IB Marsha*and RJ Whittingtonb
aElizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, Australia
bFaculty of Veterinary Science, University of Sydney, Australia *Presenting author

Posted Flock & Herd March 2015

What is a bacterial strain?

The term bacterial strain now exists in two broad contexts: bacterial taxonomy and bacterial epidemiology and the definition differs accordingly. Taxonomically, a bacterial strain is a population of bacteria that descend from a single organism or pure culture. The epidemiological definition of strain refers to a phenotypic (biochemistry, physical appearance or virulence) or genotypic (DNA-based) difference that can be used reliably to differentiate bacteria. Traditional bacteriology has focused on phenotypic differences, while modern molecular bacteriology focuses on genotypic differences. It is very useful for disease control purposes when molecular differences are matched to a property of the bacterium such as its host preference or virulence, and it is in this context that strain typing of Mycobacterium avium subsp. paratuberculosis (M.ptb) can be important. For the purpose of this paper a strain of M.ptb will be considered a genotypically distinct variant that has some correlation with a host preference.

Sheep vs Cattle strains of (M.ptb)

Johne's disease was first diagnosed in Australian cattle in 19255. Johne's disease has since been reported in goats in 19774, sheep in 19807 and alpaca in 19938.

Johne's disease presents differently in cattle and sheep with respect to clinical, pathological and epidemiological features10. Early observations on the epidemiology of the disease9 and culturability of the organism from different hosts3 suggested the existence of two strains of M.ptb. Then, DNA-based studies, looking at M.ptb isolates from a range of hosts using restriction fragment length polymorphism (RFLP) on genomic DNA, confirmed the existence of two distinct groups of M.ptb2. These are now commonly referred to as C (from cattle) and S (from sheep) strains. While specific but minor differences were observed in the genomes of the two strains, no inferences could be made on how these explained the resulting host specificity2. Comparisons of the whole genome from C and S strains have demonstrated a high degree of similarity in terms of genes and clusters of genes that are present but have demonstrated substantial genomic rearrangement (REF).

In Australia and New Zealand, most cases of BJD are caused by C strains and most cases of OJD are caused by S strains. However, host specificity of the S and C strains appears to be incomplete. C strains are promiscuous and can cause disease in most species, while S strains tend to be associated mainly with sheep. Goats and deer seem to be susceptible to both strains. However, where there has been opportunity over a long time for infected sheep, cattle and other species to mix (such as in Europe), the pattern of host preference of S and C strains is harder to observe.

Genotypic differences between the S and C strains have been well characterised and linked to host specificity using standardised IS900 and IS1311 (genes present in both S and C genomes) restriction fragment length polymorphism (RFLP) typing. The RFLP technique became the definitive tool for strain typing in the 1980s and early 1990s. Capable of differentiating between the two strains and even sub-strains within each strain, RFLP is time consuming and expensive. RFLP typing was replaced by a simpler test that could differentiate between the S and C strains directly using the IS1311 gene6. The new test, developed in an MLA-funded project, is based on IS1311, and has become the international standard for primary strain typing of M.ptb. It has been extensively used and referenced in research and diagnostic studies all over the world1.

Both the RFLP and IS1311 tests have been used to type human isolates of M.ptb, and to date all appear to be C strain.

The Bison strain

During the late 1990s Dr Robert Whitlock of the University of Pennsylvania approached Dr Richard Whittington regarding the presence M.ptb in a herd of Bison (Bison Bison) in Montana USA. DNA was obtained from the M.ptb isolated from the Bison and subjected to the new IS1311 test. The results indicated a new genetic variant which was designated the Bison strain11. The IS1311 test has been used internationally to screen for the Bison strain, which has now been found frequently in both the United States and India. Up until the discovery of the Bison strain in cattle in Queensland in 2012-2013, this strain had not been reported in Australia. Following the discovery of this strain during this investigation, Queensland researchers undertook an epidemiological investigation of M.ptb in Queensland using alternate molecular strain typing techniques to trace the movement of the disease within this state12.

IS1311, the test, the results and the implications

If you look closely at the figure below you will see the results of the IS1311 test for Mycobacterium avium subsp. avium (lane 1) and the Bison, Sheep and Cattle strains of M.ptb (lanes 2, 3 and 4, respectively). Lane 5 is for size reference only. Each strain is clearly distinguished by a unique DNA profile (fingerprint) and all three can be readily differentiated from Mycobacterium avium subsp. avium. Below the results are the DNA sequences from each strain that give rise to this outcome. The circled letters indicate the genetic variant in each strain responsible for the unique profile. Currently, the IS1311 test is the only strain typing test required to support national Johne's disease programmes. Differentiation between S and C strains may be required to support the decision on action to be taken. Whilst the genetic differences in the IS1311 gene have been validated as a marker for host specificity no evidence exists to suggest this is the actual cause of host specificity or pathogenicity.

Advanced molecular strain typing to identify the origin of outbreaks

The ability to identify subtypes within the S and C strains may make it possible to look at the spread of disease within populations and geographic regions. As knowledge of the M.ptb genome increased in the early 2000s new strain typing techniques started to emerge. This coincided with a growing interest in modern molecular epidemiology. Sabat et al, 2013 have prepared a review of molecular typing techniques for those wishing to read more13. For now, what is clear is that each typing technique must be validated prior to its use in an epidemiological investigation. This validation must include critical performance criteria such as typeability (the ability to interpret results), reproducibility (how easily the test can reproduce the same result when run in different laboratories), stability (reproducibility over time) and discriminatory power (the ability to discriminate between closely related strains, that is, the number of strains identified compared to the number of strains that exist). Importantly, a strain typing technique must be re-validated for every new application it is used for. Ultimately, failure to validate tests thoroughly may give a false or distorted sense of reality when they are applied.

What do strain typing results actually mean?

If two disease outbreaks are due to different strains of M.ptb the outbreaks are not connected. If two outbreaks are due to the same strain of M.ptb they might be connected, eg through trade in livestock, straying livestock or transfer of infection across a common property boundary fence. When MLA sponsored a large strain typing study in 1999 it was shown that BJD in dairy cattle was separate from BJD in beef cattle, and that OJD was distinct from BJD - in other words Johne's disease in these different livestock sectors were quite distinct. This was very useful epidemiological information.

The extent to which a strain typing method can separate closely related strains influences the extent to which it can be used in tracing investigations to identify the true source of infection. This issue is relevant in the context of human isolates of M.ptb - where do they really come from?

Strain typing does not tell us about the disease-causing potential of a particular M.ptb isolate in different species. This has been a research question for some years.


Strain typing in the epidemiological sense, refers to the use of genotypic variations within and between strains that can be used to reliably differentiate them. These genotypic variations must be validated as fit for purpose as was done with IS900 RFLP and IS1311 PCR-REA tests. A cautious approach to interpretation of strain typing results is required for disease tracing and in public health investigations.


The authors would like to thank the entire NSW Department of Primary Industries and University of Sydney Johne's disease research team, particularly Karren Plain, Shayne Fell, Francesca Galea, Vanessa Saunders Anna Waldron and Ann-Michele Whittington for the efforts and the following organisations who have supported John's Disease research for many years; Meat and Livestock Australia, Animal Health Australia, Sheepmeat Council of Australia, Cattle Council of Australia and WoolProducers Australia.


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