Bovine Models

16.3.1 Long-term bovine MAP challenge model

Because of the chronic nature of paratubercu­losis, bovine infection models that examined in­oculated animals for long periods (>6 months) are common (Table 16.3).

CFU, colony-forming units; IT, Intratonsillar; SC, Subcutaneous; ATCC, American Type Culture Collection; IFN, Interferon; LBT, Lymphocyte blastogenesis test; IL, Interleukin.

short-segment repeat genotype (e.g. 15g-5ggt genotype; Ghadiali et al. (2004)) or equivalent pulsed-field gel electrophoresis or amplified fragment-length polymorphism genotype may also be used (Hines et al., 2007b). This allows use of various local MAP strains and minimizes regulatory issues concerning the import of in­fectious organisms.

Types of inocula that have been ad­ministered include faeces from an infected animal, intestinal mucosal scrapings, lymph node homogenates or in vitro-cultured MAP (Table 16.3). In previous studies, MAP has been harvested from solid media (Herrold's egg yolk, Middlebrook 7H11, Middlebrook 7H10, Taylor's, Dubos) or from broth (Middlebrook 7H9). Inocula prepared from homogenized in­testinal tissue, lymphoid tissue or faeces have been used, but these are difficult to standardize and aliquot for numerous studies. To address these issues, the AMSC has suggested that the chosen strain of MAP should be propagated in vitro from a master seed stock to mid-log-phase growth. Middlebrook 7H9 broth, supplemented with Oleic AlbuminDextrose Catalase (OADC), mycobactin J and 1% glycerol, was recommend­ed as the culture medium (Hines et al., 2007b).

Storage of the inoculum at 4°C beyond 4 weeks may result in reduced viability (R. Whitlock, Philadelphia, 2008, personal com­munication) but storage up to 2 weeks at 4°C is generally considered acceptable. However, it is recommended that the stored inoculum should be incubated at 3 7°C for 2-3 h prior to admin­istration. All samples within a study should be treated in the same manner.

Oral, intragastric and parenteral routes of inoculation have been successfully used (Table 16.3). However, the oral route is rec­ommended by the AMSC as this most closely mimics natural exposure and allows MAP uptake by tonsillar tissue as well as the intes­tinal tract. The calf is induced to suckle from a syringe containing MAP mixed with a small volume of milk replacer or pasteurized milk. The mixture can also be gently expressed over the back of the tongue to induce swallowing.

Published doses range from 10-200 mg wet weight and 10⁶ to 10¹¹ CFU (Table 16.3) but were not based on standardized procedures. Doses of 5 ? 10⁸ CFU given on 2 consecutive days reliably resulted in infection in calves by 12-14 weeks post-inoculation (Sweeney et al., 2006). The AMSC recommends use of a stand­ard bovine challenge dose of approximately 10⁹ CFU/dose (100 mg wet weight) given on each of 2 successive days. Quantification of CFU should be by serial dilution and plating on solid medium known to support the organism without added antibiotics. Excessively large doses that produce clinical disease in cattle less than 18 months post-inoculation should be

Table 16.3. Bovine models for Mycobacterium avium subsp. paratuberculosis (MAP) infection.

A.M. Talaat eta∕.

Continued

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Table 16.3. Continued

A.M. Talaat eta∕.

^aND = not determined.

^bNR = not reported.

AGID₁ agargel immunodiffusion test; CFT₁CompIementfixation test; CIE₁ crossed Immunoelectrophoresis; FLOW,flow cytometry; NOX₁ nitric oxide; PI, post-inoculation; SEM,scanning electron microscopy; ÒÅÌ, transmission electron microscopy; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.

avoided as they are not typical in naturally oc­curring paratuberculosis (Hines et al., 2007b).

Previous studies reported challenge of cat­tle from 1 day of age to adulthood (Table 16.3).

Current diagnostic methods are ineffec­tive for screening of individual calves at or be­fore 8 weeks of age. Calves should be purchased from paratuberculosis-free farms to ensure lack of prior exposure. In the USA, herds that have achieved the equivalent of Status Level 3 or 4 in the National Voluntary Bovine Johne's Disease Status Control Program should be chosen as source herds (Hines et al., 2007b; Usda-Aphis, 2010). This equates to a closed herd with no his­tory of paratuberculosis in the previous 5 years, tested annually with at least one whole-herd negative serological test and one whole-herd negative faecal test (second lactation and older animals).

Calves should have received adequate quantities of colostrum. Milk replacer, if used, should be high quality and of animal origin (i.e. casein not soy protein) and high-quality rations should be fed. Routine vaccinations may be ad­ministered but should not be given on the same days as paratuberculosis vaccination or MAP administration.

Passive (pass-through) shedding occurs as early as 12 h after oral inoculation. Detection of passive shedding by culture provides addi­tional confirmation of inoculum viability and the sensitivity of the faecal culture method. Positive faecal culture results more than 14 days after inoculation should be considered shedding due to infection, except in very heav­ily contaminated environments (R. Whitlock, Philadelphia, 2008, personal communica­tion). The AMSC recommends that animals should have faecal cultures at least monthly during the course of the study. Gross exami­nation and culture of tissues for MAP as well as histopathological examination of tissues to identify acid-fast organisms and lesions char­acteristic of paratuberculosis should be per­formed in all studies. At the inoculation dose recommended, colonization of tissues can be detected by culture in most animals by 4-12 weeks after inoculation, although culture of multiple tissues (minimum of three ileum in­cluding ileocaecal valve, three jejunum, one duodenum, one spiral colon and three mesen­teric lymph nodes including ileocaecal nodes) is necessary. Although tissue samples should be culture-positive by 12 weeks, investigators should not always expect to find histologi­cal lesions at this early stage. A necropsy and histopathology scoring system should be used (Gonzalez etal., 2005; Hines etal., 2007b). The clinical status of the study animals should, at a minimum, be assessed and recorded monthly and at necropsy (Hines et al., 2007b).

The method of culturing faecal and tissue samples should permit quantification (or at least semi-quantification, e.g. by counting CFU on sol­id medium or time to positive detection in auto­mated liquid culture systems). Decontamination by incubation of faecal and tissue samples in 0.6% hexadecyl pyridinium chloride (HPC) for 14-16 h and 3 h, respectively, is strongly rec­ommended (Hines et al., 2007b).

16.3.2 Short-term bovine MAP challenge models

Experimental MAP infection models that em­ploy direct surgical access to the ileum for ad­ministration of MAP and collection of intestinal samples have been described (Allen et al., 2005, 2009). In general, these models are most use­ful for short-term study of host-pathogen in­teractions. The time frame of such studies can vary from a few hours to a few days or even a few weeks (Wu et al., 2007a). Many parameters should be the same as for long-term challenge, including strain, inoculum preparation and quantification, storage, animal selection criteria and quality control issues. Exceptions are age of administration, dose, experimental endpoints and sample collection.

Ileal cannulation model

In this model, calves are cannulated at 8 weeks of age under general anaesthesia, us­ing a modification of the method of Streeter et al. (1991), as previously described (Hines et al., 2007b; Allen et al., 2009). Calves are in­oculated in the ileum with 10¹⁰ CFU of MAP in 20 ml phosphate-buffered saline. MAP is taken up by M cells and by dendritic and epi­thelial cells of the ileum and jejunum within 30 min (Momotani et al., 1988; SigurSardottir et al., 1999, 2001). The inoculation procedure is repeated the next day. Ileal mucosal biop­sies can be obtained at various time intervals, to test for MAP infection and local immune responses.

Invasion/surgical model

Surgical incision and direct deposition of MAP into the ileum has been recently employed to establish a model for intestinal invasion to other organs within a few hours of infection (Wu et al., 2007a). Although infected calves survive the surgery and repeated biopsy sam­pling up to 10 months following infection, the focus of this model is to examine early MAP intestinal interactions. The movement of MAP from intestine to liver, spleen or mes­enteric lymph node was shown to differentiate between MAP strains with different virulence phenotypes.

Intestinal loop model

An alternative surgical approach is the ligat­ed intestinal loop model with an injected in­oculum of 3 ? 10⁹ CFU (~300mg wet weight) (Momotani et al., 1988; Khare et al., 2009). This is only suitable for studies related to bovine strain K-10, is recommended (Hines et al., 2007b).

The recommended method of inoculum preparation, quantification and storage is the same as stated above for the bovine model. The majority of studies used Middlebrook 7H9 + OADC + mycobactin J + glycerol or Tween 80 for in vitro cultivation. This medium with the addition of glycerol (1%), but without Tween or antibiotics, is considered to be the best choice (Hines et al., 2007b). The routes of administra­tion have varied (Table 16.4), and essentially all routes tested have been successful in establish­ing infection, with even the aerosol route result­ing in intestinal pathology (Harding, 1957). The oral route most closely parallels natural expo­sure and is generally considered the best route of administration.

The size of the challenge inoculum has also varied widely (Table 16.4), ranging from 2.3 7 to 200 mg and 3 ? 10⁷ to 8 ? 10¹⁰ CFU, and all were generally successful in establishing infection. Two consecutive daily doses of 10⁹ organisms (approximately 100 mg pelleted wet weight/dose and ~200 mg total dose) should establish infec­tion in most kids without overwhelming experi­mental interventions (Hines et al., 2007b).

The age at which to inoculate has var­ied from the day of birth to 10 months of age (Table 16.4). It is not known whether age-related resistance occurs in goats. The age of adminis­tration will depend on the experimental goals and endpoints, but generally the goats should be less than 4 months of age. Clinical disease is ex­pected to develop in a low percentage of animals at 9-10 months post-inoculation (Hines et al., 2007b).

The experimental endpoints will depend on the goals of the study. The minimal AMSC recommendations for goat experiments were to determine infection status through a com­bination of culture, PCR and histopathology, using quantitative or semi-quantitative meth­ods (Hines et al., 2007a, b). A lesion grading system for gross and histopathological findings (Hines et al., 2007a, b) should be used, with a sufficient range in values to allow statistical analysis. Recommendations for methods of faecal and tissue MAP culture and for sample handling are the same as for the bovine model. Like cattle, positive faecal cultures within 2 weeks or more post-inoculation should be con­sidered to be due to infection. Faecal cultures/ PCR should be performed at least monthly from all animals.

16.4.2 Short-term caprine MAP challenge models

In general, these models are used for short-term study of host-pathogen interactions. Many pa­rameters should be the same as for long-term challenge, including strain, inoculum prepara­tion and quantification, storage, animal selec­tion criteria and quality control issues.

Intestinal loop model

An intestinal loop model, as previously described in goats by SigurSardottir et al. (2001), can be used for studying initial paratuberculosis bac­terial-host interactions including bacterial attachment and internalization, bacterial locali­zation, cytokine regulation and early bacterial gene regulation. MAP strain, dose, quantifica­tion, culture medium, animal selection and sam­ple collection should be similar to the long-term challenge model (Hines et al., 2007b). Strain se­lection and quantification, animal selection and sample collection should also be similar to those for the long-term challenge model.

Table 16.4. Caprine models for Mycobacterium avium subsp. paratuberculosis (MAP) infection.

culture)

A.M. Talaat et al.

^aNR = not reported.

^bND = not determined.

ELISA, enzyme-linked immunosorbent assay; PCR₁ polymerase chain reaction; TNF₁ tumour necrosis factor; TGF₁ tumour growth factor; CFU₁ colony-forming units; AGID₁ agar gel immunodiffusion test; OFT, complement fixation test; LBT₁ lymphocyte blastogenesis test; ATCC, American Type Culture Collection; IFN, Interferon; LBT, Lymphocyte blastogenesis test; FLOW, Flow cytometry; IL, Interleukin.

Everted intestine sleeve model

An everted intestine sleeve model, as described by SigurSardottir et al. (2005), is also useful for stud­ying initial host-bacterial interactions, such as bacterial attachment and internalization, bacteri­al localization and early bacterial gene regulation, but may have limitations when evaluating host gene and early cytokine regulation in response to infection. Approximately 1.0-cm segments of small intestine are excised, everted, washed, main­tained in tissue culture and bathed in a suspension of MAP for short periods of time. Strain selection and quantification, animal selection and sample collection should be similar to those for the long­term challenge model.

16.5