Cytotoxic T lymphocytes (CTL) are associated with virus control in horses infected with equine infectious anemia virus (EIAV). Early in infection, control of the initial viremia coincides with the appearance of CTL and occurs before the appearance of neutralizing antibody. In carrier horses, treatment with immunosuppressive drugs results in viremia before a change in serum neutralizing antibody occurs. Clearance of initial viremia caused by other lentiviruses, including human immunodeficiency virus-1 and simian immunodeficiency virus, is also associated with CTL and not neutralizing antibody. In addition, depletion of CD8+ cells prior to infection of rhesus monkeys with simian immunodeficiency prevents clearance of virus and the same treatment of persistently infected monkeys results in viremia. Cats given adoptive transfers of lymphocytes from vaccinated cats were protected and the protection was MHC-restricted, occurred in the absence of antiviral humoral immunity, and correlated with the transfer of cells with feline immunodeficiency virus-specific CTL and T-helper lymphocyte activities. Therefore, a lentiviral vaccine, including one for EIAV, needs to induce CTL. Based on initial failures to induce CTL to EIAV proteins by any means other than infection, we attempted to define an experimental system for the evaluation of methods for CTL induction. CTL epitopes restricted by the ELA-A1 haplotype were identified and the MHC class I molecule presenting these peptides was identified. This was done by expressing individual MHC class I molecules from cDNA clones in target cells. The target cells were then pulsed with peptides and used with effector CTL stimulated with the same peptides. In a preliminary experiment, immunization of three ELA-A1 haplotype horses with an Env peptide restricted by this haplotype resulted in CTL in peripheral blood mononuclear cells (PBMC) which recognized the Env peptide and virus-infected cells, but the CTL response was transient. Nevertheless there was significant protection against clinical disease following EIAV challenge of these immunized horses when compared with three control horses given the same virus challenge. These data indicated that responses to peptides in immunized horses needed to be enhanced. Optimal CTL responses require help from CD4+ T lymphocytes, and experiments were done to identify EIAV peptides which stimulated CD4+ T lymphocytes in PBMC from infected horses with different MHC class II types. Two broadly cross-reactive Gag peptides were identified which stimulated only an interferon γ response by CD4+ T lymphocytes, which indicated a T helper 1 response is needed for CTL stimulation. Such peptides should facilitate CTL responses; however, other problems in inducing protection against lentiviruses remain, the most significant of them being EIAV variants that can escape both CTL and neutralizing antibody. A possible solution to CTL escape variants is the induction of high-avidity CTL to multiple EIAV epitopes.

Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral–bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the β2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1β, tumor necrosis factor-α and interferon-γ) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.

Vaccination continues to be a major tool for controlling feline infectious diseases, but numerous factors present both challenges and opportunities for maintaining and expanding the market for veterinary biologicals. Developing novel vaccines is one approach, but in most cases the market for such products will be restricted to niche populations; the comparatively smaller quantity of vaccines sold may be offset somewhat if available from a limited number of manufacturers. A second approach is to produce better formulations of existing vaccines; such products would be applicable to larger populations of cats and have greater sales potential. But veterinarians must be convinced of the improved product's superiority for it to be successful in the marketplace. To ensure that high-quality, useful and necessary vaccines reach the marketplace, communication between veterinarians, the manufacturers of biologicals, and regulatory agencies must be enhanced.

Fel-O-Vax FIV is an inactivated virus vaccine designed as an aid in the prevention of infection of cats, 8 weeks or older, by feline immunodeficiency virus (FIV). It contains two genetically distinct FIV strains. The efficacy of this vaccine was demonstrated in a vaccination–challenge study designed to meet various regulatory requirements for registering the vaccine. Eight-week-old kittens were vaccinated with an immunogenicity vaccine which contained minimal release levels of FIV antigens formulated with a proprietary adjuvant system. Twelve months later, all vaccinates and controls were challenged with a heterologous FIV strain. Following the vigorous challenge exposure, cats were monitored for FIV viremia. It was found that 16% of the vaccinated cats developed viremia while 90% of the controls became persistently infected with FIV, which demonstrated that the vaccine was efficacious and the protective immunity lasted for at least 12 months. The safety of the vaccine was demonstrated by a field safety trial in which only 22 mild reactions of short duration were observed following administering 2051 doses of two pre-licensing serials of Fel-O-Vax FIV to cats of various breeds, ages and vaccination histories. Thus, Fel-O-Vax FIV is safe and efficacious for the prevention of FIV infection in cats.

Aquaculture is the fastest growing food-producing sector, providing an acceptable supplement to and substitute for wild fish and plants. Increased production intensification, particularly in high-value species, involves substantial stress, which, as in other captive livestock species, has resulted in outbreaks of major diseases and related mortalities. Widespread use of antibiotics has led to the development of antibiotic-resistant bacteria and the accumulation of antibiotics in the environment and the flesh of fish. Thus, recently effort has been dedicated to vaccine development. Vaccination in fish is complicated by their aquatic environment. Individual injections are labor-intensive and stressful, since fish have to be removed from the water and anaesthetized. Some vaccines offer a limited duration of protection, and thus booster applications are required. In salmonid species, many commercial vaccines use oil-based adjuvants, resulting in a greatly improved duration of protection. However, oil-based adjuvants have been related to significant growth depression, internal adhesions and injection site melanization, resulting in carcass downgrading. Oral administration to aquatic species is by far the most appealing method of vaccine delivery: there is no handling of the fish, which reduces stress; and administration is easy and suitable for mass immunization. However, few oral vaccines have been commercialized, due in part to the increased quantity of antigen required to provoke an immune response, and the lack of an adequate duration of protection. For effective oral delivery, protective antigens must avoid digestive hydrolysis and be taken up in the hindgut in order to induce an effective protective immune response. Antigen encapsulation technologies have been used to protect antigen; however, such strategies can be expensive and are not always effective. Alternative approaches, currently under development, are discussed.

The ability of the fish industry to provide a continuous supply of fish protein depends on both proper biosecurity and strategies to significantly reduce the risk of infectious diseases. Vaccination is a safe and effective means to prevent disease and to increase the productivity and profitability of farmed fish. Vaccines are likely to be the prime prophylactic measure of the future because of rapidly developing advances in fish vaccine technology and producer acceptance. The efficacy of a vaccine is influenced by a variety of factors that must be considered in the development of vaccination strategies for fish. This review highlights some of these factors. The response to a modified live vaccine against Edwardsiella ictaluri is used for illustration.

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in young chickens. Two serotypes of this double-stranded RNA virus exist but only serotype 1 viruses cause disease in chickens. Detection and strain identification of IBDV is important because antigenic subtypes found within serotype 1 make it necessary to tailor vaccination programs to the antigenic type found in the bird's environment. Because conventional virus isolation and characterization are not practical for routine diagnosis of IBDV, antigen-capture enzyme-linked immunosorbent assay (ELISA) and molecular assays based on reverse transcription–polymerase chain reaction (RT-PCR) technology were developed. Compared with antigen-capture ELISA, RT-PCR assays have greater versatility and are more sensitive and specific. Strain identification has been accomplished using a variety of post-RT-PCR assays, including restriction enzyme digestion of the RT-PCR products. The resulting restriction fragment length polymorphisms (RFLP) are used to differentiate viruses into molecular groups that correlate with antigenic and pathogenic types. Recently, two types of real-time RT-PCR have been used to identify and differentiate strains of IBDV. Both methods use distance-dependent interaction between two dye molecules, known as fluorescence resonance energy transfer (FRET). The dye molecules are attached to one or more nucleotide probes that detect specific nucleotide sequences of the virus. Our laboratory has used a two-probe assay to identify single-nucleotide mutations among IBDV strains. A mutation probe is used in this assay to detect substitution mutations in a region of the viral genome that encodes a neutralizing epitope of the virus. These assays are accurate, reliable and inexpensive compared with conventional RT-PCR because they do not require RFLP or other labor-intensive post-RT-PCR assays to distinguish viral strains.

Mycoplasma hyopneumoniae, the cause of enzootic pneumonia, remains an important pathogen in the swine industry. This small, complex organism colonizes the ciliated cells of the respiratory tract, resulting in little exposure to the immune system. Confirming the presence of M. hyopneumoniae, as well as identifying its role in respiratory disease and pneumonia, remains challenging to the veterinary profession. While culture of the organism remains the gold standard for identification, the use of serology, the polymerase chain reaction and various assays to detect the presence of M. hyopneumoniae in tissue is common in diagnostic laboratories. Because of the role M. hyopneumoniae plays in increasing the severity of pneumonia associated with concurrent bacterial and viral infections, understanding the pathogenesis and diagnostic assays available is critical for developing effective intervention strategies to control respiratory disease on a herd basis.

Clostridium difficile causes pseudomembranous colitis in humans, usually after disruption of the bowel flora by antibiotic therapy. Factors mediating the frank disease include the dose and toxigenicity of the colonizing strain, its ability to adhere to colonic epithelium, the concurrent presence of organisms that affect multiplication and toxin production or activity, and the susceptibility of the host. Toxins A (an enterotoxin) and B (a cytotoxin) play the major role in pathogenesis and the detection of toxins in gut contents is the gold standard for diagnosis. Disease in horses takes the form of often-fatal foal hemorrhagic enteritis. Nosocomial, antibiotic-associated, disease is increasingly common in adult horses. Enteric clinical signs are reported in ostriches, companion animals and recently calves. Clostridium difficile colitis is now a common diagnosis in neonatal pigs in the USA and elsewhere. Clinical features include onset at 1–5 days of age, sometimes with dyspnea, mild abdominal distension and scrotal edema, and commonly with yellow, pasty diarrhea. There is mesocolonic edema grossly, with microscopic diffuse colitis, mucosal edema, crypt distension, epithelial necrosis and superficial mucosal erosion. Neutrophil infiltration of the lamina propria is common, and fibrin and numerous rod-shaped bacteria are observed on the surface. About two-thirds of litters and one-third of piglets will be affected (based upon positive toxin tests), although this appears to vary with the season. The case fatality rate is probably low if considering only direct effects of C. difficile infection. The significance of toxin-positive non-diarrheic pigs and the nature of the interaction of toxins A and B with enterocytes are unknown. Given the widespread occurrence of the disease, there is substantial effort to develop immunoprophylactic products.

Prior to the widespread use of vaccination for the control of feline immunodeficiency virus (FIV) infection, diagnosis was made by the detection of antibodies against FIV. A number of commercial animal side tests perform quite well for this determination, with positive predictive values between 91 and 100% and negative predictive values between 96 and 100%. Furthermore, results of these tests could be confirmed by western blot analysis of FIV test-positive sera. Currently, a killed whole virus FIV vaccine has been made available to practitioners. Vaccinated cats seroconvert by ELISA and western blot, making presently available diagnostic tests, which rely on antibody detection, useless in cats after vaccination. The advisory panels of the American Association of Feline Practitioners and Academy of Feline Medicine both recommend testing for feline leukemia virus antigen and FIV antibody before vaccination.