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ANIMAL PRODUCTION |
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* Department of VMPM, College of Veterinary Medicine, Iowa State University, Ames 50011;
and
ADViSYS, Inc., The Woodlands, Texas 77381
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: insulin-like growth factor I • pig • plasmid • somatoliberin • vaccination
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Recent research has demonstrated that numerous hormones affect immune function, including those of the GHRH/GH/IGF-I axis. Recent studies have demonstrated that both GHRH (Marshall et al., 2001; Siejka et al., 2004) and GH have immunomodulatory properties (Koo et al., 2001; Bozzola et al., 2003; Siejka et al., 2004). The importance of GHRH in the modulation of immune status under physiological and pathological conditions (Marshall et al., 2001) has been described, both through stimulation of the GH/IGF-I axis and directly as an immune modulator (Dialynas et al., 1999; Khorram et al., 2001). However, the mechanisms involved with GHRH mediating those effects or the impact of GHRH treatment on vaccination and pathogen challenge remain elusive.
The 3 pathogens commonly isolated from pigs exhibiting clinical disease and pneumonia consistent with porcine respiratory disease complex are porcine reproductive and respiratory syndrome (PRRS) virus, swine influenza virus and Mycoplasma hyopneumoniae (M. hyo). Mycoplasma hyopneumoniae, the causative agent of enzootic pneumonia (Stipkovits et al., 1991) induces a mild, chronic pneumonia complicated by other opportunistic bacterial infections. Pigs infected with both PRRS virus and M. hyo exhibit increased respiratory disease and prolonged viral pneumonia (Thacker et al., 1999). In addition, the presence of PRRS virus significantly reduced the efficacy of M. hyo vaccines (Thacker et al., 2000b).
This study evaluated the effect of 0.625 mg of a porcine GHRH-expressing plasmid (Draghia-Akli et al., 2003) on the clinical outcome of pigs vaccinated against M. hyo and challenged with M. hyo and/or PRRS virus. Our study showed that constitutive GHRH expression reduced the clinical disease associated with M. hyo infection as demonstrated by improved clinical scores and outcome of treated animals.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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The investigators were blinded to the identification of the plasmid treatment group. On study d 42, one animal died of apparent heart disease/vitamin E/selenium deficiency. To prevent further loss of pigs due to vitamin E deficiency, 3 mL of Vital E (Schering-Plough Animal Health, Union, NJ) was administered i.m. to all pigs in the right side of the neck.
Intramuscular Injection of Plasmid DNA
The plasmid pSP-wt-GHRH (Figure 1B
) contains a 360-bp SacI/BamHI fragment of the SPc5-12 synthetic promoter (Li et al., 1999). The porcine GHRH cDNA (1–40)OH was obtained by site-directed mutagenesis of porcine GHRH cDNA (1–44)OH using the Altered Sites II in vitro Mutagenesis System (Promega, Madison, WI) as described by Draghia-Akli et al. (1999). The GHRH cDNA is followed by the 3' untranslated region of GH. The plasmid was depleted of CpG sequences. Of the remaining motifs, not only are more than half nonimmunostimulatory, but they may even suppress the effects of the immunostimulatory CpG (so-called CpG-N motifs, including CCG, CGG, and CGCG sequences; reviewed in Krieg, 1999). The specific effect, if any, of the CpG motifs on the biological activity of the plasmid is, thus, unlikely.
The endotoxin-free plasmid (ADViSYS, Inc., The Woodlands, TX, USA) preparation of pSP-wt-GHRH was diluted in water to 2.5 mg/mL and formulated with 1% wt/wt poly-L-glutamate. Pigs were lightly anesthetized intramuscularly with ketamine (1.1 mg/kg) and telazol (2 mg/kg). A total of 0.625 mg of plasmid in a volume of 2.0 mL of water for treated animals, or 2.0 mL of saline for controls, was injected intramuscularly into the semimembranosus muscle. Eighty seconds after the injection, the injected muscle was electroporated using the ADViSYS constant current electrokinetic device, 5 pulses, 1 Amp, 52 milliseconds/pulse, as previously described (Draghia-Akli et al., 2002). To maintain a constant current, the voltage changes between 80 and 120 V/cm in response to varying tissue resistance during the electroporation procedure. For all injections, 2-cm needles were inserted through the skin into the muscle. Animals were observed immediately after injection and 24 h later for any adverse effects at the electroporation site.
Challenge
Pigs were challenged 2 wk after the second vaccination with a tissue homogenate (LI35) containing strain 232, a derivative of pathogenic M. hyo strain 11 [105 color changing units (CCU)/mL] at a dilution of 1:100 in 10 mL of mycoplasma Friis medium. The inoculum was administered intratracheally to pigs, as previously described (Thacker et al., 1998).
An inoculating dose of 1 x 104.8 of the 50% tissue culture infective dose of a high virulence PRRS virus strain, VR2385, was administered intranasally in 5 mL of minimum essential medium to pigs in the appropriate groups, as previously described (Halbur et al., 1995).
Clinical Evaluation
Pigs were weighed on d 3, and subsequently on d 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 to evaluate both BW differences by group and BW gain as measured by ADG. The ADG was determined for 3 intervals in the trial and included the interval from arrival at Iowa State University to M. hyo challenge (ADG-C), between M. hyo challenge and the first necropsy (ADG-NX1), and between the first and second necropsies (ADG-NX2).
On trial d 14, 21, and 28 of the study and daily for 10 d after challenge, respiratory and coughing scores and rectal temperatures were determined as described by Halbur et al. (1995). At each time point, each pig was evaluated for a period of at least 15 min for signs of clinical disease including changes in appetite, coughing score, respiration rate, or behavior. Rectal temperatures were measured daily for 10 d postinoculation. Respiratory scoring was performed according to published criteria (Halbur et al., 1995): 0 = normal, 1 = mild dyspnea and/or tachypnea when stressed, 2 = mild dyspnea and/or tachypnea when at rest, 3 = moderate dyspnea and/or tachypnea when stressed, 4 = moderate dyspnea and/or tachypnea when at rest, 5 = severe dyspnea and/or tachypnea when stressed, or 6 = severe dyspnea and/or tachypnea when at rest. In addition, coughing scores were recorded as 0 = no coughing or 1 = coughing. For each pig, the aggregate daily respiratory scores and the number of days coughing were calculated for the 10 d following challenge and for the entire study.
Necropsy
Necropsies were performed on 6 pigs from each group on trial d 53 and the remaining pigs on d 70. Tracheal swabs were collected aseptically for bacterial culture and isolation of M. hyo. The lungs were removed and bronchioalveolar lavage (BAL) fluid obtained by washing the bronchi with 50 mL of minimal essential medium containing antibiotics (9 µg of gentamicin/mL, 100 U of penicillin G/mL, and 100 µg of streptomycin/mL; Mengeling et al., 1995). The lungs were evaluated for macroscopic lesions. Lesions consistent with M. hyo were sketched on a standard diagram and assessed for the proportion of lung surface exhibiting lesions using a Zeiss SEM-IPS image analyzing system (Thacker et al., 1998). Macroscopic lesions consistent with PRRS virus were scored as described by Halbur et al. (1995).
A portion of each lung lobe was collected and processed for histopathological examination. Tissue was fixed in 10% neutral buffered formalin, processed, and embedded in paraffin using an automated tissue processor. Histopathologic lesions were scored as previously described (Thacker et al., 1999). Lung sections were examined and given a score (0 to 4) for peribronchiolar and perivascular lymphoid cuffing and nodule formation consistent with M. hyo-induced pneumonia lesions. Microscopic lesions consistent with PRRS virus were scored as described by Halbur et al. (1995). The severity of interstitial pneumonia lesions was scored (0 to 6). The examiner was blinded to the treatment group identification. Mycoplasmal antigens were detected in frozen tissues using a fluorescent antibody assay (Amanfu et al., 1984).
Serology and M. hyo-Specific Antibodies in BAL
Serum was collected from all pigs upon arrival, before challenge, and at necropsy. The M. hyo antibody levels were determined using a Tween-20 ELISA (Bereiter et al., 1990). Known positive and negative sera supplied by the reference laboratory were included as controls in each plate. Readings >2 SD above the mean value of the negative control were considered positive (optical density >0.22). All sera were tested for PRRS virus antibodies using a commercially available ELISA (HerdChek: PRRS virus; IDEXX Laboratories, Westbrook, Maine) according to the procedures provided by the manufacturer. Samples were considered positive if the calculated sample-to-positive control ratio was 0.4 or greater.
The M. hyo-specific antibodies in BAL were measured by ELISA as previously described (Thacker et al., 2000a). Plates were coated with a membrane preparation of M. hyo clone 232-2A3. The M. hyo-specific antibody isotype was determined using peroxidase-labeled goat anti-swine immunoglobulin A (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD), immunoglobulin G, and immunoglobulin M (Bethyl Laboratories, Inc., Montgomery, TX), all heavy-chain specific.
Hematology
Before plasmid administration and at the first necropsy, whole blood was collected in Microtainer brand tubes with EDTA (Becton Dickinson, Franklin Lakes, NJ) for complete blood count analysis (Iowa State University, Department of Veterinary Pathology).
IGF-I Radioimmunoassay
Serum was aliquoted and stored at –80° C until analysis. All samples were analyzed in a single assay using a heterologous human IGF-I kit (Diagnostic Systems Laboratories, Inc., Webster, TX). The cross-reactivity between the human and porcine IGF-I was 100%. The intraassay variability was 3.6%.
Statistical Analysis
Data were analyzed by an independent statistician (Synergos, Inc., The Woodlands, TX), and reviewed by the investigators. Data were analyzed using SAS (SAS Inst., Inc., Cary, NC) to examine if there were any significant differences among the groups for each variable at different time points. Comparisons were adjusted by the Wilcoxon Rank-Sum Test or Fisher’s Exact test. Mean values were compared with Students t-test, ANOVA, or linear regression, with < 0.05 taken as the level of statistical significance. Rectal temperatures were also analyzed using area under the curve (AUC) to compare the extent and duration of fever, using the trapezoid rule.
Antibodies.
The antibody levels were compared by ANOVA between the experimental groups at each time point. Additionally, an AUC analysis was performed to determine if the rate at which the antibodies were produced was different between the experimental groups. The antibody AUC was compared between all of the experimental groups using ANOVA.
Lung Lesions.
The proportion of lung surface affected by lesions at the time of necropsy was compared for each group using ANOVA. Pair-wise comparisons between the experimental groups of interest were made using a 2-sample t-test to examine the effect of the GHRH plasmid and vaccine.
Coughing.
The number of coughs per pen per 5 min (cough scores) was compared between the experimental groups using ANOVA. Additionally, pair-wise comparisons between the experimental groups of interest were made using a 2-sample t-test to examine the effect of the GHRH plasmid and vaccine. The proportion of pens with worsening counts from prechallenge at any time postchallenge was also compared between the experimental groups. Fisher’s Exact test was used to examine the effect of the GHRH plasmid and vaccine.
Rectal Temperature.
Rectal temperature was analyzed using an AUC > 40° C using ANOVA. Additionally, the experimental groups were compared in a pair-wise fashion using a 2-sample t-test to examine the effect of the GHRH plasmid and vaccine. The proportion of animals that experience fevers at any time postchallenge was also compared between the experimental groups using the Fisher’s Exact test. The duration of fevers was defined as the number of days from the first temperature measurement > 40° C to the last temperature measurement > 40° C and was compared using ANOVA.
Respiratory Disease.
For each animal, the total postchallenge respiration score was calculated by adding the scores from each daily assessment and compared between the experimental groups using ANOVA. Additionally, pair-wise comparisons between the experimental groups of interest will be made using a 2-sample t-test to examine the effect of the GHRH plasmid and vaccine and plasmid/vaccination schedule. The duration of respiratory disease was also examined by ANOVA.
Body Weight.
The values and percentage change from prechallenge in BW was compared between the experimental groups at each time point by ANOVA, and pair-wise comparisons between the experimental groups of interest were made using a 2-sample t-test. Additionally, an AUC analysis was performed to determine if the rate at which BW is gained was different between the experimental groups.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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No differences were observed in BW or ADG between the treatment groups within each challenge/treatment group at any time. In addition, ADG was determined when all challenge/treatment groups were combined and groups differed by only the plasmid treatment. In this case, the treatment improved ADG-NX1 (between M. hyo challenge and the first necropsy), 0.65 ± 0.03 kg/d in plasmid GHRH-treated group, versus 0.54 ± 0.04 kg/d in controls (P = 0.04; Table 2).
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). The AUC analysis of rectal temperatures was borderline significant (0.4 ± 0.5 in MHp versus 1.2 ± 1.4 in MHs, mean ± STD). The median AUC for the MHp group was 0.1 degrees * d, whereas the median AUC for the MHs group was 0.7 degrees * d. At the end of the study, the difference in the proportion of pigs that experienced fever was greater in the MHs group than in the MHp plasmid-treated pigs (P < 0.05, Fisher’s Exact Test).
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None of the pigs experienced symptoms of respiratory disease before being challenged. More severe respiratory disease occurred in all PRRS virus-challenged pigs. No statistically significant differences were observed among any of the PRRS virus-challenged pigs. After challenge with M. hyo, 55.6% (5/9) in MHs experienced respiratory problems, as compared with 22.2% (2/9) in the MHp. At the end of the observation period, 33.3% (3/9) in the MHs group still had respiratory problems, whereas none of the pigs in the MHp group had any respiratory problems. Overall respiratory score was reduced in the MHp group as compared with the MHs group, (0.22 ± 0.15 in MHp versus 1.1 ± 0.48 in MHs, P = 0.1; Table 3).
As previously demonstrated, the groups infected with both pathogens had significantly increased pneumonia at each time point compared with the groups challenged with only one pathogen (Table 4). Macroscopic lesions associated with only M. hyo (Figure 2) were similar between the challenged groups at the first necropsy. At the second necropsy, the number of visible lung lesions was 0.95 ± 0.34 in MHp versus 2.3 ± 0.88 in MHs (P = 0.24) due to some interanimal variability. No significant differences were found in the groups challenged with PRRS virus, although PRRS-MHp (plasmid) had less pneumonia than PRRS-MHs, 1.5 ± 0.47 compared with 4.4 ± 1.6 (P = 0.17) at the second necropsy.
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Hematology, Serology, and M. hyo-Specific Antibodies in BAL
Hematological endpoints were within the normal range for pigs, and no differences were observed between groups. Both the GHRH-treated groups and controls showed a significant increase in white blood cells and circulating lymphocytes from the baseline values before treatment, vaccination, and challenge (baseline—white blood cells: 12.83 ± 0.48 for the GHRH-treated and 12.67 ± 0.66 for saline-treated; lymphocytes: 6.76 ± 0.3 for the GHRH-treated and 6.5 ± 0.32 for saline-treated) to necropsy (white blood cells: 19.73 ± 0.67 for the GHRH-treated and 19.6 ± 0.54 for saline-treated; lymphocytes: 11.84 ± 0.47 for the GHRH-treated and 12.13 ± 0.34 for saline-treated). Pigs in both groups vaccinated with the mycoplasma bacterin seroconverted by challenge day and remained seropositive for the remainder of the trial. Until the second vaccination, the antibody titer was similar between the test groups. However, after the second vaccination, M. hyo antibodies were greater in the MHp (plasmid) group than the MHs (saline) group: 0.43 ± 0.06 in MHp group versus 0.29 ± 0.05 in MHs (P = 0.1). No differences in M. hyo antibody levels were observed between the groups challenged with both pathogens although as with the groups challenged only with M. hyo, antibody levels tended to be greater in the group that received the plasmid (PRRS-MHp). Following challenge, the antibody levels between groups reached a plateau at the same level (Figure 3).
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All groups challenged with M. hyo had greater levels of M. hyo-specific IgG and IgA in the BAL at both necropsies. An increase in IgG levels in BAL was observed in plasmid-treated pigs at the second necropsy (Table 5): 0.88 ± 0.04 in MHp vs. 0.69 ± 0.05 in MHs (P < 0.03). In the groups infected with M. hyo, all but one pig in each group was positive for antigen as detected by ELISA assay.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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GHRH can be delivered as recombinant protein and can be used for immunomodulation purposes in conjunction with vaccination. However, this method is not practical, due to the short half-life (6 to 12 min) of the GHRH molecule in vivo (Evans et al., 1985; Thorner et al., 1986) and lack of orally bioavailable formulations. Intravenous or subcutaneous administration of the recombinant GHRH 1 to 3 times per day would be required. A plasmid-mediated approach can overcome this primary limitation of recombinant GHRH therapy (Draghia-Akli and Fiorotto, 2004). Earlier animal studies suggest that a single plasmid injection into the animal’s skeletal muscle, followed by electroporation to enhance uptake (Muramatsu et al., 2001), could ensure transgene expression for a substantial length of time (Tone et al., 2004).
Plasmid-mediated GHRH supplementation has been shown to have a variety of immunostimulatory effects in animals with depressed immune systems due to illness or various treatment regimens (Dorshkind and Horseman, 2001). Studies indicate that cells of the immune system produce GHRH, GH, and IGF-I (Burgess et al., 1999), suggesting that immune function might be regulated through autocrine and paracrine mechanisms. It has also been suggested that increased morbidity in the elderly associated with respiratory disease may be due to the age-related decreases in GH/IGF-I production and reduced IGF-I availability (Gelato, 1996; Krishnaraj et al., 1998). Conversely, administration of GHRH or its analogs to the elderly resulted in short- and long-term immuno-enhancing effects, with increased number of lymphocytes, monocytes, B-cells as well as 
ß and 
T-cells (Khorram et al., 1997). Similar findings have been demonstrated in animal models of disease and vaccination, showing that in vivo administration of GH can enhance the phagocytic activities of macrophages and increase resistance to pathogens (Sakai et al., 1997).
Recently, we determined that plasmid-mediated GHRH supplementation resulted in positive changes in the immune system in a large animal model (i.e. pregnant cows; Brown et al., 2004). The GHRH-treated animals had increased numbers of CD2+ ß T-cells and increased CD25+CD4+ and CD4+CD45R+ cells compared with controls. These increases were maintained long-term after treatment and correlated with plasmid expression. At 300 d postGHRH therapy, CD45R+/CD45R0-naïve lymphocytes were significantly increased in frequency. Natural killer lymphocytes were also increased. As a consequence of improved health status, BCS of treated animals improved significantly, and morbidity and mortality of heifers were decreased. However, the animals in this experiment were not studied after specific vaccination or pathogenic challenges (Brown et al., 2004).
The IGF-I levels were increased by 5 to 9% in GHRH-treated animals compared with controls (not statistically significant) and within the normal range in this study. These results are consistent with previous studies in large mammals (Brown et al., 2004; Tone et al., 2004) and not unusual. Low-dose GH treatment used in recent human studies also induces normal IGF-I levels with similar increases, and thus physiological changes, compared with high-dose therapy. Low-dose therapy has been associated with similar positive clinical changes as the classic high dose regimens, but a longer treatment time is usually necessary (Abrahamsen et al., 2004).
Previous studies using this vaccination-infection model found that the differences between BW and ADG of the treated and control pigs were small (Thacker et al., 1999; Thacker et al., 2000b). The results of this study were consistent with earlier studies, and no significant differences among the average BW of the treatment groups were observed. When the BW data of all groups were pooled and evaluated, a significant difference in ADG between challenge and the first necropsy was observed with the plasmid-treated groups having a significantly greater ADG than the saline-treated groups. This finding is significant because it indicates that the plasmid-treated pigs maintained a better growth curve despite the pathogenic challenge. These findings also correlate to the slight decrease in days with fever and AUC for fever for the M. hyo-challenged animals. These results support previous findings that suggest increased GHRH levels may maintain anabolic processes even in acute and chronic phases of critical illness (Van den, 2003).
It is difficult to isolate the actual impact of the apparent vaccine reaction that occurred following the second M. hyo vaccination. The percentage of pigs challenged only with M. hyo with pneumonia observed at both necropsies suggests that this adverse reaction might have reduced vaccine efficacy. However, the percentage of the lung surface with pneumonia consistent with M. hyo infection in the groups infected with both pathogens was significantly greater than the group challenged only with M. hyo. These results, combined with other M. hyo studies in which the percentage of pneumonia is < 1%, suggest that the mycoplasma vaccine was efficacious (Thacker et al., 1998, 2000a). On average 3% pneumonia was observed in this particular trial. Groups of nonvaccinated M. hyo-infected pigs were not included in this study. Therefore, we compared plasmid treatments within challenge/vaccinated groups. Nevertheless, the study was informative regarding the effect of plasmid treatment within the groups. The average percentage of M. hyo lung lesions was decreased in GHRH-treated animals from the first necropsy to the second. Whereas the improvement did not reach statistical significance, the median percentage of lesions in the saline-treated group was the same at each time point and did not exhibit any reduction in the percentage of pneumonia.
Respiratory disease remains a significant problem in both humans and animals (Thibodeau and Viera, 2004). In addition, an increase in community-acquired pneumonias, with otherwise less common pathogens such as Mycoplasma pneumoniae (M. pneumoniae), has been noted in the elderly, young, or immunodeficient patients (Kobashi et al., 2001). Recent studies have found the prevalence of M. pneumoniae in adults with pneumonia to range from 1.9 to > 30% (Hammerschlag, 2001). Thus, better animal models are needed for the evaluation of vaccination, immune stimulation, and treatment modalities (Maes et al., 1996).
The efficacy of the plasmid in reducing pneumonia was further supported by the reduction in respiratory scores and coughing scores observed in the GHRH-treated animals challenged with M. hyo. The pigs in the plasmid-treated group experienced a median of only 1 d of coughing, whereas controls coughed for a median of 3 d. There was a reduction in the AUC temperature of M. hyo-challenged pigs as well as the number of days treated pigs had rectal temperatures > 40°C. However, fever is not typically observed in association with M. hyo infection, so the biological significance of this finding in the context of this particular model is unclear. Other studies with GHRH-plasmid in conjunction with other pathogens may be necessary to elucidate this point. Plasmid treatment did not appear to have a significant influence on the clinical disease associated with PRRS virus. However, it may be that the immunomodulatory effects of GHRH are more directed toward the immune response to bacterial pathogens than a viral pathogen that infects macrophages, or that the treatment better mitigates challenge in conjunction with specific vaccination.
Interestingly, microscopic scores in the saline-treated group had lower lesion scores than the plasmid-treated groups, specifically in the PRRS virus-infected groups. Again, this suggests a strong impact on bacterial pathogens as compared to a viral agent. This may have reduced our ability to observe statistical significance, and future studies will include larger numbers of animals in each group.
Differences between groups were observed in serum antibodies to M. hyo and local antibodies in the respiratory tract specific for M. hyo. Usually, the systemic serum IgG against M. hyo is evoked after a second immunization in vaccinated pigs. Whereas it is important that the circulating antibody levels were increased before challenge in the GHRH-treated group, there is no correlation between serum antibodies against M. hyo and protection against disease (Thacker et al., 2000a). Local humoral immunity appears to play a role in controlling the disease associated with M. hyo infection (Sarradell et al., 2003). In the study reported here, IgG levels in the BAL fluid were increased by the second necropsy and appeared to be correlated with reduced clinical scores in the plasmid-treated animals. Other studies in aging humans have shown that GHRH analog treatments increased T- and B-cells and their responsiveness to antigens (Khorram et al., 1997). The observed increase in circulating and local antibodies might have allowed the GHRH-treated group to respond more effectively to the challenge, which might explain the increased cellular infiltrate in the airway, increasing immune responsiveness, and decreased clinical pathology in this group.
The results of this study suggest that plasmid-based GHRH supplementation before vaccination may enhance the protection against M. hyo-induced pneumonia, and that a single dose of GHRH-expressing plasmid is able to elicit an improved clinical outcome in this animal model of disease challenge.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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3 Corresponding author: ruxandradraghia{at}advisys.net (regarding GHRH technology).
2 Corresponding author: ethacker{at}iastate.edu (regarding Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus models).
Received for publication December 14, 2004. Accepted for publication October 26, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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