Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

Chicken-type lysozyme in channel catfish: expression analysis, lysozyme activity, and efficacy as immunostimulant against Aeromonas hydrophila infection.

To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with A. hydrophila by bath immersion. Quantitative PCR revealed that the transcription levels of Lys-c in infected catfish were significantly (P < 0.05) induced in all five tissues tested as well as in blood cells. Recombinant CC-Lys-c produced in Escherichia coli expression system (R-CC-Lys-c) exhibited significant (P < 0.05) lytic activity to Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-c (pcDNA-Lys-c) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-c offered significant (P < 0.05) protection to G1B against A. hydrophila infection. When channel catfish were intraperitoneally injected with QCDCR adjuvant formulated pcDNA-Lys-c and challenged with a highly virulent A. hydrophila strain AL-09-71 at 1-, 2-, 14-, and 28-days post treatment, pcDNA-Lys-c offered 75%, 100%, 60%, and 77% protection to channel catfish, respectively. Macrophages of fish treated with pcDNA-Lys-c produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish treated with pcDNA vector alone. Taken together, our results suggest that pcDNA-Lys-c could be used as a novel immunostimulant to protect channel catfish against A. hydrophila infection.

Evaluation of an alternative dosing regimen of a J-5 mastitis vaccine against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows.

The objective of the study was to evaluate the efficacy of an alternative vaccination regimen of a J-5 bacterin against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows. The parameters analyzed to assess the effect of vaccination were milk yield, milk conductivity, somatic cell count, J-5-specific serum IgG titers, and clinical signs. Twenty multiparous Holstein cows from the Cornell teaching and research dairy herd were paired by days in milk and were randomly selected to receive either the alternative off-label regimen of commercial J-5 bacterin or act as nonvaccinated controls. Cows received a first dose of bacterin 15 d before dry off, a second dose with the same product at the day of dry off, and the third dose 2 wk after dry off. The cows in both groups were challenged 10 d before the expected calving date. Serum IgG (total, IgG1 and IgG2) levels were higher in vaccinates compared with control cows. Eighty-five percent of challenged quarters became infected between both groups of animals. Eight of the 10 vaccinated and 9 of the 10 control cows showed signs of clinical mastitis postfreshening. A non-severe clinical mastitis was observed 24 to 48 h postparturition, characterized by flakes or clots in milk and mild swelling or pain. Off-label vaccination did reduce the clinical severity of clinical mastitis in the vaccinated group of cows as evidenced by reduced California mastitis test score, fewer flakes and lower overall clinical mastitis score. No significant differences between vaccinated and control groups were detected for rectal temperature. In conclusion, the alternative off-label vaccination scheme used in our study and evaluated in a novel E. coli challenge model did not prevent new intramammary infections but reduced clinical severity of experimentally induced E. coli mastitis.

Evaluation of novel adjuvant Eimeria profilin complex on intestinal host immune responses against live E. acervulina challenge infection.

The effects against avian coccidiosis of two novel adjuvants, Quil A/cholesterol/dimethyl dioctadecyl ammonium bromide/Carbopol (QCDC) and QCDC/Bay R1005 (R)/cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides (CpG ODN [T]) (QCDCRT) emulsified with profilin, a conserved Eimeria recombinant protein, were determined in broiler chickens. Chickens were subcutaneously immunized with isotonic saline (control group), profilin (P), profilin emulsified with QCDC (P-Q), or profilin with QCDCRT (P-QR) at 2 and 9 days post-hatch and orally challenged with 1.0 x 10(4) sporulated oocysts of Eimeria acervulina (EA) at 7 days postimmunization. All profilin-immunized groups showed increased body weight gain when compared to the control group, and the P-QR group had significantly higher body weight gain than did those of the P and P-Q groups following EA challenge infection. All groups immunized with profilin showed significantly decreased intestinal lesions compared with the control group, with the P-QR group showing the lowest intestinal lesions among the profilin-treated groups. Finally, the P-QR group showed greater CD4+/CD8+ and TCR1+/TCR2+ splenocytes and higher antiprofilin serum antibody titers compared with the P and P-Q (or both) groups following EA challenge infection. These results further suggest that vaccination of chickens with profilin, in combination with the QCDCRT adjuvant, may provide a novel control strategy against EA infection in commercial flocks.

Effects of novel vaccine/adjuvant complexes on the protective immunity against Eimeria acervulina and transcriptome profiles.

SUMMARY. This study investigated the ability of two novel adjuvant formulations, QCDC (Quil A/cholesterol/DDA/ Carbopol) and QCDCR (QCDC/Bay R1005), in combination with a recombinant profilin vaccine, to modulate host protective immunity and to alter gene expression during experimental avian coccidiosis. Vaccination with profilin plus QCDCR significantly reduced the severity of intestinal lesions and increased mitogen-induced lymphocyte proliferation in infected chickens compared with immunization with profilin alone or profilin plus QCDC. Immunization with profilin plus QCDC or profilin plus QCDCR increased body weight gain but had no effect on fecal oocyst shedding of chickens infected with Eimeria acervulina compared with birds vaccinated with profilin alone. The results of global gene expression analysis revealed that, compared with PBS controls, (a) chickens vaccinated with profilin alone had 71 up-regulated and 56 down-regulated mRNA transcripts, (b) chickens immunized with profilin plus QCDC had 198 up-regulated and 247 down-regulated mRNAs, and (c) birds immunized with profilin plus QCDCR had 210 up-regulated and 267 down-regulated mRNAs. Compared with birds vaccinated with profilin alone, (a) chickens given profilin plus QCDC had 60 up-regulated and 104 down-regulated transcripts and (b) chickens immunized with profilin plus QCDCR had 103 up-regulated and 130 down-regulated mRNAs. Finally, chickens vaccinated with profilin plus QCDCR had 193 up-regulated and 204 down-regulated transcripts compared with birds given profilin plus QCDC. Biological function and network analysis revealed that the majority of altered transcripts were encoded by immune-related genes.

Efficacy of QCDCR formulated CpG ODN 2007 in Nile tilapia against Streptococcus iniae and identification of upregulated genes.

The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P<0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P<0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity.

Embryo vaccination of chickens using a novel adjuvant formulation stimulates protective immunity against Eimeria maxima infection.

Our previous study demonstrated that chickens immunized subcutaneously with an Eimeria recombinant profilin protein vaccine emulsified in a Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant developed partial protection against experimental avian coccidiosis compared with animals immunized with profilin alone. Because in ovo vaccination is presently used in commercial applications worldwide throughout the poultry industry, the current study was undertaken to investigate chicken embryo vaccination with profilin plus QCDC adjuvant. Eighteen day-old embryos were immunized with isotonic saline (control), profilin alone, QCDC alone, or profilin plus QCDC, and orally challenged with live Eimeria maxima at 7 days post-hatch. Body weight gain, fecal oocyst output, and intestinal cytokine transcript levels were assessed as measures of protective immunity. While immunization with profilin alone or QCDC alone did not alter body weight gain of infected chickens compared with the saline control group, vaccination with profilin plus QCDC increased body weight gain such that it was equal to the uninfected controls. Immunization with profilin plus QCDC also reduced fecal oocyst shedding compared with unimmunized controls, although in this case QCDC failed to provide an adjuvant effect since no difference was observed between the profilin-only and profilin/QCDC groups. Finally, increased levels of transcripts encoding IL-1ß, IL-15, and IFN-γ were seen in the intestinal tissues of animals given profilin plus QCDC compared with the profilin-only or QCDC-only groups. In summary, this study demonstrates an adjuvant effect of QCDC on body weight gain and intestinal cytokine responses following in ovo vaccination of chickens with an Eimeria profilin vaccine.

The effects of a novel adjuvant complex/Eimeria profilin vaccine on the intestinal host immune response against live E. acervulina challenge infection.

The effects of a novel adjuvant composed of Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC) on protective immunity against avian coccidiosis following immunization with an Eimeria recombinant protein were determined. Broiler chickens were subcutaneously immunized with isotonic saline (control), Eimeria recombinant profilin alone, or profilin emulsified with QCDC at 1 and 7 days post-hatch, and orally challenged with live Eimeria acervulina at 7 days following the last immunization. Body weight gains, gut lesion scores, fecal oocyst outputs, profilin serum antibody titers, lymphocyte proliferation, and intestinal cytokine transcript levels were assessed as measures of protective immunity. Chickens immunized with profilin plus QCDC showed increased body weight gains and decreased intestinal lesion scores compared with the profilin only or control groups. However, no differences were found in fecal oocyst shedding among the three groups. Profilin serum antibody titers and antigen-induced peripheral blood lymphocyte proliferation in the profilin/QCDC group were higher compared with the profilin only and control groups. Finally, while immunization with profilin alone or profilin plus QCDC uniformly increased the levels of intestinal transcripts encoding all cytokines examined (IL-1ß, IL-10, IL-12, IL-15, IL-17F, and IFN-γ) compared with the control group, transcripts for IL-10 and IL-17F were further increased in the profilin/QCDC group compared with the profilin only group. In summary, this study provides the first evidence of the immunoenhancing activities of QCDC adjuvant in poultry.

Biopolymer mediated trehalose uptake for enhanced erythrocyte cryosurvival.

A biopolymer has been shown to facilitate efficient delivery of trehalose, a bioprotectant normally impermeable to the phospholipid bilayer, into ovine erythrocytes. Cellular uptake of trehalose was found to be dependent on polymer pendant amino acid type and degree of grafting, polymer concentration, pH, external trehalose concentration, incubation temperature and time. Optimization of these parameters yielded an intracellular trehalose concentration of 123 +/- 16 mM and concomitant improvement of erythrocyte cryosurvival of up to 20.4 +/- 5.6%. Intracellular trehalose was shown to impart cellular osmoprotection up to an external osmolarity of 230 mOsm and increased osmotic sensitivity above this threshold. Biopolymer mediated membrane permeability was shown to be rapidly and completely reversible via washing with phosphate buffered saline.

Identification of multiple linear epitopes of the plasminogen activator A (PauA) of Streptococcus uberis with murine monoclonal antibodies.

Streptococcus (S.) uberis is a common cause of mastitis in cattle. A protein (PauA) secreted by this bacterium is capable of activating plasminogen from sheep and cattle. The PauA first binds to bovine plasminogen (b-plg) to form a PauA-plasminogen complex that subsequently binds to and activates b-plg to form plasmin. We have identified several linear epitopes of PauA that are recognized by murine monoclonal antibodies to PauA. Two of the monoclonal antibodies which neutralized the enzymatic activity of PauA, EC3 and 2.22, recognized common linear peptide sequences with similar charge and spacing patterns. These neutralization epitopes are located in the predicted alpha-domain of the PauA molecule. Further, these same epitopes are in critical structure/function domains identified in other studies. These characterizations may facilitate the design of an efficacious vaccine for streptococcal mastitis in the dairy cow.