The application of molecular biology to determine the genomic structure, classification and pathogenesis of disease-causing agents in poultry was a consistent theme of the presentations at the 2008 Southern Conference of Avian Diseases. This meeting in Atlanta formed part of the International Poultry Scientific Forum organised by the US Poultry and Egg Association.
Research has been intensified by the recognition that highly pathogenic avian influenza (HPAI) is endemic in Asia and West Africa. Sporadic cases of both low and high pathogenicity in Europe are an increasing cause of concern. Developing more effective vaccines to reinforce current inactivated products is a high priority of many research teams.
Scientists at the University of Arkansas described a modified Salmonella enteritidis (SE) strain as a vector of two M2e epitope sequences. This vaccine candidate was evaluated both by in-ovo and sub-cutaneous post-hatch administration. Within one day of administration, the SE vector could be isolated from liver, spleen and from caecal tonsils. After 28 days, it could not be isolated from liver and spleen tissues. Hatchability was unaffected by in-ovo administration into the air cell. The modified SE vector stimulated an antibody response detected at seven days but waned through the four-week post-vaccination study period.
Another recombinant AI vaccine was described by workers at Auburn University. They inserted genes from an H10N7 field isolate into Pichia pastoris. Preliminary results suggest this yeast vector may offer opportunities for commercial flocks.
The mode of action of HPAI viruses was investigated in a joint programme conducted at the Southeast Poultry Research Laboratory and Ohio State University. Reverse genetics was applied to evaluate the pathogenicity of a range of recombinant viruses. Single genes from a highly virulent virus isolated from egrets in Hong Kong were substituted in a chicken strain isolated in Indonesia in 2003. Substitution of genes coding for haemagglutinin proteins that are antigenic increased mortality, viral propagation and shedding. Increased virulence was noted with substitution of genes coding for NS, NP and N proteins as compared to the original virus.
Studies conducted at the University of Georgia, USA, established that infectious laryngotracheitis (ILT) strains can be classified into nine categories based on an analysis of four regions of the ILT genome. Biotype IV corresponds to Chick Embryo Origin (CEO) vaccine. Biotypes V and VI represent the majority of field isolates. This observation confirms the prevailing opinion that variants of CEO vaccines are responsible for field cases of LT in broilers in the south-eastern and Delmarva states of the USA. Biotypes VII through IX are mainly derived from backyard flocks and are rarely encountered in commercial broilers. Additional techniques to differentiate biotypes include clinical effects and the propensity to form plaques on avian cell tissue culture.
An additional study from the University of Georgia laboratory evaluated TCO LT vaccine against a prevalent field strain. Studies were conducted in isolation chambers using vaccinated and non-vaccinated, challenged and contact chicks. Vaccination was carried out at four weeks, and challenge at eight weeks of age. Effectiveness of the vaccine was assessed by clinical signs, bodyweight and shedding of LT virus as demonstrated by RT-PCR and viral isolation. Tissue culture origin vaccine is mildly pathogenic to both recipients and contact birds but reduces the intensity of shedding. It was also determined that the presence of antibodies is not a direct indication of protection.
A study conducted at Auburn University confirmed that darkling beetle (Alphitobius) in both adult and larval form serve as reservoirs of LT virus as detected by real-time polymerase chain reaction (RT-PCR). This investigation reinforces results of previous studies in which beetles were shown to be reservoirs of avian reovirus, Newcastle disease virus, infectious bursal disease virus and Salmonella spp.
The application of RT-PCR to demonstrate the presence of avian reovirus was investigated in a study conducted at Auburn University. Chicks derived from parents immunised against reovirus showed lower viral replication and shedding than infected specific-pathogen-free chicks. Viral RNA can be detected within a day of infection with peak release after two days, with a progressive reduction in dissemination of virus. This study used a known enteric strain 2408, which has been implicated in malabsorption syndrome. The response to maternal vaccination confirms the need to carry out diligent and comprehensive vaccination of parent flocks.
Genetic drift among live attenuated infectious bronchitis (IB) vaccine strains is, in all probability, responsible for clinical signs noted in flocks in specific areas. Analysis of the S1 gene sequence carried out at the University of Georgia on 39 field isolates over a five-year period showed specific changes in the S1 gene sequence. These are associated with viruses that were originally derived from vaccine strains. Sequence analysis of the S1 gene can now be used to distinguish between field IB and vaccine-derived strains.
Random polymorphic DNA amplification PCR (RAPD) was used by scientists at the University of Georgia to differentiate between isolates of Salmonella enteritidis (SE). This technique was used since conventional pulse field gel electrophoresis lacks specificity. Applying RAPD demonstrated differences according to geographic region. It is anticipated that this technique will be of value in future epidemiologic investigations relating to the source and dissemination of SE.
E. coli vaccination
The effect of an E. coli vaccine administered either in-ovo or subcutaneously on the subsequent appearance of airsacculitis was evaluated in a joint study conducted by Auburn University and the manufacturers of the vaccine. The vaccine (Poulvac E. coli vaccine; Fort Dodge Animal Health) was administered to specific-pathogen-free chicks, which received either gentamicin or Naxcel, which are routinely administered in-ovo or subcutaneously to suppress bacterial infection. The vaccine was effective in reducing air sac lesions compared to controls subjected to challenge at 42 days of age with a known pathogenic E. coli O78 strain. There was no adverse effect associated with prior administration of either of the two commonly used antibiotics on vaccine efficacy.
The effect of a mannan-oligosaccharide (Bio-Mos; Alltech) and a combination of oligosaccharides and botanicals (Natustat; Alltech) on subsequent incidence of necrotic enteritis (NE) and salmonella colonisation were evaluated at a commercial research laboratory. Natustat significantly reduced NE lesion score and mortality compared to controls. The combination of products reduced salmonella shedding.
A trial conducted in a commercial laboratory found that, in flocks affected by gangrenous dermatitis, 37% of isolates were identified as Clostridium perfringens and 63% as Cl. septicum. Isolates had been subjected to RAPD-PCR assay to ascertain the genetic profile of 295 strains of Clostridium shown to be toxigenic. A significant observation from the trial was the relation between isolates derived from the intestinal tract and from skin lesions within affected flocks. This presumes that production of toxins facilitates dissemination of virulent Clostridium spp. from the lumen of the intestine, through the bloodstream, to the sub-cutis where small lacerations predispose to gangrenous dermatitis.
Scientists at the United States Department of Agriculture research centre at Mississippi State developed a polymerase chain reaction assay to differentiate between Mycoplasma gallisepticum strains ts-11, 6/85 and F-strain from challenge strains of the pathogen. Specific primers were developed and annealing temperatures were specified to detect a unique protein common to vaccine strains but absent in pathogenic challenge strains.
Mississippi State's research team identified factors involved in achieving effective immunisation against M. gallisepticum. These include appropriate water temperature to reconstitute the vaccine, adjusting pH and osmolarity of water and the droplet size and pressure used in administration. Delivery rate was a significant factor in achieving acceptable protection.