5 Diagnostic Challenges for the Detection of Emerging Pathogens

Transcript

Hello!! Welcome to the last talk of the week that is Diagnostic challenges for the detection of emerging practices. We all know that it is very hard to diagnose a new problem when it emerges in a new location, because lot of data associated with it is not available by the time and because of those it is very hard for the investigators to establish the causal agent of a particular disease which is considered to be a new or emerging problem in a new locality. Let us see the example of Pseudomonas syringae pv. actinidae which is the causal agent of bacterial canker of kiwifruit and it was an emerging pathogen in New Zealand somewhere in 2010. By the time of its outbreak the kiwi fruit orchards were not seen this particular type of disease earlier. And therefore there investigators were not able to associate it the problem and the cause associated with it and it was a big challenge for the investigators to establish the causal agent because it is only then corrective control measures can be adopted and lost due to this pathogen outbreak could be checked in a shorter period of time. Little had been known or published about the Diagnostics ecology and epidemiology of this problem prior to 2010 in New Zealand so it was a challenge for the diagnosticians to diagnose the disease and stops its spreading. An introduction of Psa into New Zealand underscored a number of challenges in the use of molecular diagnostics to detect and characterize the exotic pathogenic bacteria. Normally, what is preferred for use of our application of molecular diagnostics is that the epidemiology of the pathogen is well understood and reliable and specific diagnostic assays are available for that particular pathogen. But these were not available in case of Psa outbreak in New Zealand. So the time of outbreak there was significant knowledge gaps in the biology of PSA and a lack of validated diagnostic tools for the high throughput detection of Psa in kiwifruit plant material was also not available. The timeframe to validate the identification of Psa was further hampered by the need of culture of the causal agent and to conduct pathogenicity test to demonstrate Koch postulates. All these steps normally takes significant amount of time and it was not very preferred at that time to have that much time to establish that particular causal agent. 0o methods for identifications of Psa 0adopted at the time were definitely diagnosis for combination of molecular, biological, chemical and pathogenicity tests and colonies on nutrient agarplates showed little evident that they are of Pseudomonas origin. Like rounded, convex, glittering, translucent and creamy white colonies. But they lacked a fluorescent pigment on King’s B medium which again baffled the investigators to establish the actual causal agent. Psa proved to be gram-negative and exhibited characteristics of Pseudomonas syringe on the LOPAT group a test. But by the time of Psa outbreak in New Zealand, PCR assays to identify Psa had been developed however the primers used for PSA diagnosis was not PSA specific as in some situations the primers used to amplify DNA from other Pseudomonas found on the Kiwi fruit as well. So it was only sequencing that was helpful to distinguish PSA from other leaf spotting Pseudomonas on kiwifruit. So these were certain challenges that existed during the outbreak of Psa in New Zealand. Further large-scale testing of Psa requires DNA extraction from PCR testing protocols or detection in large quantities of samples which was not established during the New Zealand outbreak. So this required an immediate evaluation of DNA extraction methods and PCR techniques for high-throughput detection of kiwi fruit plant material.

PHEL that is the Public Health and Environment laboratory developed rapidly a SYBR Green qPCR assay using the primers Psa F 1 and R 2 to detect Psa in leaf and pollen tissue that the use of DNA-binding dye SYBR green for the detection of PCR amplifications allowed rapid conversion of conventional q peas to qPCR and enable the high-throughput detection of Psa in response setting. So lot of work had been to be done in a very short period of time to establish or to save time and to correctly identify the Psa strains that are present in different plant tissues.

Further Genetic Diversity of Psa was again as challenge because all Psa strains isolated from the earlier Italian outbreaks shared the same repetitive PCR fingerprint and MLST profile but were different to the strains previously isolated in Japan. So this was again a confusion that existed at the time that the profiles generated from the Italian strain is different from the Japan strain. So none of the strains from the Italian outbreaks presents a possessed genes coding for phytotoxins phaseolotoxin or coronatine, further differentiating these strains for those in Japan, Italy and Korea. So analysis of the cts gene of these strains consistently detected two haplotypes that differed by two base pairs Psa strains from Japan, Korea and the Italian 1994 outbreak belong to one haplotype whereas Italian strains isolated from the epidemics in 2008-2009 belong to another haplotype. So this tools has been able to differentiate that the Japan,Korea and Italian earlier outbreak strain has been put into one group whereas, the new strain of Italian Psa outbreak has been grouped into the different haplotype. These studies concluded that epidemics in Italy during 2008 and 2009 appear to have been caused by different Psa population then those previously recorded in Japan, South Korea and earlier Italian outbreaks.

Molecular Characterization of the Psa: that causes the New Zealand outbreak the identification of Psa was the causal agent of the kiwifruit disease was quickly obtained during the response investigation. However the identification of the outbreak strain was more challenging. Although, the pathogen is established but looking into the genetic diversity of the pathogen existed throughout the world it was very difficult to categorize which strain that is causing the outbreak in New Zealand whether it falls into the Japan haplotye or it is fall into the new Italian outbreak haplotype.

Genetic differences have been detected among overseas PSA strains and the question was raised whether the New Zealand strain was similar to those strains causing an epidemic of bacterial canker in Italy.

So the role of NGS came into play in establishing that fact and through application of NGS technology the strainer type was established for the Psa that was responsible for New Zealand Psa outbreak. The original outbreak strain was sequence using Roche 454 GS Junior sequencing platform within the first weeks of the outbreak and the draft genome sequence was assembled within three days. It did allow an analysis of effector and toxin genes known to be very key virulence determinants in Pseudomonas syringe complex. This analysis of drafts genome sequence quickly validated the identification of the New Zealand Psa isolate and provided significant insight into possible strain type. The draft sequence enabled key effector and phytotoxic genes to be screened and revealed some commonalities with New Zealand Psa strain and the more aggressive Italian strain.

So with the use of NGS platforms finally the investigators were able to establish a relationship between the New Zealand outbreak PSA with the modern Italian outbreak PSA strain to be have some similar characteristics. So this is how technologies help to identify or detect the emerging pathogens in a new location and with these technologies we can now go up to identification of the strain in level of the pathogen that is causing the particular damage to the crops associated with and finally we can then practice the recommendations that is required for management of that particular strain of the particular pathogen.

So with this we have come to an end of the week poll and in the week 5 we’ll be talking about special applications of plant disease diagnostics where we will be talking about recent developments and some special applications that are being deployed for plant disease diagnosis. Till then have a good time.

Thank you very much.