CASE NOTES

Diagnostics and Technology update from Microbiology and Parasitology

Mark Hazelton, Pathology Resident, Department of Primary Industries, Elizabeth Macarthur Agricultural Institute (EMAI), Menangle NSW

Posted Flock & Herd December 2019

Introduction

There are many factors that influence a diagnostic outcome. Some factors include (but are not limited to) identification of the true problem, understanding of pathogenesis, collection of representative samples, sample quality, appropriate test selection and skill required to interpret test results. Test limitations, including (but not limited to) sensitivity and specificity, pathogen viability, sample type and size, test duration and cost, all play a role in the value of a test result in reaching a diagnosis. It is important to remember that many infectious disease test results only reflect the point in time of sampling. Therefore, the biology of the disease of interest needs to be strongly considered prior to sampling and testing. For herd-level diagnoses, epidemiology is often required to extrapolate test results of a pre-determined cohort of individual animals to represent the disease status of a group of animals. All this considered, there are a number of factors a clinician needs to take into consideration when attempting to reach a diagnosis and ultimately make decisions with respect to disease therapy, prevention or control.

Diagnostic technology is rapidly and constantly evolving in an attempt to overcome the influencing factors and test limitations described. As new diagnostic test technologies become available, they may either replace or complement existing technologies. Routine diagnostic tests such as culture and Gram stain still have application today, and may be used to provide diagnostic information in complement to new diagnostics. For example, a Gram stain can be utilised to quickly assess the bacterial characteristics of a sample such as type, abundance, pure vs mixed population, and the presence of polymorphonuclear cells. This can sometimes be used to guide initial treatment while a culture is set up, as a result often takes several days to obtain.

Polymerase Chain Reaction (PCR) is a diagnostic tool that has become very popular in recent years. It was first developed in the early 1980s, however PCR techniques and procedures have been developed and refined to include different types such as real-time/quantitative, nested, multiplex, and arbitrary-primed. The development of these different PCR types enhances sensitivity and specificity and can provide greater diagnostic information from a single test with a faster turn-around time. A good example is the development of a quantitative multiplex PCR assay for the detection of Theileria orientalis and differentiation of clinically relevant subtypes (Bogema et al. 2015). The quantitative aspect of this PCR classifies a high-, moderate- or low-level infection based on clinical thresholds, while the multiplex component describes the presence of three different genotypes that have variable pathogenicity—a much more sensitive test when compared to evaluation of a routine blood smear.

There are several new PCRs now available at EMAI. Some of these new PCRs have been developed in response to demand from the field with respect to important disease outbreaks, new disease presentations and/or NSW prohibited matter incursions. Other PCRs have been upgraded from a conventional method to real time/quantitative and multiplex methods. New tick-associated PCRs available include Babesia bovis, Babesia bigemina, Anaplasma marginale and Anaplasma centrale (vaccine strain). In relation to recent Salmonella enteriditis outbreaks in poultry, PCRs are now available for Salmonella sp., Salmonella Group D sp., and Salmonella enteriditis. A Salmonella Group D antibody ELISA is now available. In response to enquiries regarding the NZ Mycoplasma bovis incursion, new Mycoplasma PCRs are available for Mycoplasma sp., Mycoplasma bovis, M. bovigenitalium and M. californicum in cattle. PCRs are available for Mycoplasma meleagridis, M. gallisepticum and M. synoviae in poultry. A Bordatella sp. PCR is also available for poultry. A number of Chlamydia PCRs are now available for Chlamydia sp., C. psittaci and C. pecorum.

There are also a number of PCRs undergoing development or upgrade for Trictrichomonas foetus, Campylobacter fetus subspecies venerealis, Mycoplasma ovis, Anthrax, American foulbrood and Lawsonia sp. Some serology tests under development include a Q-Fever multispecies ELISA and a Brucella suis ELISA.

Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS). One of the major recent advancements in microbiology is the creation of MALDI-TOF MS. In a nutshell, a matrix is applied to biomolecules (e.g. DNA, proteins, peptide and sugars) and large organic molecules to create ions that then pass through a field-free vacuum until they reach a detector and generate a mass spectrum unique to the substrate, which is then queried against a database for an identification. There are numerous scientific applications for MALDI-TOF MS technology, of which, bacterial and fungal identification are very relevant to veterinary diagnostics. Whilst the use of this technology commonly requires a cultured isolate, it can dramatically decrease the labour, cost and time requirements for biochemical analyses for routine bacterial identification while increasing the sensitivity. At present, there are some limitations with respect to the range of identifiable organisms within approved reference databases and some species can be misidentified. In a 2015 study, 89.4% of 180 aerobes and 90.1% of 152 anaerobes were identified by MALDI-TOF MS, concluding that this technology is adequate for the majority of routine field isolates encountered in a veterinary diagnostic laboratory (Randall et al. 2015). An example of the use of MALDI-TOF MS in cattle diagnostics is for identification of bacterial isolates from cows affected by clinical and subclinical mastitis, especially mixed bacterial growths (Barreiro et al. 2010).

Next Generation Sequencing is another laboratory technology that is becoming popular. Today's technology enables rapid determination of the complete DNA sequence of an organism's genome at a single time. The Ion Torrent™ is an example of a machine that is capable of Next Generation Sequencing. While this technology is only really used at a research level at the moment due to expense, research findings can have practical application to our understanding of pathogenesis of certain diseases. For example, Next Generation Sequencing performed on 82 Australian Mycoplasma bovis isolates from different sample types collected over a nine year period from clinical and carrier animals, demonstrated there was minimal variation in virulence genes, suggesting host and environmental factors are more likely to effect the disease manifestation (e.g. mastitis or arthritis) as opposed to different strains (Parker et al. 2016). Veterinary application of this technology is likely to include diagnosis of inherited and infectious diseases into the future.

Conclusion

There is always exciting new technology being developed every day, however, even with significant advancements, there is often no 'silver-bullet' test and skilled interpretation of results or a combination of tests may often be required to reach a diagnosis. Practitioners should remain aware of test limitations. A comprehensive understanding of the biology of the disease or diseases of interest is required to guide the correct diagnostic pathway so the appropriate samples can be collected, the appropriate tests are performed and results are interpreted correctly. The value of a thorough case history and complete examination of the animal/s and their environment will never be replaced by a diagnostic test.

References

Barreiro, J.R., C.R. Ferreira, G.B. Sanvido, M. Kostrzewa, T. Maier, B. Wegemann, and V. Böttcher. 2010. Short communication : Identification of subclinical cow mastitis pathogens in milk by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. J. Dairy Sci. 93:5661-5667. doi:10.3168/jds.2010-3614
Bogema, D.R., A.T. Deutscher, S. Fell, D. Collins, G.J. Eamens, and C. Jenkins. 2015. Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant. J. Clin. Microbiol. 53:941-950. doi:10.1128/JCM.03387-14
Parker, A.M., A. Shukla, J.K. House, M.S. Hazelton, K.L. Bosward, B. Kokotovic, and P.A. Sheehy. 2016. Genetic characterisation of Australian Mycoplasma bovis isolates through whole genome sequencing analysis. Veterinary Microbiology 196:118-125. doi:10.1016/j.vetmic.2016.10.010
Randall, L.P., F. Lemma, M. Koylass, J. Rogers, R.D. Ayling, D. Worth, M. Klita, A. Steventon, K. Line, P. Wragg, J. Muchowski, M. Kostrzewa, and A.M. Whatmore. 2015. Evaluation of MALDI-ToF as a method for the identification of bacteria in the veterinary diagnostic laboratory. Res. Vet. Sci. 101:42-49. doi:10.1016/j.rvsc.2015.05.018