This review seeks to provide an up-to-date view on the diagnosis and prevention of two major bacterial diseases of poultry - fowl cholera and infectious coryza. The review will focus on key issues and recent advances. More detailed and historical data are already covered in a range of standard texts that are widely available e.g. Diseases of Poultry (Blackall and Soriano 2008; Glisson et al. 2008)) and Poultry Diseases (Blackall and Hinz 2008; Christensen et al. 2008).
Infectious coryza - Laboratory tools to aid diagnosis
As the causative agent of infectious coryza - Avibacterium paragallinarum - is a demanding organism in terms of growth in laboratory media, the usual culture media are not appropriate for this disease. The main growth requirement is a need for V-factor (NADH). However, diagnosticians need to be aware that there are some isolates of Av. paragallinarum that do not need NADH (ie they will grow on blood agar) (Garcia et al. 2004). Further, it has recently been found that some Australian isolates of Av. paragallinarum won´t grow on a complete medium that meets the NADH requirements of other typical Av. paragallinarum isolates (Blackall et al. 2011). The most universally used isolation medium for Av. paragallinarum remains 5% sheep blood agar with a cross-streak of a nurse culture of Staphylococcus epidermidis to provide the required NADH. This medium aids in the recognition of Av. paragallinarum as the organism will show satellitism (except if NADH independent!!). Complete media that do not show satellitism have been used e.g. CLBA medium (Terzolo et al. 1993). The use of CLBA medium does allow the incorporation of selective antibiotics that reduce the level of contamination due to Gram positive organisms (Terzolo et al. 1993). Regardless of the medium used, there is a need for a 5% carbon dioxide atmosphere.
Following initial isolation, there is a need for a level of biochemical testing as there are a number of satellitic organisms present in chickens. At a minimum, the following test results are required:- Gram negative organism, catalase negative, satellitic organism from birds showing clinical signs. This level can be achieved in most routine diagnostic laboratories.
More extensive phenotypic characterisation (antimicrobial sensitivity testing, detailed biochemical characterisation, serotyping) requires access to suitable media that can support the growth of the organism. Typically, this level of characterisation is available only in a small number of laboratories.
The well recognised difficulties with conventional diagnosis - specialised, expensive media - have lead to the development of two DNA-based assays. The conventional PCR for Av. paragallinarum has been validated for use on isolates and directly on birds ((Chen et al. 1996; Chen et al. 1998a; Chen et al. 1998b). A real-time PCR has also been described and validated for use directly on the bird (Corney et al. 2008). For those laboratories that lack experience in handling NADH requiring organisms, the use of PCR is an attractive alternative. Indeed, in regions where NAD-independent Av. paragallinarum is known to occur (South Africa and Mexico), these two PCR assays are the only practical tools for the confident identification of this form of Av. paragallinarum as distinct from Ornithobacterium rhinotrachealae. The main drawback with the use of the PCR tests is that there is no way of obtaining knowledge of serovar or antibiotic resistance.
Fowl cholera - laboratory tools to aid diagnosis
The robust nature of the causative agent of fowl cholera (Pasteurella multocida) means that a simple isolation medium (5% sheep blood agar) is the medium of choice. Selective media for use on laryngotracheal and cloacal swabs (Muhairwa et al. 2001) as well as samples from the alimentary tract (Lee et al. 2000) have been described. Confirmatory biochemical testing can be done with relative ease. A key point for diagnosticians is that some of the key biological characters associated with the typical P. multocida - indole positive, failure to ferment maltose, presence of ornithine decarboxylase and no requirement for NADH - can vary (Krause et al. 1987; Christensen et al. 2004).
AA range of species-specific PCR assays for P. multocida have been described - with the two assays being well validated for use on cultures (Townsend et al. 1998; Miflin and Blackall 2001). Neither of these tests has been validated for direct use on tissues. However, a recently developed real-time PCR has been shown to be both species-specific and suitable for use directly on swabs (Corney et al. 2007).
Infectious coryza - Prevention by vaccination
In recent years, there has been a significant focus on the cross-protection (within a serovar) provided by inactivated infectious coryza vaccines. There is now widespread acceptance that the Page serovars (A, B and C) do not cross-protect. The focus in recent times has been on the issue of cross-protection within the A, B and C serovars. For some time, it has been known that not all serovar B isolates are cross-protective ((Yamaguchi et al. 1991). This has reached the stage where some commercial vaccines now include multiple B strains (Jacobs et al. 2003).
The capacity of the Kume serotyping scheme to subtype within the Page A and C has meant that there have been questions on whether these Kume sub-types represent immunotypes i.e. will a Kume C-1 based vaccine provide protection against a Kume C-2 challenge? A large study from Mexico has provided the answer (at least for single reference strain for each serovar) (Soriano et al. 2004). Essentially, this study has shown good cross-protection within the four A subtypes but less within the C subtypes (Soriano et al. 2004). What is not still clear is whether these results (ie a single strain of each sub-type) are typical of all isolates within the sub-type.
While only killed whole cell coryza vaccines remain the only current commercially available vaccines, there has been active research on possible alternative approaches. Several studies have shown that recombinant haemagglutinin antigens can provide protection (although only tested to date for homologous serovar protection) (Noro et al. 2008; Wu et al. 2011). Additionally, a peptide has been shown to be capable of providing homologous protection (Wang et al. 2007). Whether these research results can be converted into commercial products remains an open question.
Fowl cholera - Prevention by vaccination
Unlike infectious coryza, both killed whole cell and live fowl cholera vaccines have been available for some time have been available in the USA. In Australia, a new vaccine has been just released - the Vaxsafe live fowl cholera vaccine - the first live vaccine in that market. The generally accepted situation is that the killed vaccines provide protection against the serovars in the vaccine (typically serovars 1, 3 and 4) while the live vaccines (typically a single strain) provide cross-serovar protection.
In many regions, there remains a high use of autogenous vaccines - isolates from a farm are used for a farm-specific vaccine. While not preferred by some vaccine manufacturers, this approach is based on the belief that the best protection for a killed vaccine is given by the isolate responsible for the challenge.
There are a number of research advances in recent years that may herald the dawn of a new era in fowl cholera vaccines. As an example, it has recently been shown that a single nucleotide change in a nucleoid-associated protein (the FIS protein) is an explanation for spontaneous capsule loss (Steen et al. 2010). In addition, this gene (the fis gene) seems to play a central role in the regulation of virulence factors and surface components of the bacterium (Steen et al. 2010). Clearly, his developing understanding of key virulence regulation mechanisms could lead to next generation fowl cholera vaccines
Laboratory Support Tools
For both infectious coryza and fowl cholera there are now a range of tools to help guide disease investigations and improve prevention and control programs. For infectious coryza, the tools currently available are limited - restriction endonuclease analysis (Blackall et al. 1990), ribotyping (Miflin et al. 1997) and ERIC-PCR (Morales-Erasto et al. 2011). While these tools have proven useful they have not yet been widely adopted or used.
For fowl cholera, there is now an extensive range of typing tools - with a critical review identifying restriction endonuclease analysis and REP-PCR as most suitable at that time (Blackall and Miflin 2000). The major addition to the array of typing tools is multi-locus sequence typing (MLST) (Subaaharan et al. 2010). MLST is now widely accepted as the definitive typing tool. A Website that allows access to the typing results of over 450 strains is now fully functioning and being extended on a regular basis (http://pubmlst.org/pmultocida_rirdc/). The data-base is currently being used to address questions of host specificity - are certain clones of P. multocida found only in cattle and not in poultry? As well, the original study defining the MLST scheme showed the methodology had a good capacity to investigate the epidemiology of fowl cholera outbreaks (Subaaharan et al. 2010).
There have been significant new technologies developed for laboratory based tests to help in the diagnosis and prevention of both fowl cholera and infectious coryza. While these new technologies have relevant roles, the traditional approach of culture and phenotypic characterization remains a valid and important option for diagnostic investigations. As with all diseases, a diagnosis and a prevention and control program requires input from the full range of expertise and skills - field veterinarians, laboratory diagnosticians and pathologists.
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Watch Dr. Pat Blackall lecture at the XXII Latin American Poultry Congres