Characteristics of Infection and Disease

12.3.1 Clinical signs and clinical pathology

The clinical signs of paratuberculosis in sheep are limited to chronic weight loss, which may oc­cur from 2 years of age, with most animals suc­cumbing to disease at 3-5 years of age.

Most sheep that die of paratuberculosis have normal faecal pellets. Diarrhoea is not considered to be a feature of paratuberculosis in small ruminants, except in the terminal stages of disease. In a study of 50 sheep with clinical paratuberculosis, most were emaciated, half had normal faecal pellets, 30% had soft-formed faeces and 20% had severe diarrhoea (Carrigan and Seaman, 1990).

12.2.2 The strain of MAP

The strain of MAP that infects sheep will vary with the predominant strain type in the geo­graphical region and whether or not sheep co­habit with other species (Begg and Whittington, 2008; Sohal et al., 2019). It is becoming clear from experimental infections that the MAP strain can have a significant impact on infection rates, the severity of pathology and the num­ber of clinically affected animals (Verna et al., 2007). Sheep develop more severe lesions when experimentally infected with MAP-S strains rather than MAP-C strains (Fernandez et al., 2014). Different isolates of MAP-C inoculated into sheep can also result in different pathologi­cal and immunological outcomes (Verna et al., 2007; Fernandez et al., 2014). These factors may be responsible for the differences in dis­ease outcome and prevalence between flocks in different geographical regions or countries. Quarantine authorities in endemic areas should consider excluding animals that harbour par­ticular strains of MAP that are not already found in that area. MAP strains are described in more detail elsewhere (see Chapter 6, this volume).

12.3.2 Pathology - gross and microscopic lesions

Advanced cases of paratuberculosis in sheep typically have thickening of the mucosa of the terminal ileum; the wall may be oedematous and the mucosa thrown up into transverse ridges; there may be cording of the subserosal lymphat­ics, and these are clearly visible and palpable. The caecum and colon may also be involved. In the cases described by Carrigan and Seaman (1990), lymphadenomegaly was present in 38% of the sheep. Histologically, there was moder­ate to severe granulomatous enteritis (94% of sheep), typhlitis (74%) and colitis (14%); the rec­tum was involved in 2%; lymphocytic infiltrates were also present in most sheep.

In tissue sections from the same animals, 88% of sheep had abundant acid-fast bacilli (AFB) and 12% had few AFB (Carrigan and Seaman, 1990). This dichotomy led others to describe two distinct forms of disease in sheep: paucibacillary and multibacillary (Clarke and Little, 1996). Clarke et al. (1996) used a lesion grading system: sheep with a mean of 0-10 AFB per macrophage were called paucibacil- lary, and those with >10 were multibacillary. Animals in both groups were emaciated and had carcass oedema; the multibacillary animals were more likely to have detectable gross lesions in the intestine and associated lymph nodes. Histologically, the paucibacillary group tended to have a lymphocytic infiltrate with fewer mac­rophages compared with the multibacillary group, in which macrophages dominated the infiltrate (Clarke and Little, 1996). A more de­tailed lesion-grading system with five categories (1, 2, 3a, 3b, 3c) was proposed by Perez et al. (1996). Mycobacteria were not visible in most sections from sheep with Type 1 (mild) and Type 3c (severe) lesions but were abundant in Type 3b. Although the authors were far from certain, an ordinal progression of lesions from Type 1 to Type 2 and beyond was implied. However, the time of infection and the influence of a success­ful immune response in hindering progression and AFB numbers were factors that could not be accounted for (Perez et al., 1996).

The prevalence of the various types of pa­thology within an infected flock is unclear due to biases in sampling. The progression of lesions is also unknown. It is clear, however, that the first lesions develop in the ileal Peyer's patches and then spread to the surrounding mucosa (Perez et al., 1996).

12.3.3 Route of infection and transmission between individuals

The most common route of infection is faecal- oral. Infection can also be spread by intrauter­ine and transmammary transmission (Lambeth et al., 2004; Verin et al., 2016). The intratonsil- lar route of infection may also play a role (Begg et al., 2005), although this is unlikely to be of significance in the natural infection process, mainly because the large number of bacteria that are ingested and swallowed will heavily outweigh those that may become lodged in the tonsil. Experimental infection data have shown that sheep infected by alternative routes de­velop altered immunological response profiles compared with those infected orally (Begg et al., 2005). Lambs infected via the intratonsillar route compared with oral-challenged animals had increased interferon gamma (IFN-γ), lym­phocyte proliferation and antibody levels. This may partly explain the large variation in im­mune responses recorded in groups of naturally infected sheep. This has implications for the de­sign of experimental infection models and the outcomes observed from this type of research.

Ileal Peyer's patches in sheep, unlike those in humans, show prenatal maturation, with antigen-independent lymphopoiesis and a rate of lymphocyte production greater than in the thymus. The ileal Peyer's patches in sheep are unique in that they are required for the develop­ment of B cells and like the thymus they involute with age (Landsverk et al., 1991).

2001). The lymphoid follicles of ileal Peyer's patches contain large numbers of B cells but few T cells (Landsverk et al., 1991). While jeju­nal Peyer's patches persist in adult animals, they contain clusters of B cells and have numerous CD4+ T cells in the associated lymphoid follicle (Landsverk et al., 1991). In sheep, the ileal and jejunal Peyer's patches appear to have different functions. The jejunal Peyer's patches are essen­tial for producing mucosal responses, while the ileal Peyer's patches seed the systemic immune organs with B cells (Mutwiri et al., 1999). An intestinal loop model in lambs has shown that higher numbers of MAP translocate across the intestinal wall from sections of ileum than jeju­num (Ponnusamy et al., 2013).

It is generally accepted that the entrance of MAP through the intestinal wall is via M cells overlying the Peyer's patches from where the bacteria are engulfed by macrophages (Momotani et al., 1988). Enterocytes may also allow MAP translocation, but less so than M cells (Ponnusamy et al., 2013). After this, very little is known about the host-pathogen inter­action. There is a period of clinical latency be­tween when the animal is infected, typically as a lamb, and the clinical signs of disease. During the time immediately after oral challenge, most of the bacteria are passively shed into the envi­ronment (Reddacliff and Whittington, 2003). Presumably, organisms taken into the intes­tines are in such low numbers that animals may remain unaffected for months or years.

12.3.4 Immunopathobiology

MAP dissemination and propagation are possi­bly due to decreased cellular immunity allowing infection to develop into clinical disease (Stabel, 2000, 2006). Containment and restriction of replication of the bacteria within the mac­rophage are critical, as these cells can tolerate low numbers of bacteria, whereas higher num­bers of MAP are cytotoxic and result in apopto­sis (Merkal et al., 1968; Bannantine and Stabel,

2002). As bacterial numbers increase within the gut wall, the number of intracellular bac­teria sloughed off into the lumen will increase. This results in larger numbers of bacteria within faecal material and an increased chance of de­tection by faecal culture. It is therefore unsur­prising that sheep with multibacillary lesions are more likely to shed MAP in faeces than animals with paucibacillary lesions (Whittington et al., 2000a). A sheep with multibacillary disease will shed on average 10⁸ bacteria per gram of faeces or up to 10¹⁰ bacteria per day (Whittington et al., 2000b).

Major histocompatibilty complex (MHC) processing and antigen presentation may be downregulated in MAP infections (Berger and Griffin, 2006). Production and expression of MHC class I and II molecules may be reduced in MAP-infected macrophages (Alzuherri et al., 199 7; Berger and Griffin, 2006). Expression of lymphocyte function-associated antigen, a molecule involved with cell-to-cell interactions, is also reduced in MAP-infected macrophages (Alzuherri et al., 199 7).

Other innate pathways that are activated during a MAP infection include the pattern recognition receptors (PRR). These include the Toll-like receptor family (TLR), involved in recog­nition of binding of pathogens by macrophages and other cells of the innate immune system.

Paucibacillary and multibacillary disease states in sheep correlate broadly with the pre­dominant pathway of immunity (Clarke, 199 7). Sheep with paucibacillary lesions are likely to have an associated cell-mediated immune (CMI) response, with large numbers of lymphocytes at the site of disease (Clarke et al., 1996). Lesions found in the small intestine show an increase in the numbers of CD4+ T cells and gamma delta (γδ) T cells, and the ratio of CD4+ to CD8+ is >1 (Little et al., 1996; Reddacliff et al., 2004). Increased antigen-specific lymphoproliferative responses can be seen from blood and gut cells isolated from sheep with paucibacillary dis­ease (Kurade et al., 2004; Kurade and Tripathi, 2008). Increased levels of T helper-1 (Th1) cy­tokines are produced within the intestinal tissues by leucocytes in animals with paucibacillary le­sions. The predominant cytokines are IFN-γ, in­terleukin 2 (IL-2) and IL-12 (Clarke et al., 1996; Burrells et al., 1999; Smeed et al., 2007; Rossi et al., 2009). Th17 cells may also play a role in the development of paucibacillary lesions, with upregulation of the master regulator T-cell tran­scription factors TBX21, RORC2 and RORA be­ing observed (Nicol et al., 2016). CMI responses, as measured by lymphocyte transformation and IFN-γ assays, can be variable along the chain of mesenteric lymph nodes. The lowest level of re­activity is found in leucocytes in the intestinal lamina propria (Burrells et al., 1998; Begg and Griffin, 2005; Begg et al., 2005).

In contrast to paucibacillary lesions, multibacillary lesions typically contain larger numbers of macrophages, and sheep with these lesions typically have a strong Th2 (humoral an­tibody) response. Animals with multibacillary lesions have a significantly reduced lymphocyte proliferation response (Burrells et al., 1998; Kurade and Tripathi, 2008). The number of CD4+ T cells in the lesion decreases and the ra­tio of CD4+ to CD8+ T cells changes from >1 to of choice for detecting MAP-infected sheep flocks in Australia (Sergeant, 2001). MAP culture is described in more detail elsewhere (see Chapter 18, this volume).

12.4.2 PCR

In recent years, the sensitivities of PCR-based as­says for the detection of MAP from both faecal and tissue samples have greatly improved. This is primarily due to improved DNA extraction techniques and the use of quantitative or real­time PCR assays. The sensitivity of PCR for fae­cal samples was reported to be equivalent to that of culture with a high specificity due to primers designed to avoid detection of environmental bacteria (Kawaji et al., 2007). The sensitivity of the PCR assays appears to be as good as if not better than faecal culture (Plain et al., 2014; Bauman et al., 2016a; Sonawane and Tripathi, 2016; Arsenault et al., 2019), although valida­tion studies for the assays following the World Organisation for Animal Health (OIE) and other guidelines (Gardner et al., 2011) are few and far between for sheep, for example Bauman et al. (2016a) and Plain et al. (2014) An extensive review of the benefits and disadvantages of PCR is given elsewhere (see Chapter 19, this volume).

Diagnosis by PCR and culture for faecal samples are dependent on the stage of infection: late-stage disease is typically characterized by persistent heavy shedding in most affected ani­mals. In the first months after exposure, intermit­tent or transient shedding can occur. Detection of this early shedding does not indicate the final outcome of the exposure (disease or resilience) (Begg et al., 2015), although animals that shed higher numbers of MAP are more likely to de­velop disease (de Silva et al., 2013).

12.5