c) by recombinant DNA technology.

5. The composition of claim 4 wherein the PACAP (1) is in a concentration of 50 to 750 g/Kg of feed if the composition is applied as a formulated feed, (2) produces a concentration of 0.1-10 g per gram of body weight if it is applied by injection, or (3) produces a concentration of 50-1000 g per liter of water if the composition is applied by immersion baths.

6. The composition of claim 1 wherein the PACAP is obtained by administering a vector comprising cDNA coding for the PACAP.

7. A vaccine combination comprising a pituitary adenylate cyclase activating peptide (PACAP) and at least one vaccine antigen specific for an infectious agent, wherein said PACAP and said vaccine antigen are present in an amount sufficient for producing protection to an infectious disease caused by the infectious agent in fish and are administered in a same immunization schedule.

8. The combination of claim 7 wherein the PACAP and the antigen are formulated to be administered simultaneously, separately or sequentially during the same immunization schedule.

9. The combination of claim 8 wherein the PACAP (1) is in a concentration of 50 to 750 g/Kg of feed if the combination is applied as a formulated feed, (2) produces a concentration of 0.1-10 g per gram of body weight if it is applied by injection, or (3) produces a concentration of 50-1000 g per liter of water if the combination is applied by immersion baths.

10. A method for increasing immune response to a vaccine antigen in a fish comprising administration of the composition of claim 1 to the fish.

11. The method of claim 10 wherein the composition is administered simultaneously, separately or sequentially during the same immunization schedule.

12. The method of claim 10 wherein the PACAP peptide is obtained by a) isolation from its natural source, b) by chemical synthesis, or c) by recombinant DNA technology; or as a nucleic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Titles of total immunoglobulin G (IgG) (A), IgG1 (B) and IgG2a (C) induced in mice by immunization with OVA co-administered with the neuropeptide PACAP38. Two experimental groups of 6 animals each one were used. The negative control group (phosphate buffered saline (PBS)/OVA) was inoculated intraperitoneally on day 0 and 7 with a dose of 6 g of OVA in 0.2 mL of PBS. The group that also received PACAP38 (PBS/OVA+peptide) was inoculated intraperitoneally on day 0 and 7 with a dose of 6 g of OVA+0.5 g of PACAP38 in 0.2 mL of PBS. Different letters indicate significant differences.

(2) FIG. 2. Assessment of the cellular immune response in pigs vaccinated with the vaccine composition based on E2-PACAP. The cellular immune response was measured in pig lymphocytes isolated on day 5 after vaccination with E2 (group 1), with E2+PACAP (group 2) and with placebo (group 3). (A) lymphoproliferation assay: the results are expressed as stimulation index (SI), defined as the ratio between counts per minute (cpm) of the stimulated culture (cpm) and cpm of the untreated control group. The lymphoproliferative response with an SI2 was considered positive (B) IFN- secretion determination by real time PCR. Values are expressed as the arithmetic mean of 2.sup.Ct. (**) p<0.01.

(3) FIG. 3. Titers of hemagglutination inhibition in chickens immunized with HA or HA-PACAP (A). The arithmetic mean of the antibody titers were expressed as the log.sub.2 of the reciprocal value of the highest dilution of serum which produced hemagglutination inhibition. (B) Cellular immune response in immunized chickens. The IFN- secretion was determined by real-time PCR. Values are expressed as the arithmetic mean of 2.sup.Ct. (**) p<0.01.

(4) FIG. 4. Expression analysis by real time PCR of the in vitro effect of PACAP38 over the transcription of IL 1 in peripheral blood leukocytes (A) and head kidney (B) of nave rainbow trout. The effects of PACAP administration at a concentration of 10.sup.10 M, 10.sup.9 M and 10.sup.8 M was evaluated at 48 h post-treatment. The experiment was repeated 4 times. The leukocyte cultures were treated in duplicates and the PCRs were performed in triplicate. Data are expressed as mean of relative expression of IL 1 related to the endogenous elongation factor EF1standard deviation (SD). (*) p<0.05). * (p<0.05) indicates that the relative expression of the gene of interest was statistically higher compared to its relative expression in untreated leukocyte cultures (negative control).

(5) FIG. 5. Expression analysis by real time PCR of the in vitro effect of PACAP38 over the transcription of IL 15 in peripheral blood leukocytes (A) and head kidney (B) of neve rainbow trout. The effects of PACAP administration at a concentration of 10.sup.10 M, 10.sup.9 M and 10.sup.8 M was evaluated at 48 h post-treatment. The experiment was repeated 4 times. The leukocyte cultures were treated in duplicates and the PCRs were performed in triplicates. Data are expressed as the mean of the relative expression of IL 15 related to the endogenous elongation factor EF1standard deviation (SD). (*) p<0.05.

(6) FIG. 6. Agglutinating antibody titers against A. hydrophila in common carp. Values in the Y axis represent the arithmetic mean of the antibody titersstandard error. (*) p<0.05, (**) p<0.01. Group 1: injected with PBS, Group 2: injected with formalin inactivated A. hydrophila cells, Group 3: injected with formalin inactivated A. hydrophila cells and PACAP (1 g/fish).

(7) FIG. 7. Effect of the co-administration, by intramuscular injection, of the DNA vaccine (pP-IPNV) at a dose of 1 g/fish with a plasmid DNA having the Clarias gariepinus PACAP cDNA, under the control of the immediate early promoter of the human cytomegalovirus (pCMV-PACAP). At day 30 post-vaccination, fish were exposed to the virus by intraperitoneal injection of 100 L of IPNV (110.sup.7 (TCID.sub.50) ml.sup.1 per fish). At day 7 post-infection, 10 fish per group were sacrificed in order to evaluate the viral load in head kidney by real time RT-PCR. Values represent the meanstandard deviation. Different letters in superscript represent statistically significant differences between groups. Group 1: injected with PBS, Group 2: injected with pP-IPNV at a dose of 1 g/fish, Group 3: co-injected with pP-IPNV (1 g/fish) and pCMV-PACAP (0.5 g/fish), Group 4: co-injected with pP-IPNV (1 g/fish) and pCMV (0.5 g/fish), Group 5: co-injected with pP-IPNV (1 g/fish) and pCMV-PACAP (0.05 g/fish), Group 6: co-injected with pP-IPNV (1 g/fish) and pCMV (0.05 g/fish).

(8) FIG. 8. Cumulative mortality of rainbow trout immunized intramuscularly with a DNA vaccine based on the gene encoding to the glycoprotein G of the VHSV (pG-VHSV) and the peptide PACAP. At week 4 post-vaccination fish were exposed to the virus (110.sup.5 TCID.sub.50 mL.sup.1 per fish) by immersion baths. Cumulative mortality was assessed during 4 weeks post-challenge. Group 1: injected with PBS. Group 2: injected with pG-VHSV (0.01 g/fish), Group 3: injected with pG-VHSV (0.01 g/fish) and PACAP (0.1 g/fish), Group 4: injected with pG-VHSV (0.01 g/fish) and PACAP (0.5 g/fish).

EXAMPLES

Example 1

Effect of the Co-Immunization with OVA and PACAP on the Humoral Immune Response in Mice

(9) BALB/c mice (n=12) with a body weight of 20 g were separated into two experimental groups of 6 animals each one. The negative control group (PBS/OVA) was intraperitoneally injected, on days 0 and 7 with a dose of 6 g of OVA in 0.2 mL of PBS. The group treated with PACAP38 (PBS/OVA+peptide) was intraperitoneally injected on days 0 and 7 with a dose of 6 g of OVA+0.5 g of PACAP38 in 0.2 mL of PBS. On day 15 of the immunization protocol, blood from each fish was taken in order to evaluate total IgG, IgG1 and IgG2a titers.

(10) The FIGS. 1A, B and C show the total IgG, IgG1 and IgG2a titers, respectively, induced by immunization of mice with OVA co-administered with PACAP38. Animals in the group PBS/OVA+peptide showed a specific total IgG titer against OVA statistically superior compared with the control group (FIG. 1A). Similarly, we observed that the titles of IgG1 and IgG2a specific against OVA in the group immunized with OVA+PACAP were significantly higher than those observed in the group immunized with OVA only (FIGS. 1B and C).

Example 2

Evaluation of Cellular Immune Response in Pigs Vaccinated with the Vaccine Composition Based on E2-PACAP

(11) The antigen E2 is the major envelope glycoprotein of the CSFV. In order to evaluate the cellular immune response in pigs vaccinated with the vaccine composition based on E2-PACAP, and to compare its response with that produced by the E2 antigen we selected 18 healthy pigs of 20 kg average weight and serologically negative to CSFV, from a farm with no history of this disease and non-vaccinated against CSFV in the 3 preceding years. The pigs were located into 3 different groups of 6 animals each one, with water and food ad libitum. Each vaccine composition was applied in single immunization as follow: 25 g of recombinant E2 (Group 1) and 25 g of E2 co-administered with a similar amount of PACAP38 (Group 2). Group 3 was established as a placebo group. The immunogens were formulated in an emulsion of oil adjuvant and inoculated by intramuscular injection at a final volume of 2 mL. The animals were challenged on day 8 post-immunization by intramuscular injection of the CSFV (10.sup.5 LD.sub.50 of the Margarita isolation). We conducted a daily analysis of clinical signs and blood samples were taken on days 3 and 5 to assess lymphocyte proliferation and the expression of the IFN- as indicators of cellular immune response. In the animals immunized with E2-PACAP was detected an increase in the lymphocyte response (FIG. 2A) and the highest levels of IFN- on day 5, as compared with the other groups (FIG. 2B). This results show that the co-administration of the E2 antigen with PACAP produces a cellular immune response against CSFV in pigs.

Example 3

Effect of the Co-Immunization of the HA of the Avian Influenza Virus (Virus A/VietNam1203/2004) and PACAP on the Humoral and Cellular Immune Response in Chickens

(12) For immunization were used white leghom chickens of 3 weeks of age. Three experimental groups of ten chicks each were employed.

(13) Group 1: injected with 20 g of HA

(14) Group 2: injected with 20 g of HA+0.5 g of PACAP27

(15) Group 3: injected with 20 g of bovine serum albumin

(16) The HA was obtained by recombinant expression in mammalian cells. The vaccines were formulated with Montanide 888 and were administered in a final volume of 0.5 mL by subcutaneous route. The animals were re-immunized at day 28 post-immunization. Blood samples were taken at days 0, 14, 28, 35, 42 and 49, in order to evaluate the titers of protective antibodies using a haemagglutination inhibition assay and also to isolate leukocytes to evaluate the cellular immune response produced by the vaccines in vitro.

(17) The chickens immunized with HA-PACAP showed a superior antibody response compared with the group immunized with HA, from day 35 of the re-immunization (FIG. 3A). The group treated with HA-PACAP showed an increase in the cellular immune response, measured as stimulation of IFN-, in comparison to the groups immunized with HA or the control group (FIG. 3B).

Example 4

Effect of PACAP on the Expression of IL1- and IL15 in Peripheral Blood and Anterior Kidney Leukocytes of Neve Rainbow Trout (Oncorhynchus mykiss)

(18) We used juvenile rainbow trout (O. mykiss) of approximately 50 g, free of the VHSV and IPNV. Fish (n=5) were anesthetized with methanesulfonic acid salt (Sigma, USA) and peripheral blood from the tail vein was taken aseptically of each fish and then the head kidney was collected.

(19) Peripheral blood and the head kidney leukocytes were isolated following the method described by Graham and Secombes (Graham and Secombes (1998) Immunology 65:293-7).

(20) The cells were resuspended in L-15 with 5% FCS at a concentration of 510.sup.6 cells per ml and dispensed in 24-well plates at 1 mL per well. The leukocytes were treated with 3 doses of Clarias gariepinus PACAP38 (10.sup.10 M, 10.sup.9 M and 10.sup.8 M) obtained by chemical synthesis, in duplicate. Untreated leukocytes dispensed in duplicate were used as negative control.

(21) The levels of IL 1 and IL 15 were evaluated 48 hours after treatment. In order to achieve this objective, total RNAs were purified from leukocyte cultures treated with PACAP by the method described by Chomczynski and Sacchi (Chomczynski and Sacchi (1987) Anal. Biochem. 162:156-9).

(22) Because of the designed primers to amplify IL 1 and IL 15 do not discriminate between cDNA and genomic DNA, total RNAs purified from different tissues were treated with a DNA nuclease, specifically with the RQ1 RNase-free DNase (Promega).

(23) For cDNA synthesis was used a commercial kit that use the SuperScript III reverse transcriptase (Invitrogen). Finally, for quantitative PCRs (qPCRs) was used a commercial mixture of PCR: Power SYBR Green PCR Master Mix (Applied Biosystems). The results of qPCR were normalized against an endogenous gene of constitutive expression, specifically against the elongation 1 factor (EF 1) and were performed in triplicate. The results were expressed as 2.sup.Ct, where Ct is equal to the remainder of the Ct value of the target gene minus the Ct value of the normalized gene EF 1.

(24) The IL 1 protein levels increased after 48 hours of treatment in peripheral blood leukocytes treated with 10.sup.10 M of PACAP38. At a dose of 10.sup.9 M, there is also a stimulatory effect of PACAP38 on the transcription of IL 1 compared with the negative control group, but the expression levels detected in this case were below to those obtained with the dose of 10.sup.10 M. These results show a positive effect of PACAP38 on the transcription of IL 1 at low concentrations (10.sup.9 M and 10.sup.10 M) (FIG. 4A). In head kidney leukocytes, the effect of PACAP 38 on the IL1 transcription was moderate compared to those obtain in peripheral blood leukocytes, showing a stimulatory effect only at a dose of 10.sup.9 M (FIG. 4B).

(25) Additionally, we observed a stimulatory effect of PACAP38 on the expression of the IL 15 in peripheral blood leukocytes stimulated with PACAP at a doses of (10.sup.10 M and 10.sup.9 M). The highest values were obtained at a dose of 10.sup.10 M (FIG. 5A). In head kidney leukocytes, as it was previously observed with the IL1transcription the effect was moderate. The IL 15 expression levels in the PACAP treated group were statistically higher than the negative control group at the dose of 10.sup.9 M (FIG. 5B).

Example 5

Controlled Challenge Test with Caligus Rogercresseyi in Salmo Salar Previously Immunized Intraperitoneally with the Antigen My32, and My32 Co-Administered with PACAP

(26) My32 protein was obtained in recombinant form, in the pellet of broken BL21(DE3)-pET28a-my32 transformed E. coli cells. It is known that this protein, administered intraperitoneally (IP), produced a 57% inhibition of infection in the second generation of parasites in a challenge test with C. rogercresseyi in S. salar (Carpio et al. (2011) Vaccine. 29 (15):2810-20).

(27) To demonstrate the adjuvant effect of PACAP on this protein, we designed a vaccination-challenge experiment in S. salar under controlled conditions. In this experiment were established six experimental groups of 25 animals (average weight 80 g) each one:

(28) Group 1: Injected IP with PBS

(29) Group 2: Injected IP with PACAP38 at the dose of 1 g/fish

(30) Group 3: Injected IP with my32 at the dose of 3 g/g of body weight (gbw)

(31) Group 4: Co-administration by IP injection of my32 at 3 g/gbw and PACAP at the dose of 1 g/fish

(32) Group 5: Fish injected IP with my32 at the dose of 3 g/gbw and fed with 250 g of PACAP/Kg of feed daily, for one week before and after de immunization with my32.

(33) During the course of the experiment, fish were fed twice daily with a commercial formula without PACAP at the rate of 1% of their body weight, with the exception of the group 5, which was provided one week before and after immunization the same commercial formula with PACAP, as described by Adelmann et al. ((2008) Vaccine 26, 837-844) for oral administration of an attenuated strain of the virus VHSV.

(34) After 500 arbitrary thermal units, fish were adapted to seawater for two weeks. Subsequently, they were challenged with 2000200 copepodites to each tank. The fish were kept without water flow in the dark and under conditions of temperature, salinity, and oxygen suggested by Stone et al. (Dis Aquat Organ, 2000, 41: 141-149) for 24 days. Turnover and filtration was performed manually every 48 hours. At 24 days, fish were anesthetized and killed for parasite counting under a stereomicroscope. The results in Table 1 show a reduction in infestation levels in the groups treated with PACAP, my32 and PACAP+my32. The largest reduction occurred in groups 4 and 5, which shows the adjuvant effect of PACAP on the antigen.

(35) TABLE-US-00001 TABLE 1 Count of parasites 24 days post-challenge with C. rogrecresseyi Number of Inhibition of the Experimental groups parasites/fish infestation (%) PBS 37 3.sup.a PACAP 29 5.sup.b 22 my32 17 8.sup.c 55 PACAP-IP + my32-IP 8 3.sup.d 79 PACAP-oral + my32-IP 5 2.sup.e 86 Letters indicate statistical significant differences Percent of inhibition of infestation calculated as (1 T/C) 100 (T: parasite number per fish in vaccinated group, C: parasite number per fish in placebo group).

Example 6

PACAP Adjuvant Effect on the Humoral Immune Response in Common Carp (Cyprinus Carpio) Versus Aeromonas Hydrophila

(36) The experiment was performed with carp (C. carpio) 4010 g. These aquariums were maintained in 600 L at a temperature of 282 C. Three experimental groups of 10 carps each one were established and they were injected intraperitoneally with the following immunogens:

(37) Group 1: PBS

(38) Group 2: Inactivated cells of A. hydrophila

(39) Group 3: Inactivated cells of A. hydrophila plus 1 g per fish of PACAP38.

(40) The fish were injected on day 0 and 14, and blood was collected from the caudal vein on days 0 and 21. The results showed that agglutinating antibody titers were significantly higher in the group immunized with the bacterium plus PACAP compared with the group immunized with the bacteria alone (FIG. 6). These results demonstrate the effect of PACAP as a molecular adjuvant. The cell preparation of A. hydrophila and the antibody titers measurements were made according to Yin et al. ((1996) Fish & Shellfish Immunology 6, 57-69).

Example 7

Controlled Challenge Test with A. Hydrophila in Common Carp (C. Carpio) Previously Immunized Intraperitoneally with Inactivated Bacteria and Inactivated Bacteria Co-Administered with PACAP

(41) The experiment was performed with common carp (C. carpio) 305 g. The fish were maintained in 250 L aquariums at a temperature of 302 C. Three experimental groups were established with 20 carps each one, which were injected intraperitoneally.

(42) Group 1: PBS

(43) Group 2: Inactivated cells of A. hydrophila

(44) Group 3: Inactivated cells of A. hydrophila plus 1 g of PACAP38 per fish.

(45) The fish were injected on day 0 and 14. On day 21 the challenge was performed by injecting IP LD.sub.50 of the bacterium and the mortality was recorded during 7 days. We calculated the relative percent survival (RPS) as:
RPS(%)=(% mortality controls% mortality-treated)/(% mortality controls)100

(46) The result was 65% in the Group 2 and 95% in the Group 3, which demonstrates that administration of PACAP increases the survival rate of vaccinated and challenged fish to the pathogen.

Example 8

Effect of the Co-Administration of PACAP with a DNA Vaccine Based on the Gene Encoding the 106 kDa Polyprotein (VP2-VP4VP3-NH2-COOH) of IPNV, in the Rainbow Trout (Oncorhynchus Mykiss) Experimentally Challenged with this Virus

(47) An experiment was conducted to evaluate the effect of the intramuscularly co-administration of PACAP with a DNA vaccine based on the gene encoding the 106 kDa polyprotein (VP2-VP4VP3-NH2-COOH) of the IPNV in rainbow trout challenged experimentally with this virus. Six experimental groups were conformed of 15 fish each one (121 g) and were kept in water at 10-12 C.:

(48) Group 1: Fish injected with PBS.

(49) Group 2: Fish injected with the DNA vaccine (pP-IPNV) at a dose of 1 g/fish.

(50) Group 3: Fish co-injected with the DNA vaccine (pP-IPNV) at a dose of 1 g/fish and a plasmid having the cDNA sequence of C. gariepinus PACAP under the control of the immediate early promoter of the human cytomegalovirus (pCMV-PACAP) at a dose of 0.5 g of pCMV-PACAP/fish.

(51) Group 4: Fish co-injected with pP-IPNV vaccine (1 g/fish) with a negative control plasmid at a dose of 0.5 g pCMV/fish.

(52) Group 5 Fish co-injected with the DNA vaccine (pP-IPNV) at a dose of 1 g/fish and the plasmid having the cDNA sequence of C. gariepinus PACAP (pCMV-PACAP) at a dose of 0.05 g of pCMV-PACAP/fish.

(53) Group 6: Fish co-injected with pP-IPNV vaccine (1 g/fish) with a negative control plasmid at a dose of 0.05 g pCMV/fish.

(54) At 30 days post-vaccination, the fish were exposed to the virus by intraperitoneal injection of 100 L of IPNV (110.sup.7 TCID.sub.50 mL.sup.1 per fish).

(55) At day 7 post-infection, 10 fish were sacrificed per group and the viral load was assessed in head kidney.

(56) To determine the viral load, RNA was isolated from individual samples and it was performed the RT-PCR from 1 g of RNA. Detection of VP1 gene expression was assessed by real time PCR. The results are shown in FIG. 7.

(57) Co-administration of pP-IPNV and pCMV-PACAP decreased the viral load significantly, compared with the group that was co-injected with pP-IPNV and empty vectors. There were no differences between the doses of pCMV-PACAP tested.

Example 9

Effect of the Co-Administration of PACAP with a DNA Vaccine Based on the Gene Encoding the G Glycoprotein of VHSV, in Rainbow Trout (O. mykiss) Experimentally Challenged with Said Virus

(58) An experiment was conducted to evaluate the effect of the co-administration by intramuscular injection of C. gariepinus PACAP with a DNA vaccine based on the gene encoding the G glycoprotein of VHSV (pG-VHSV) in rainbow trout experimentally challenged with this virus. Four experimental groups were formed, each of 20 fish (102 g) and were kept in water at 10-12 C.:

(59) Group 1: Fish injected with PBS.

(60) Group 2: Fish injected with the DNA vaccine pG-VHSV (0.01 g/fish).

(61) Group 3: Fish co-injected with the DNA vaccine pG-VHSV at a dose of 0.01 g/fish and C. gariepinus PACAP at a dose of 0.1 g of PACAP/fish.

(62) Group 4: Fish co-injected with the DNA vaccine pG-VHSV at a dose of 0.01 g/fish and C. gariepinus PACAP at a dose of 0.5 g of PACAP/fish.

(63) At 4 weeks post-vaccination the fish were exposed to the virus by immersion baths. The challenge was performed for 2 h in water containing a VHSV infective dose (110.sup.5 TCID.sub.50 per fish mL.sup.1). Cumulative mortality was assessed 4 weeks post-challenge and the results are shown in FIG. 8.

(64) The co-administration of the peptide with the DNA vaccine reduced the mortality at 15% and 29% compared with the administration of the DNA vaccine alone, at the doses of 0.1 and 0.5 g of PACAP/fish, respectively.

Example 10

Effect of the Administration of PACAP on Survival of Channel Catfish (Ictalurus Punctatus) after Immunization with Theronts of Ciliated Parasite Ichthyophthirius Multifiliis

(65) An experiment was conducted to evaluate the effect of PACAP administration, after immunization with ciliated parasite theronts I. multifiliis, on survival of channel catfish (I. punctatus). The vaccination and challenge procedure was performed as proposed by Wang and Dickerson ((2002) Clinical and Diagnostic Laboratory Immunology 9 (1), 176-181). Four experimental groups of 25 fish (125 g) each one were established:

(66) Group 1: Fish immunized on days 1 and 35 with PBS.

(67) Group 2: Fish immunized on day 1 with 8000 live theronts I. multifiliis and at day 35 with 10,000 living theronts parasite.

(68) Group 3: Fish immunized on day 1 with 8000 live theronts I. multifiliis and at day 35 with 10,000 living theronts parasite. These fish also received by immersion baths the neuropeptide PACAP at a dose of 100 g of PACAP/L of water. The treatment was performed for 1 hour, on alternate days, in the two weeks prior to challenge (six immersion baths).

(69) Group 4: Fish immunized on day 1 with 8000 live theronts I. multifiliis and at day 35 with 10,000 living theronts parasite. These fish were also subjected to immersion baths for 1 hour without PACA, by a procedure similar to Group 3.

(70) Prior to the immunizations fish were treated with formalin to remove existing ectoparasites. The fish were maintained at 232 C. and continuous water flow. At day 84, the fish were challenged with 15 000 theronts. Mortality data were obtained within 30 days after challenge. The results are shown in Table 2. These results show a 27% increase in survival of the immunized fish which was simultaneously treated with PACAP.

(71) TABLE-US-00002 TABLE 2 Survival of I. punctatus immunized and challenged with live theronts of I. multifiliis, after treatment with PACAP by immersion baths. Challenged fish/ Experimental groups surviving fish Survival (%) PBS 25/0 0 Live theronts 25/15 60 Live theronts + PACAP ib 25/22 87 Live theronts + ib 25/14 56 ib: These fish were subjected to 6 immersion baths for 1 hour, on alternate days in the two weeks prior to challenge