Progression pathways

A number of studies support the existence of two possible pathological pathways following infection with MAP: one leading to recovery and the other to progressive disease. These distinct pathways can be evaluated by multiple testing strategies identifying infection load within host tissues, damage to the host and shedding into the environment. Recovery can be

illustrated by a variety of outcomes, e.g., a lack of progression to clinical disease, a regression

of specific lesions (recovery from disease) and the potential clearing of MAP infection from the tissues (recovery from infection).

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4.3.2.1

Immunological response

The feature of immuno-pathology of PTB is a polarization of the immune response for a given animal at a given point in time. The host immune response is either predominantly cell mediated (Th1 oriented) in paucibacillary animals, or predominantly humoral (Th2 oriented) in multibacillary animals (Chiodini et al., 1984; Pérez et al., 1996). This polarisation of immunity is controlled by the activation of distinct cellular receptors and cytokines (Abendaño et al., 2014). This determines two differing patterns of T cell activation in the two pathological types (Smeed

et al., 2007; Smeed et al., 2010). Cytokines such as IFNJ and interleukin-10 (IL-10) are thought

to be pivotal in the establishment or the failure, respectively, of a Th1 mediated protective immune response.

Pro-inflammatory IFNJ mediates the adaptive immune response against intra-cellular

pathogens (Koets and Gröhn, 2015). High levels of IFNJ are observed in early stages of

infection, whereas a decline of IFNγ is predictive of progression to severe pathology and

disease (de Silva et al., 2013). Genetic expression of IFNJ is up-regulated in intestinal tissues of

sheep presenting paucibacillary disease compared to multibacillary (Smeed et al., 2007). On the other hand, IL-10 is an immuno-suppressive cytokine induced by MAP to evade the cell- mediated adaptive immune system, thus allowing the persistence of MAP in the macrophages (de Silva et al., 2013; Koets and Gröhn, 2015). In the early days of infection, a correlation was noted between an increased anti-apoptotic, anti-destructive response by macrophages infected in vitro, and the survival of MAP in these macrophages (Abendaño et al., 2014). This was mediated by differential cytokine regulation, in particular upregulation of anti- inflammatory IL-10 and down-regulation of pro-inflammatory IL-2. The level of IL-10 increases progressively in infected animals as the disease progresses (de Silva et al., 2011; de Silva et al., 2013) However, the precise role of IL-10 is not yet fully elucidated. The cytokine is thought to be associated with the failure of Th1 mediated adaptive immune response and hence is used as a marker of disease progression. However, an elevation of IL-10 levels in peripheral blood can be observed as early as four months post-inoculation in experimentally infected sheep (de Silva et al., 2011) and, is associated with resistance to disease later in the course of progression (de Silva et al., 2013). This suggests that the immunosuppressive effect of IL-10 might have a protective effect at the animal level by limiting the damage of the intestinal tissues (de Silva et al., 2013; Koets and Gröhn, 2015), at least in animals that control the infection. It is not clear however, whether measures of the adaptive immune response adequately reflect the local intestinal immunity (Koets and Gröhn, 2015). High peripheral blood levels of IL-10 could also

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result from a failure act locally. A variation of the adaptive immune response and particularly the expression of IL-10 can be observed at various sites (blood versus mesenteric lymph nodes versus ileal tissue). The existence of distinctly different immune pathways can also be illustrated by the clear differences in the IL-10 response pattern between paucibacillary and multibacillary sheep. IL-10 is upregulated in the ileal wall of sheep with multibacillary disease presenting a Th2 dominant response (Smeed et al., 2007). On the other hand, de Silva et al. (2011) observed that the secretion of IL-10 in the mesenteric lymph nodes was lower in multibacillary sheep compared to paucibacillary.

This immune polarization was thought to be relatively antagonistic (Clarke, 1997). Thereupon the cell-mediated immune response would correspond to a “controlled” infection observed in early stages and paucibacillary disease. A later switch to a non-protective humoral response would then be associated with multibacillary disease, determining the onset of clinical disease. An excessive cell-mediated immune response reportedly led to advanced inflammation of the intestine walls in severe paucibacillary cases referred to in 4.3.1.2. (Chiodini et al., 1984; Clarke, 1997). Thus, paucibacillary infection can also be at the end stage of PTB in sheep unlike cattle (Smeed et al., 2007). A switch between Th1 and Th2 responses could be triggered by various factors: T cell exhaustion, MAP exposure dose, macrophage bursting size and other host-level metabolic triggers (Koets and Gröhn, 2015).

However, recent research suggests a more complex immunologic response to MAP infection than the classical switch hypothesis (early predominance of Th1, then Th2 response). This was based on the observation that half the sheep experimentally infected with MAP actually

presented a combined antibody and INFJ response at an early stage of infection (Begg et al.,

2011). Simultaneous cellular and humoral responses were also observed (Fernandez et al., 2015), as well as a lack of early interferon gamma production in sheep with only focal intestinal lesions. A transient early elevation of IL-10, as well as B cells, was noted in cattle (Koets and

Gröhn, 2015). Moreover, recent research on bovine tuberculosis suggests that IFNJ may be

more correlated with bacterial load and lesion severity, which can fluctuate over time within an individual, rather than a marker of protection (Koets and Gröhn, 2015). These recent breakthroughs in immuno-pathology of mycobacterial infection conclude that progression to disease could result from a generalised failure of the immune system where the Th1 response fails first, rather than switches.

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4.3.2.2

Presence of MAP in intestinal tissues

The presence of MAP organisms in intestinal tissues of infected sheep can be demonstrated directly by culture, or indirectly by the presence of microscopic lesions characteristic of PTB. These methods, based on serial biopsies or sequential necropsies, can show the onset of infection or pathology. This is followed by two possible pathways, a progressive infection towards disease in some sheep or, a recovery (resolution of lesions and/or clearing of infection) in others.

According to the experiment of Begara-McGorum et al. (1998), neonatal lambs can become histologically positive in their Peyer’s patches as early as 18 days after inoculation, although such early lesions may not harbour visible MAP. Those authors proposed early MAP distribution as an indication of initial local propagation within intestinal tissue followed by a dissemination phase.

Similarly, one month after the end of a 10-week challenge period in which 1.8 x 108 total CFU

was administered, MAP could be detected by culture in the intestinal mucosa of 13/14 sheep

slaughtered early in the study. MAP was detected in quantities estimated between 1.6 x 104 to

106 viable MAP/g of mucosa, thus indicating an intestinal location of MAP with likely active

division of the organism (Gilmour et al., 1965b). In a second group slaughtered nine months

post challenge, 9/14 were intestinal culture positive, suggesting a decrease5 of infection load in

the guts over time for some of the sheep. A second experiment in the same paper, using the same inoculum dose, suggested a possible recovery from pathology. While 8/10 sheep culled at any point in the first year after inoculation harboured MAP specific intestinal lesions, only

3/8 sheep culled 18 months after inoculation did5.

A 1968 study involved three-week-old lambs (n=18) given a single oral challenge of 1010 _{to 10}11

MAP (cattle strain) from a tissue homogenate. The aim was to describe the chronology of infection and pathological processes using sequential necropsy of animals at one, four, eight,16 days and one, two, four, eight, and 16 months post inoculation (Kluge et al., 1968). The first AFB were identified within macrophages in lymph follicles of the intestinal wall at 1- month post challenge, along with the first intestinal lesions. The peak of lesions in the intestine corresponded with a sudden peak in the number of AFB detected in tissues, between four and eight months after inoculation. This period corresponded with the onset of clinical signs at five and six months post-inoculation. After 16 months, the authors noted that surviving lambs were

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recovering from disease, with histological recovery of tissue lesions in which very few AFB could be detected and few viable MAP could be grown. The fast onset of extensive multibacillary lesions and clinical signs could be related to the high inoculum dose of likely potent (not laboratory attenuated) MAP from a tissue homogenate. It is also noticeable that despite onset of relatively severe pathology in the first year, the sheep that were not culled during this time appeared to be recovering from disease around 16 months post-inoculation. This propensity to recover from extensive lesions was noted elsewhere as a characteristic of PTB in sheep infected with the cattle strain of MAP (see 4.5.4) although at the time of the study, technologies to allow such distinction were not available.

Gilmour et al. (1977) inoculated 30 sheep with an oral challenge with a total dose estimated at

109 _{MAP over 10 weeks. Post-mortem examinations were performed with histology and tissue}

culture on a proportion of animals at regular intervals over 27 months. Necropsies performed in the first year after inoculation demonstrated more frequent and higher concentrations of bacteria and more histologic damage than those performed throughout the second year. Of those sacrificed between 22 and 27 months post inoculation (n=11), six had no lesions and five harboured mild lesions suggestive of healing of previously active lesions. Five of the sheep underwent serial biopsies, allowing a follow up of the evolution of infection status. At five and 11 months post challenge, all five were infected according to either histology or culture of intestinal wall. Between 17 and 27 months, two sheep became consistently negative to both tissue culture and histological lesions up until necropsy. One sheep apparently cleared

infection from its tissues although microscopic lesions were seen at post-mortem after 27

months and two sheep developed more severe pathology. The authors conclude that sheep infected orally become colonised initially. Some then totally recover from infection while others become permanent carriers (with lesions), among which a further proportion become clinical while the rest recover from sub-clinical infection.

In a more recent experiment, 30 weaned Merino sheep were inoculated with a moderate to

high dose (5 x 108 viable MAP) of an ovine strain obtained directly from a tissue homogenate

(Begg et al., 2005). Sequential culling was performed at different times. All of a random subset of six animals were tissue culture positive 13 months after inoculation and five presented histological lesions. In the following year, 10 of the sheep became clinically affected, six with severe histological lesions, though only three were tissue culture positive. At the end of the trial (22 months post inoculation), the six animals that had survived were all tissue culture negative, and only half presented minor lesions. Since the infection rate one year post- challenge was nearly 100%, it is likely that this level of experimental challenge achieved active

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infection of all the sheep. These results suggest that infected sheep could experience a primary phase of active infection where MAP invades the tissues, causing lesions in most animals. Some sheep harbouring severe lesions expressed the disease clinically. All the clinically affected animals in this experiment were identified based on weight loss between 11 to 21 months post challenge, with a median time of 15 months (Begg et al., 2005). The six sheep without clinical signs after 22 months recovered and even cleared (or controlled) the infection below detection limits.

An observational study of natural infection with MAP (Dennis et al., 2010), followed 77 sheep from a flock with clinical PTB from 10 to 36 months of age. Serial biopsies of intestinal tissues and mesenteric lymph nodes were performed by laparotomy. Among the 46 (60%) infected sheep detected by tissue culture, six cleared MAP from their tissues by the end of the trial and 12 died of clinical disease. Thus, the proportion which recovered from infection was at least 13%. As sheep were naturally infected rather than experimentally challenged, we do not know when infection took place. If infection took place early in life, there is no way to tell how many of the 31 sheep had already been infected but recovered before the study began. All 12 sheep dying of clinical PTB had progressed in less than two years from having no intestinal lesions to more advanced intestinal pathology, with 11/12 severe multibacillary lesions at the time of death. Serial biopsies are very rare in the literature of ovine PTB. By allowing disease progression within an animal to be monitored, this technique is well suited to establish a progression pattern although the stress of anaesthesia/surgery might alter the natural course of the disease.

4.3.2.3

MAP in faeces

Faecal shedding is more difficult to detect than histo-pathology due to the sub-optimum sensitivity of faecal culture and the intermittence of shedding (possibly confounded by shedding levels below the limit of detection). However, shedding can be monitored by repeatedly collecting samples over time.

Stewart et al. (2004) inoculated either at six months of age with a cattle-type (10 sheep) or at 10 months of age with an ovine-type of MAP (10 sheep), and monitored them for 54 and 35 months, respectively, equivalent to a typical productive life of sheep. Faecal culture revealed that shedding started as early as two months post inoculation in nearly all of the 12 shedders. Thereafter, two patterns were apparent: ten sheep shed intermittently and transiently at some point between two and 16 months after challenge and thereafter stopped shedding permanently. The other two sheep were persistent shedders and were the only animals that

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developed clinical disease, occurring at 20 and 32 months after challenge (Stewart et al., 2004). Although the evidence is tenuous, this study is the only one to follow the sheep long enough to suggest a long-term pattern. Findings suggested that only a proportion of infected sheep progressed towards disease, while all those that did not progress shed only transiently before stopping shedding for an extended period compatible with the rest of their productive life. These animals might either eliminate MAP infection or control it below the level of detection by faecal culture.

In another trial (Kawaji et al., 2011), 38 four-month-old sheep were inoculated and followed up to 13 months after challenge. Faecal shedding was detected via direct quantitative PCR. At eight months post-challenge, all 38 animals shed detectable MAP. At thirteen months, qPCR failed to detect MAP in faecal samples from five sheep. This suggests that these were transient shedders, although intermittent shedding could not be ruled out. At the first sampling at four months, all shedders excreted low levels of MAP DNA, while subsequent samplings allowed the distinction of low and high shedders. At the last sampling 13 months after challenge, there was a clear difference between low and high shedders that were several orders of magnitude apart in MAP DNA quantities, plus a proportion that stopped shedding as described by Stewart et al. (2004). Sheep shedding high loads of MAP at 13 months post-challenge had concomitant

signs of disease progression, i.e. advanced pathology at post-mortem.

4.3.2.4

Conclusions: progression pathways

- All measurable outcomes of PTB infection (histology, tissue infection and faecal

shedding) display a similar pattern of an early transient active infection stage followed by either control/remission or irreversible progression to severe disease/high shedding.

- This dichotomy in the disease is mediated by a polarization of the underlying immune

response.

- This can be modelled by two possible pathways following infection with MAP: a non-

progressor pathway leading to recovery and a progressor pathway leading to disease.