Data extraction and standardisation

Individual sheep data were retrieved from all the studies (reconstructed from summary data if necessary), with the aim of matching the experimental protocol and the observed individual outcome. Outcomes that could not be inferred at the individual sheep level were dismissed. To avoid duplication, care was taken to identify and exclude cases where the same experiment was reported several times in different papers by the same authors. Any extra information in those cases was retained when, for example, the experimental outcome was different and also used to double-check the data. Each individual sheep was only included once (avoiding duplicates). When the information from the paper was confusing (example: numbers of sheep not adding up, uncertainty about the strain type of MAP) or some of it missing (example: age/breed of the sheep) authors were contacted by email to clarify. We tried to contact eleven authors and reached nine, of which eight gave useful information and sometimes additional data. The literature search and data extraction was performed by the primary author and the entire process was repeated twice several months apart.

To evaluate the effect of MAP inoculum dose on various pathological outcomes, the total dose administered singly or over successive challenges over a period of time was determined for all sheep from all the papers included. The time of inoculum is defined as when the first dose of MAP was administered. The concentration of MAP in the inoculum (dose) was expressed

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- McFarland turbidity scale for a suspension of MAP,

- Direct microscopic count,

- Most probable number (MPN) in liquid culture ,

- Plate count,

- Pelleted bacterium weight (dry or wet weight): these were retro-converted into CFU

equivalents using the equivalence published in recent guidelines (Hines et al., 2007),

- Mucosa weight of intestine tissue: in one study (Stewart et al., 2004) a subset of the

inoculated sheep (10 animals) were dosed with a given weight of intestinal mucosa from a clinical case of paratuberculosis, with no estimation of MAP numbers. These observations were kept for the descriptive analysis but removed in all regression models where the effect of dose was evaluated.

These enumeration methods were divided into two categories: culture-based methods enumerating viable MAP (MPN, plate count, CFU equivalent of bacterium pelleted weight) or total viable and non-viable MAP organisms (turbidity, direct microscopic count).

6.3.2.1

Outcome definitions

The following outcomes were subjected to meta-analysis:

- Presence of MAP in the tissues (yes/no), either from small intestines or regional mesenteric

lymph nodes, detected by bacteriology (tissue culture), immuno-histo-chemistry or tissue PCR. This outcome is a marker for the colonisation of the intestinal tract with MAP with or without tissue lesions.

- Presence of MAP-specific lesions of any grade in the wall of the small intestine by

histological examination: this is a marker of active infection where the cellular immunity responds to the presence of MAP in tissues. The severity of lesions was determined when possible from the described individual histology.

- Mild-moderate lesions (type 1 and 2) were considered markers for early disease

stages from which it was possible to recover or progress to more severe disease.

- Severe lesions presented a diffuse cellular infiltrate of macrophages and/or

lymphocytes involving structures beyond the lamina propria in areas of the sub- mucosa and mucosa, such that the ultra-structure of the intestinal villosities was altered (atrophy). They were grade 3 lesions according to the Perez score (Pérez et al., 1996), also called lepromatous lesions in some early publications. The presence of these lesions was considered an early marker for onset of a clinical stage (sub- clinically affected, then clinically affected).

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- Progressor: Binary covariate representing a marker of progression for every sheep.

Progression was defined as presenting severe histological gut lesions or clinical signs. The presence of clinical signs was usually monitored by a loss of weight or low body condition score. If both conditions (histology with recording of lesion severity and assessment of clinical status of the animals) were fulfilled, and the individual correspondence could be established, the progressor covariate was set to 1 when the sheep was positive to either test (versus 0 if negative to both). If only one test was performed, this partial information informed the progressor status. Some sources did not provide detailed results with sheep ID but pooled results instead, so that both tests were performed but no formal correspondence at the individual level could be established between the different outcomes. In this case, the test that identified the largest number of sheep as progressors was used, and the information of the other test was dismissed to minimize sources of bias.

- Faecal shedding: for a small subset of animals, faecal shedding was monitored serially

(more than once). We recorded the results of faecal shedding assessed by faecal culture and faecal PCR. Results obtained by faecal smears were dismissed due to the lack of sensitivity of this technique.

6.3.2.2

Covariate definitions

The covariates thought to have an impact on the outcome (from Chapter 4) and retrieved for each sheep were as follows:

- Time to post-mortem (ttpm): most outcomes were evaluated at post-mortem, hence the

time from first challenge to post-mortem was a variable of interest.

- Dose: the log of the total estimated inoculum dose received by each sheep during the

challenge process.

- Enumeration method: a two-level covariate representing the enumeration method used in

the paper to establish the inoculum dose, either MAP CFU using culture-based methods (a proxy for viable MAP or cultivable MAP) or enumerating the total number of MAP organisms in the inoculum irrespective of viability (qPCR, direct microscopic counts, turbidity estimates). A potential differential bias may exist between these because the enumeration of total MAP cells likely overestimated viable MAP counts (see Chapter 4). Models were fitted using the reported dose or a dose calculated according to guidelines of Hines et al. (2007). The potential bias was evaluated in all final models by including an interaction term between the dose and a binary variable representing the enumeration method, viable or total MAP.

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- Inoculum type: a two-level covariate representing whether the inoculum was made of

laboratory passaged MAP (pure culture of MAP) or directly from tissue homogenate of a clinical case of paratuberculosis.

- Strain: a two-level covariate representing the strain type (type I or “ovine strain” versus

type II or “bovine strain”). The strain type was evaluated in a proportion of studies using PCR of the IS1311 fragment, which is considered perfectly specific. For studies not using molecular diagnostic tests, we determined the most likely strain by an educated guess using a combination of information about the cultural and biological characteristics of the strains, epidemiological information and in some cases by contacting the authors of the studies,

guided by expert advice (Karen Stevenson8 and Marian Price-Carter9).

- Age of the sheep at dosing: four mutually exclusive methods of coding the effect of age in

each model were used to find the best way to account for age in each distinct model (corresponding to a distinct dataset):

- age as a continuous variable,

- age category: lamb = 0 to 3 months old, hoggets = 4 to 12 months old, two-tooth = 13

to 24 months old, adult = more than 24 months old.

- lamb: 1 if the sheep was less than 3 months old (0 otherwise).

- young: 1 if the sheep was in its first year of age (0 otherwise).

- Breed: there was a great diversity of breeds (17 breeds and crosses plus unknown), hence

they were categorised as “Romney and crosses”, “Merino and crosses”, “Other British breeds”, “Mediterranean and Middle East breeds”, or “others and unknown”.

A general assumption of all experimental challenge models included in this meta-analysis was

that all enrolled sheep had not been infected by MAP prior to the time of inoculation (t0). To

take that into account, each animal in the dataset had two observations: one at t0 with all

outcome variables set to 0, and one at the actual time of post-mortem with the outcome

variables observed at post-mortem, and with the same exposure variables at both time points.

8

Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, Scotland, UK

9

Hopkirk Research Institute, AgResearch Grasslands, Tennent Drive, Private Bag 11008 Palmerston North 4442, New Zealand

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