Intrauterine immunizations trigger antigen-specific mucosal and systemic immunity in pigs and passive protection in suckling piglets.

We assessed whether vaccines administered to the uterus at breeding can lead to sufficient colostral antibodies to protect suckling piglets against Porcine Endemic Diarrhea Virus (PEDV). An antigen from Lawsonia intracellularis, a disease that impacts weanling intestinal health, was also included because we have extensive knowledge on the pig immune response to this antigen. Gilts were mock-bred at 2nd estrus with killed sperm including an intrauterine (i.u.) vaccine comprised of recombinant (r) PEDV Spike protein (rPEDVS1) and L. intracellularis flagellin (rFliC) formulated with poly I:C, host defense peptide, and polyphosphazene (TriAdj). Gilts returned to estrus within 3 weeks and they were inseminated with killed sperm (3rd estrus) or live sperm (4th estrus) with rPEDVS1-TriAdj vaccine. They also received an i.m. injection of rFliC-TriAdj at 3rd and 4th estrus to establish whether i.u. vaccination primes systemic immunity without inducing mucosal tolerance. Control gilts were administered semen alone at 2nd estrus which allowed us to compare litter weights and sizes to industry standards. Colostrum from gilts challenged with low dose PEDV plus alum was used as positive reference samples for neutralizing antibodies and passive protection. Thirteen weeks later, the i.u.-vaccinated gilts showed significant PEDVS1-specific serum, colostral, and uterine antibody titers and colostral PEDVS1-neutralizing antibodies but poor cell-mediated immunity. Piglets born to i.u. vaccinated gilts received partial passive protection from PEDV infection 3 days after birth but eventually succumbed to the disease. Immunization by the i.u./i.m. route triggered significant anti-FliC cell-mediated immunity and colostral FliC antibodies that remained high in weaned piglet serum. This trial and a repeat trial wherein gilts were immunized at 1st estrus without semen and at 2nd estrus with live semen showed that intrauterine immunization did not impact fertility, number of live births or piglet growth kinetics. Further optimization is needed to promote robust passive protection in suckling offspring.

In vivo immunostimulatory effects of CpG oligodeoxynucleotide in cattle and sheep.

Oligodeoxynucleotides (ODN) containing cytosine-phosphate-guanosine (CpG) motifs have been shown to activate the innate immune system and protect mice and chicken from bacterial and viral infections. Unfortunately, similar studies in other veterinary species are lacking. In this study we assessed the in vivo immunostimulatory effects of CpG ODN 2007, an ODN with previously demonstrated in vitro biological activity. The in vivo effects of ODN 2007 were compared in two closely related outbred species, sheep and cattle, to determine if there were common biological responses. We demonstrated that subcutaneous (s.c.) injection of the CpG ODN induces an acute phase response in the form of a transient fever, a mild transient increase in circulating neutrophils and elevated serum haptoglobin in both sheep and cattle. Sheep injected with CpG ODN also exhibited increased serum 2'5'-oligoadenylate (2'5'-A) synthetase activity, but no increase in serum 2'5'-A synthetase was detected in cattle. The ODN-induced responses were stronger in animals injected with CpG ODN formulated in 30% emulsigen than phosphate buffer saline (PBS) alone. These in vivo data demonstrate for the first time that a CpG ODN induces acute phase immunostimulatory responses in sheep and cattle. However, CpG ODN-induced antiviral effector molecule 2'5'-A synthetase was detected only in sheep but not in cattle.

Cloning non-transformed sheep B cells.

The capacity to clone B cells and establish permanent B cell lines has greatly facilitated a wide variety of studies characterising the growth, differentiation, and gene expression of murine and human B cells. Similar investigations of B cell biology for other species have been severely restricted by an inability to culture or clone B cells. This is the first report of a method to clone non-transformed sheep B cells using a culture system based on murine CD154 and a combination of human gamma chain-common cytokines. Sheep Peyer's patch B cells were cultured for 120 days and then cloned by limiting dilution culture. The parental B cell culture contained both surface immunoglobulin (sIg)M(+) and sIgG1(+) B cells and both types of B cell were cloned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and light chain (LC) expression and DNA sequencing of HC V genes. There was agreement between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for sheep lambda and kappa LC did not react with all clones. Soluble Ig was detected in the culture supernatant of sIgG1(+) clones but not sIgM(+) clones. The B cell clones remained dependent upon CD154 and gamma chain-common cytokine co-stimulation for sustained growth and maintained stable Ig expression. The cloning of non-transformed sheep B cells should provide a valuable tool for studying sheep B cell biology, establishing Ig HC- and LC-specific monoclonal antibodies, analysing the B cell Ig repertoire, and may be used to produce sheep monoclonal antibodies.

Cytokine profiles following interaction between bovine alveolar macrophages and Pasteurella haemolytica.

Pasteurella haemolytica is a gram negative bacterium frequently isolated from the lungs of calves suffering from a fibrinous pneumonic condition known as shipping fever. To understand the pathogenesis of this disease, we investigated the induction of cytokin gene expression in cultures of bovine alveolar macrophages (BAM) stimulated with heat-killed P. haemolytica. Northern blot analysis of total RNA showed that P. haemolytica induced early, abundant, and consistent synthesis of IL-1, TNF-alpha, and IL-8 mRNA. Cytokine mRNAs were detected within 1 hr post-stimulation with heat-killed P. haemolytica. IL-1 and IL-8 mRNA accumulated to high levels with increase in stimulation time, whereas TNF-alpha mRNA clearly declined by 4 and 8 h post stimulation. IL-1, TNF-alpha, and IL-8 proteins were also secreted into the culture medium of BAM stimulated with heat-killed P. haemolytica. All three proteins were detected at high levels 8 and 12 h post stimulation with P. haemolytica. BAM cells treated with bovine interferon-alpha and then stimulated with P. haemolytica produced higher amounts of IL-1, IL-8 and TNF-alpha proteins compared to BAM stimulated with P. haemolytica alone. These findings demonstrate the powerful and selective induction of cytokine mRNA and protein synthesis in BAM stimulated with heat-killed P. haemolytica and may explain certain aspects of shipping fever pathogenesis.

Molecular cloning and expression of bovine interleukin-8.

Interleukin-8 (IL-8) is a neutrophil and T-lymphocyte chemotactic and activating factor. This cytokine is produced by many cell types including macrophages in response to a variety of microbial and non-microbial agents. In the present study, we determined the nucleotide sequence for bovine IL-8 cDNA. The amino acid sequence encoded by this cDNA shares 76 and 87% homology with the human and swine IL-8 proteins, respectively. Bovine IL-8 cDNA was expressed in Escherichia coli as a beta-galactosidase fusion protein. Western blotting demonstrates that this fusion protein, but not beta-galactosidase cross-reacts with monospecific anti-human IL-8 antiserum. We also studied the induction of IL-8 mRNA synthesis in bovine alveolar macrophages (BAM) stimulated with heat-killed Pasteurella haemolytica. IL-8 mRNA was induced in BAM as early as 1 h and was detectable at high levels 12 h post-stimulation with P. haemolytica. A dose titration of P. haemolytica and E. coli endotoxins showed that a relatively low level of P. haemolytica endotoxin induced high levels of bovine IL-8 mRNA. The significance of these findings in the pathogenesis of bovine pneumonia caused by P. haemolytica is discussed.

Lymphocyte proliferative responses to recombinant bovine herpes virus type 1 (BHV-1) glycoprotein gD (gIV) in immune cattle: identification of a T cell epitope.

The lymphocyte proliferative response to BHV-1 in immune cattle was compared to recombinant wild-type gD and truncated gD produced from recombinant vaccinia viruses. The response exhibited by recombinant proteins was comparable to the response induced by BHV-1 suggesting that gD is the major target structure for stimulation of bovine lymphocytes. Analysis of the proliferative response using vaccinia virus vectors expressing various modified forms of gD identified a region between residues 165 and 216 recognized by T-lymphocytes of immune cattle. Further analysis by overlapping peptides in this region localized the T cell epitope to residues 161-172. Antibody-blocking studies demonstrated that lymphocytes responding to this epitope are CD4+. In addition, lymphocytes stimulated with gD or peptide 77 (residues 161-172) also produced IFN-gamma and IL-2.

Effect of chronic administration of recombinant bovine tumor necrosis factor to cattle.

Cachectin/tumor necrosis factor-alpha (TNF), a protein produced by macrophages upon stimulation, has been implicated as an important mediator of inflammatory processes and of clinical manifestations in chronic infectious diseases. In order to study further the potential role of TNF in infectious diseases, a homologous system was employed in which recombinant Escherichia coli (E. coli) derived bovine TNF (rBoTNF) was injected in cattle, either as a single bolus or in a repetitive treatment-regime. No clinical signs were observed, although changes occurred in hematologic and immunologic parameters when less than 0.5 mg of TNF/100 kg body weight was administered twice daily for 18 days. Prolonged treatment with 0.05-0.5 mg/100 kg induced histologic but no gross changes in the kidneys and liver. When doses were increased above 0.5 mg/100 kg, depression, anorexia, cachexia, and diarrhea appeared rapidly. Pathologic changes were apparent in various tissues including liver, kidneys, and lymphoid organs; body fat depots were depleted. Most of these changes appeared to be reversible; return to normal tissue-morphology occurred within 3 weeks of withdrawal of rBoTNF. The clinical and pathologic changes induced by prolonged rBoTNF administration resembled those observed in some chronic parasitic and viral infections of cattle in which macrophage-activation characteristically occur. Our finding may be relevant to the elucidation of the pathogenesis of these and other chronic infections.