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published online 6 February 2013Vet PatholFerreras and V. Pérez

L. Delgado, J. F. García Marín, M. Muñoz, J. Benavides, R. A. Juste, C. García-Pariente, M. Fuertes, J. González, M. C. paratuberculosisSubspecies Mycobacterium avium Doses of

Pathological Findings in Young and Adult Sheep Following Experimental Infection With 2 Different  

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Pathological Findings in Young and AdultSheep Following Experimental InfectionWith 2 Different Doses of Mycobacteriumavium Subspecies paratuberculosis

L. Delgado1, J. F. Garcıa Marın1, M. Munoz1, J. Benavides1,R. A. Juste2, C. Garcıa-Pariente1, M. Fuertes1, J. Gonzalez1,M. C. Ferreras1, and V. Perez1

AbstractMycobacterium avium subsp paratuberculosis (Map) is assumed to infect young ruminants; however, little is known concerning thepossibility of adult animals becoming infected. An experimental infection was conducted to establish the effect of age and doses ofMap on susceptibility to paratuberculosis in sheep. Sixteen of twenty-four 1.5-month-old Churra lambs and 23 of 30 adult ewes(from 2–11 years old) were orally challenged with an ovine field strain of Map. Thirteen ewes and 8 lambs were infected with ahigh dose (HD) and 10 adult sheep and 8 lambs with a low dose (LD) of Map. The remaining animals were unchallenged controls.Animals were euthanized at 110 to 120 and 210 to 220 days postinfection. Histological, bacteriological, and nested polymerasechain reaction (PCR) studies were conducted in samples of intestine and related lymphoid tissue (Peyer patches, lymph nodes).Animals were classified according to their lesions. The number of granulomas was counted in 3 tissue sections from each sample.Only the HD groups showed lesions associated with paratuberculosis (92.3% of ewes and 100% of lambs). Adults had lesionscharacterized by few small demarcated focal granulomas restricted to the lymphoid tissue, whereas granulomas were morenumerous and larger, appearing in the lamina propria unrelated to lymphoid tissue, in the lambs. Only HD-infected lambs werepositive to culture, whereas nested PCR also detected positive HD ewes and some LD animals. These results suggest that adultsheep can become infected by Map, as seen by the development of lesions, but they are focal and restricted to the lymphoid tissue.

Keywordsage, granulomas, intestine, mycobacteria, sheep, paratuberculosis

Paratuberculosis or Johne disease is a chronic granulomatous

inflammation of the intestine and related lymph nodes caused

by Mycobacterium avium subsp paratuberculosis (Map) that

affects both domestic and wild ruminants. It occurs worldwide

and is responsible for heavy economic losses to domestic

livestock industries. This infectious disease is characterized

by a long incubation period, which leads to weight loss with

or without diarrhea and eventual death.6

The pathological response of the infected animals varies

widely, and different lesional classifications have been estab-

lished in ovine, caprine, and bovine species depending on the

presence, intensity, and distribution of granulomatous lesions

as well as the number of bacteria.10,19,33 Briefly, these patholo-

gical forms are divided into focal forms, characterized by small

granulomas restricted to the lymphoid tissue of the Peyer

patches; multifocal forms, where the granulomas spread into

the lamina propria related or not to the lymphoid tissue; and

diffuse forms, related to clinical signs, where wide areas of the

mucosa are severely affected. It has been proposed that these

different pathological responses represent different stages of

the disease, where the immunological status of the animal plays

an important role.10,19,33

In flocks with endemic paratuberculosis, it is common to

find animals showing focal lesions, similar to those observed

in the initial stages of experimental Map infection.21,31 This

observation raised the hypothesis that they could be considered

1 Departamento de Sanidad Animal (Anatomıa Patologica), Instituto de Gana-

derıa de Montana (CSIC-ULE), Facultad de Veterinaria, Universidad de Leon,

Leon, Spain2 Departamento de Sanidad Animal, NEIKER-Tecnalia, Bizkaia, Spain

Corresponding Author:

V. Perez, Departamento de Sanidad Animal, Instituto de Ganaderıa de Montana

(CSIC-ULE), Facultad de Veterinaria, Universidad de Leon, Campus de Vega-

zana s/n, 24071 Leon, Spain.

Email: vperp@unileon.es

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latent lesions of adult animals infected earlier in life or as initial

lesions of recently infected adult individuals.10,19,33

The concept of ‘‘age-related’’ resistance to paratuberculosis

has been proposed in bovine25,32,43 and in red deer,28 but it has

not yet been deeply investigated in other species such as ovine.

Several studies carried out in cattle have confirmed that

animals become more resistant to the infection as they get

older,20,34,40 but bacteriological and/or histological evidence

of infection in adult cattle has also been reported.15,25,32,34

These findings would be of great importance, as one of the

main hygienic measures in paratuberculosis remains the

removal of the young animals from their dams within the first

months of life to prevent the transmission of Map within them,

and these recommendations were based on the assumption that

animals become less susceptible to infection as they age.6,43

Several doses of Map inoculum have already been used in

experimental infections, leading to disparate results. Reddacliff

and Whittington36 could not induce any detectable infection in

Merino sheep challenged with doses lower than 104 colony-

forming units (CFU) of Map, whereas 6 of 12 lambs infected with

103 CFU of Map developed detectable lesions in another study.31

An experiment was designed to study the role of the age of

infection on the pathogenesis of paratuberculosis, where young

and adult sheep were experimentally challenged. The first part

of the experiment, focusing on the differences in the peripheral

immune response, already has been reported.13 Both the

humoral and specific cell-mediated immune responses

appeared earlier and more intensely in the adult sheep than in

the lambs, probably due to previous contact with Map or other

mycobacteria, whereas the Map-challenged lambs showed a

more immature immune response, unable to control the

progression of the infection.13

The main objective of the present work is to study and char-

acterize the pathological response of Map infection in the same

experiment, by comparing the differences observed in the

pathological evolution of these experimentally infected lambs

and ewes and evaluating the severity of the lesions with a

detailed count of the granulomas formed. The effect of the

challenge inoculum dose in the establishment and developmen-

tal stage of the lesions in these animals is also assessed.

Materials and Methods

Experimental Animals

Thirty adult ewes 2 to 11 years old (73.3% of them 6–9 years

old) as well as 24 one-month-old lambs of the Churra breed

were used in this study. All of them were randomly selected

from a flock without clinical cases of paratuberculosis in the

past 5 years and in which no vaccination against paratubercu-

losis was practiced before.13

After a period of adaptation once in the experimental

facilities of the University of Leon, the animals were housed

in separate barns to prevent direct contact between the groups.

Both adult and young ovine were randomly divided into 3

groups: one infected with the high dose of Map inoculum

(HD group; n ¼ 13 ewes and 8 lambs), the second one with the

low dose (LD group; n ¼ 10 ewes and 8 lambs), and a third

group of uninfected control animals, challenged with saline

solution (n ¼ 7 ewes and 8 lambs). None of the sheep was bred

during the experiment. Animals followed a diet based on com-

mercial feed appropriate for each age.

Map Inoculum

The strain of Map used in this study was isolated from intestinal

(ileal) mucosa scrapings of 3 sheep from the same flock, all

clinically affected with paratuberculosis and shedding large

number of bacilli, using the Ratnamohan and Spencer35 tech-

nique with slight modifications consisting of the elimination

of deoxyribonuclease and lysozyme treatments. Briefly, sam-

ples of ileum were rinsed with phosphate-buffered saline

(PBS), and the mucosal surface was removed from the submu-

cosa by scraping. Then, 20 g of tissue was mixed with 2 ml of a

solution containing 150 mg/ml ampicillin (Sigma-Aldrich,

Madrid, Spain) per 5 g of mucosa and homogenized using a tis-

sue grinder. The suspension was digested with a solution with

1% trypsin (Sigma-Aldrich) in PBS (pH 7.5–8) at 23�C for

30 minutes. Finally, and after centrifugation, the sediment was

resuspended in 10 ml PBS.

The field strain of Map was then determined to be IS900 pos-

itive by polymerase chain reaction (PCR)18 and typed as an

‘‘ovine’’ strain by IS1311 PCR-restriction enzyme analysis.38

Subsequently, the inocula were subjected to culture on 7H11 and

Lowenstein-Jensen medium supplemented with mycobactin J

(Allied Monitor, Fayette, MO) as described previously,13 leading

to counts of 4 � 106 CFU Map/ml for the high-dose suspension

and 1� 102 CFU Map/ml for the lower dose. For ease of under-

standing, these 2 different doses hereafter will be referred to as the

higher dose (HD) and lower dose (LD) inoculum of Map.

Each challenged animal received a 40-ml oral dose of the

respective inoculum, divided into 4 doses of 10 ml adminis-

tered at 3-day intervals.

Experimental Design

The experimental procedures were performed in accordance

with Spanish Royal Decree 1201/2005 for the protection of

animals used for experimental and other scientific purposes.

At 110 to 120 days postinfection (dpi), 6 ewes and 3 lambs

from the HD group, 4 ewes and 3 lambs from the LD group, and

2 ewes and 3 lambs from the control group were humanely eutha-

nized by intravenous injection of a veterinary euthanasia drug

(T61; Intervet, S.A., Salamanca, Spain), followed by exsanguina-

tion. The remaining animals were euthanized at 210 to 220 dpi.

Pathological Studies

Complete necropsies were performed, and tissues were grossly

inspected with special attention to the gut and related lymph

nodes. Samples for histopathologic examination consisted of

ileocecal valve (ICV), ileum (IL) (three 5-cm samples, taken

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20, 40, and 60 cm from the ileocecal valve), jejunum (JJ) and

jejunal Peyer patches (JPP) (at least 3 patches from each of the

proximal, medium, and distal zones), the caudal mesenteric

lymph node (MLN), 1 jejunal lymph node (JLN), and ileocecal

lymph nodes (ICLN). All these tissues were fixed in 10%neutral buffered formalin and processed to routine histological

sections that were stained with hematoxylin and eosin (HE) and

by the Ziehl-Neelsen (ZN) technique for acid-fast bacilli (AFB)

and examined by light microscopy for histological changes.

For the immunohistochemical study, selected sections hav-

ing representative granulomatous lesions were immunolabeled

using an EnVision þ HRP visualization kit (Dako North

America, Carpinteria, CA). The sections were incubated with

specific rabbit anti-Map serum at a dilution of 1/9000. This

polyclonal primary antibody was kindly gifted by Dr Balseiro,

from SERIDA (Gijon, Asturias, Spain), and previously used in

bovine3 and fallow deer.2 Technique specificity was controlled

by omission of the primary antibody and substitution by a

normal rabbit serum, omission of the EnVision polymer, and

omission of diaminobenzidine. All these controls gave negative

results. To evaluate the specificity of the anti-Map antibody,

tissue samples of ileum from a sheep with clinical paratubercu-

losis were used as positive controls.

All the lesions observed in the digestive tract were classified

following the guidelines previously proposed by Perez et al33

for paratuberculosis lesions in the ovine species, according to

the presence and location of the granulomas in the different

gut-associated lymphoid tissue compartments and the number

of AFB detected by ZN. Sections were blinded and scored by

the same observer into 5 categories, according to the amount

of AFB and Map antigens present in the lesions: 0 (no AFB

or Map antigens), þ/– (doubtful presence of bacteria), þ (soli-

tary or very few bacteria), andþþþ (high load of AFB or Map

antigens in the cytoplasm of the macrophages).

Granuloma Count

After the conventional histopathological examination, the num-

ber of granulomas per tissue section was quantified in different

areas of the intestinal tract and related lymph nodes. For this pur-

pose, 10 different intestinal sites were studied (ie, the ICV) and 3

samples of the IL, JJ, and JPP (proximal, middle, and distal).

Three different lymph nodes (MLN, JLN, and ICLN) were also

analyzed. Three tissue sections were randomly selected from each

intestinal site and 2 tissue sections from each lymph node, so that a

total of 30 intestinal and 6 lymph node tissue sections were

examined from each animal. Sections were blinded, and the mean

number of granulomas per tissue section in each site was recorded

by the same observer, distinguishing those granulomas located in

the lymphoid tissue from those in the associated lamina propria

(LP) or in the mucosa not related to lymphoid tissue.

Tissue and Fecal Culture

Fecal samples were taken at 15 and 90 dpi, as well as on the day

of the scheduled postmortem examination, from each animal.

They were frozen at –20�C until cultured for Map. Bacteriolo-

gical studies were also carried out from tissue samples of the

ICV, a 5-cm sample of the distal ileum (dIL) and of the medial

jejunum Peyer patch (mJPP), and a piece of the caudal MLN.

Two grams of tissues or feces was processed for culture onto

tubes containing Lowenstein Jensen and Middlebrook 7H11

solid medium, supplemented with mycobactin J and previously

decontaminated with hexadecylpyridinium chloride (Merck,

Darmstadt, Germany), as described by Aduriz et al.1 The

procedure is explained in detail in a previous publication.13

Nested PCR

A nested PCR method was performed from paraffin-embedded

tissues. In total, 10 mm of ICV, dIL, mJPP, and MLN tissue

sections adjacent to those used for the immunohistochemical

study was cut twice, and the nested PCR was carried out as pre-

viously described,13 using primers to detect the presence of

Map-specific IS900 DNA.

Statistical Analysis

Data on granuloma count were subjected to analysis of var-

iance using the general linear model procedure of the SAS sta-

tistical package (version 9.1; SAS Institute, Cary, NC) for the

evaluation of age, treatment, time of killing, and location main

effects and interactions. Previously, tissue section granuloma

count figures were logarithmically transformed to submit them

to normal distribution-based tests of significance. Thus, the

mean numbers of granulomas per tissue section were compared

among the groups at each time of killing using the Student t test

for pairwise comparisons with the Tukey-Kramer correction

for multiple comparisons, at the 95% significance level. Group

PCR-positive frequencies were compared and tested for signif-

icance by w2 analysis.

Results

Three animals died during the course of the experiment from

causes unrelated to Johne disease (1 ewe and 1 lamb from the

uninfected control group and 1 ewe from the LD group) and

were excluded from this study.

Pathological Findings

No clinical signs or gross lesions related to paratuberculosis

were seen in any of the animals during the whole experiment.

Microscopically, lymphoid tissue was always found in all

the ileum samples from the lambs, besides the ICV and JJP

samples. In the ewes, no lymphoid tissue was detected in the

samples taken from the ileum, at any level.

When examined histologically, granulomatous lesions typi-

cal of paratuberculosis were only found in the tissue sections

from the HD groups, regardless of the age of infection (ewes

and lambs). However, the type of lesion varied between adult

sheep and lambs.

Delgado et al 3

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Among the HD-challenged lambs, all 8 animals (100%)

showed histologic changes consistent with paratuberculosis.

Lesions were classified in several categories, according to their

location and pathological features, following the guidelines

provided by Perez et al.33 For categorizing each animal, the

final classification was based on its more severe intestinal gran-

ulomatous lesion.

Focal lesions appeared in 2 HD-infected lambs. They were

composed of small, well-defined granulomas formed by homo-

geneous macrophages with a large pale cytoplasm, large nuclei,

and scattered with a few lymphocytes within them, and they

were found exclusively restricted to the interfollicular area of

the Peyer patch lymphoid tissue (Figs. 1, 2). In some areas,

these granulomas were coalescent and partially altered the nor-

mal structure of the Peyer patches. Lesions considered multifo-

cal a were detected in 2 other HD-infected lambs. In this case,

granulomas were more numerous and larger in size, and

besides those seen in the lymphoid tissue, granulomatous

lesions were also detected in the LP always related to the Peyer

patch lymphoid tissue (Fig. 3). In the last 4 lambs, lesions were

classified as multifocal b. In them, many granulomas were also

found in the LP not related to the lymphoid tissue, invading and

thickening the apex of the villi, and in the more severely

affected areas, they partially disrupted the normal architecture,

causing the enlargement of the villi with separation of the

intestinal glands (Fig. 4). In those cases, the granulomas were

not very well demarcated from the adjacent tissue.

Among the HD-challenged ewes, 12 of 13 animals (92.3%)

showed microscopic changes consistent with paratuberculosis.

Eleven of the sheep had focal lesions, with granulomas restricted

to the intestinal lymphoid tissue of the ileocecal and jejunal

Peyer patches, mostly in its basal area. In contrast with the

lambs, the granulomas were fewer, smaller, round shaped, and

well defined from the adjacent tissue (Figs. 5, 6). No differences

were observed in the type and intensity of the lesions among the

ewes, irrespective of their age. Only 1 sheep showed multifocal a

lesions, with a few granulomas located in the mucosa adjacent to

the lymphoid tissue of the ileocecal and distal jejunal Peyer

patches (Fig. 7). This adult sheep also showed a mild granuloma-

tous lymphangitis in the serosa of the ileocecal valve.

When considering the time of euthanasia, 2 of the 3 lambs

examined at 110 to 120 dpi had multifocal b lesions, and the

remaining one was classified as multifocal a. All the sheep

showed focal forms except for 1 that had no lesion. At 210 to

220 dpi, multifocal b lesions were seen in 2 lambs, focal lesions

in 2, and a multifocal a form in the remaining animal. Among

the sheep, all but 1 (with a multifocal a lesion) showed focal

forms.

In those 4 HD-challenged lambs with the more severe intest-

inal lesions (multifocal b), granulomatous lesions were also

seen in the ICLN and MLN, especially spread throughout the

interfollicular and paracortical areas, with a multifocal distri-

bution. In these locations, granulomas were numerous and coa-

lescent, formed by 20 to 100 macrophages with a large pale

cytoplasm, morphologically similar to those observed in the

intestine.

On the contrary, the ewes and lambs with focal intestinal

lesions only occasionally showed a focal granulomatous lym-

phadenitis characterized by a few small granulomas composed

of less than 10 macrophages, scattered within the interfollicular

part of the cortical area of some MLN.

No AFB or Map antigens were detected in any of the control

or LD-challenged animals or in the HD-infected ewes. How-

ever, different grades of bacterial load were demonstrated in

the HD-infected lambs by ZN or IHC, varying from hardly any

AFB or Map antigens in the intestinal focal lesions (grade þ),

to a scant positivity by both techniques in the multifocal a

lesions (gradeþþ), and to the highest bacterial load in the mul-

tifocal b lesions (grade þþþ), especially in the mucosa of the

ileal and jejunal Peyer patches (Fig. 4). Only occasional AFB

or Map antigens were appreciated in the MLN or ICLN from

3 HD-infected lambs with multifocal b lesions.

Granuloma Counts

The mean granuloma counts corresponding to the different

intestinal compartments examined in the lambs and ewes at

110 to 120 dpi and 210 to 220 dpi are shown in Fig. 8. Varia-

bility among individuals of the same group was observed. The

statistical analysis showed that the total mean number of gran-

ulomas was significantly higher in the lambs (85.55) than in the

ewes (19.37) at 110 to 120 dpi (P < .01) and also at 210 to 220

dpi (42.05 vs 24.03, respectively) (P < .05). It is worth high-

lighting that granulomatous lesions in the LP were only

detected in the lambs, whereas in the ewes, granulomas were

only observed in the lymphoid tissue (except for 1 adult sheep).

In the lambs, a significant decrease was noted in the number of

granulomas located in the lymphoid tissue from 110 to 120 dpi

and the end of the experiment (P < .05), whereas those located

in the LP slightly increased. However, the number of granulo-

mas in the organized lymphoid tissue remained similar in the

ewes within the same period (Fig. 8).

When considering the lesions in each different intestinal site

examined (Fig. 9), the mean number of granulomas per tissue

section was always higher in the lambs compared with the

ewes, especially in the mJPP and in the IL (P < .05). Although

granulomas appeared to be slightly more numerous in the ICV

in the ewes than in the lambs (Fig. 9), differences were not sta-

tistically significant. In both adult and young animals, most of

the granulomatous lesions were associated with the lymphoid

tissue in the JPP, IPP, or the ICV (Fig. 9). Specifically, in the

ewes, the granulomas were significantly more numerous in the

ICV than in the rest of the lymphoid tissue (P < .05). Concern-

ing the lambs, the granuloma count was higher in the mJPP

compared with the remaining JPP or IPP (P < .05), but no

differences were detected with the ICV.

In addition to the lesions in the intestine, some granulomas

were also noted in the associated lymph nodes. These were

more numerous in the lambs than in the ewes (P < .05) and

were counted more frequently in the MLN, in both the adult

and young animals.

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Figure 1. Jejunal Peyer patch, high-dose (HD)–challenged lamb. Focal lesion composed of small granulomas (*) exclusively located in the inter-follicular area of the Peyer patch lymphoid tissue. Hematoxylin and eosin (HE). Figure 2. Jejunal Peyer patch, HD-challenged lamb. Highermagnification of a focal granuloma in the interfollicular area of the Peyer patch lymphoid tissue. The granuloma is formed by homogeneousmacrophages with an abundant and pale cytoplasm and a few lymphocytes scattered within them. HE. Figure 3. Jejunal Peyer patch, HD-challenged lamb. Multifocal a lesion. Besides the severe granulomatous lesion in the lymphoid tissue, with large granulomas partially invadingthe interfollicular areas, some of the granulomas are also affecting the lamina propria related to this lymphoid tissue. HE. Figure 4. Medium

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Fecal and Tissue Culture Results

No isolations from feces were obtained at 15 or 90 dpi. The only

positive feces cultures were isolated on the day of the postmor-

tem examination from 4 of the 8 HD-infected lambs, 1 eutha-

nized at 110 to 120 dpi and 3 at 210 to 220 dpi. Apart from 1

lamb classified as focal (euthanized at 210–220 dpi), the 3 others

had multifocal b lesions, with high bacterial load in the intestine.

All the tissue samples from the control, the LD group, and

the HD-infected ewes remained negative (Table 1). Instead,

mycobacteria were isolated, only in 7H11 medium, from the

dIL and/or ICV of 4 of the 8 HD-infected lambs: 1 multifocal

b animal, euthanized at 110 to 120 dpi, and 3 euthanized at the

end of the experiment, 1 with multifocal b lesions and the 2 oth-

ers with focal lesions. All but 1 of these animals were also pos-

itive in the fecal samples.

Nested PCR

IS900 Map-specific sequence was demonstrated by nested PCR

in all 8 HD-infected lambs (100%) from at least 2 of the 4

studied tissue sections (Table 1). The ICV was positive in all

these cases. Seven of the 8 LD-challenged lambs (87.5%), in

which no granulomatous lesions were detected in any of the

examined tissue sections, were also positive from at least 1 tissue

section. The negative lamb was euthanized at 110 to 120 dpi.

Map DNA was also detected in 12 of the 13 HD-infected

adult sheep (92.3%) in at least 1 tissue section. The negative sheep

was examined at 210 to 220 dpi and showed a focal lesion. ICV

and MLN were the most frequent tissue sites with a positive result,

although differences were not statistically significant (Table 1).

Among the LD-infected ewes, in which no histological lesions

were appreciated, the IS900 sequence was demonstrated in 7 of

the 9 animals (77.8%) from at least 1 tissue section. Negative

ewes were euthanized at 210 to 220 dpi.

All analyzed tissue sections from the uninfected lambs and

ewes were negative to PCR.

Discussion

According to the results of this study, the experimental inocu-

lation with Map was able to infect both the ewes and lambs, but

Figure 9. Total granuloma count from the tissues of the high-dose–infected lambs and ewes, regardless of the time of slaughter. Mean of thetotal number of granulomas per section and animal, according to their intestinal location. ICV, ileocecal valve; IL, ileum; JJ, jejunum; JPP, jejunalPeyer patches. d, distal; m, medium; p, proximal. Error bars: standard deviation. Lambs¼ black column. Ewes¼ white column.

Figure 8. Count of granulomas from the tissues of the high-dose–infected lambs and ewes euthanized at 110 to 120 dpi (a) and 210 to 220 dpi (b).Mean number of granulomas per animal, corresponding to their location in the different intestinal compartments. dpi, days postinfection. Error bars:standard deviation. Peyer patch (PP) lymphoid tissue¼ black column. Lamina propria (LP) associated with PP¼ grey column. LP not associated withPP¼white column.

Figure 4. (continued)ileum, HD-challenged lamb. Multifocal b lesion. Granulomas located in the lamina propia not related to the lymphoidtissue partially thicken the apex of the villi. HE. Inset: Presence of numerous acid-fast bacilli positive in the cytoplasm of the macrophages.Ziehl-Neelsen stain. Figure 5. Ileocecal Peyer patch, HD-challenged ewe. Focal lesion composed of few small well-defined granulomas (*)restricted to the basal area of the interfollicular area of the Peyer patch lymphoid tissue. HE. Figure 6. Ileocecal Peyer patch; HD-challengedewe. Focal lesion. Higher magnification of small and focal granulomas (*) composed of macrophages and some lymphocytes. Note that thegranulomas are round and well demarcated from the adjacent tissue. HE. Figure 7. Ileocecal valve, HD-challenged ewe. Multifocal a lesioncomposed of granulomas located both in the lymphoid tissue and in the associated lamina propria. HE.

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detectable lesions and immune response13 were observed only

in those animals challenged with a high dose of the mycobac-

teria. Adult HD-infected sheep developed lesions consistent

with Map infection, with clear differences in the pathological

features when compared with the HD-infected lambs. Several

studies have reported the presence of focal lesions in adult

sheep, similar to those found in our study, considered to be

latent lesional forms originating from infections that took place

early in the life of the animals.10,17,19,33 Although this possibil-

ity cannot be completely ruled out in this study, the fact that

these focal lesions were not found in the LD-challenged ewes

or in the controls would indicate that these granulomas would

be rather the direct consequence of the dose of Map challenge

or the short duration of the examination period.

Lesions consistent with paratuberculosis were found in 12 of

the 13 (92.3%) adult sheep infected with a high dose of Map,

associated with a specific peripheral immune response.13 How-

ever, no similar lesions or an immune response were detected

in any of the ewes from the control uninfected group, suggest-

ing that adult sheep, older than 2 years of age, can get infected

with high doses of Map. In bovine, it was shown that calves

older than 4 to 6 months were more resistant to the infection

with Map,20,40 although other studies have also found that Map

was able to cause lesions in cattle experimentally infected,

older than 3 years32 and even with a 9-year-old,25 although the

lesions were more restricted than in the young calves infected

under the same conditions.25,32 These findings would be in

agreement with our results. In sheep, no experimental studies

have been carried out to date, but in a longitudinal field study,

it was shown that sheep exposed to Map as lambs had higher

odds to shed Map and to develop more severe lesions than those

exposed for the first time as adults.30

The development and features of the lesions in the

HD-challenged groups were different between lambs and ewes.

In the latter, the lesions were, in most cases, classified as focal,

always restricted to the intestinal lymphoid tissue, but small,

round shaped, and well defined, even with initial fibrosis,

suggesting a regressive character. Similar granulomas have

also been described in vaccinated animals.21 On the contrary,

in the HD-infected lambs, the granulomas were more numer-

ous, larger, and spread into areas of the mucosa related or not

(multifocal a or b lesions) to this lymphoid tissue. This fact has

been related to more advanced stages of the infection.10,19,21,33

This is confirmed in our study by the marked decrease in the

number of granulomas found in the lymphoid tissue from 110

to 120 and 210 to 220 dpi, whereas those in the LP partially

increased (Fig. 8), as well as by the high mycobacterial load,

evaluated by ZN and IHC, in the multifocal b lesions.

Payne and Rankin,32 in an experimental infection of adult

and young cattle, found that granulomatous lesions were fewer

and better demarcated in adult animals than in calves at 4 and 6

months postinfection (mpi); however, in a previous postmor-

tem examination carried out at 2 mpi, lesions were more severe

in the adult animals. It was then hypothesized that paratubercu-

losis infection could have reached its higher development in the

adults at 2 mpi, but their immune response could have been

able to react more efficiently and control the progression of the

lesions more quickly, unlike what happened in the calves.32,34

Although in our experimental design, the animals were not

examined until 4 mpi (110–120 dpi), and thus it is not possible

to compare both studies, the fact that granulomatous lesions

were focal and well demarcated in the adult sheep, together

with the earlier and more intense peripheral cell-mediated

immune response mounted by the ewes compared with the

lambs,13 would further support this hypothesis.

One feasible factor that could explain the higher resistance

of the adult animals could be the previous expositions to myco-

bacteria in the adult sheep. Sensitization experimented by the

immune system of the animals after previous and repeated

contacts with the mycobacteria, its antigens, or some other

environmental mycobacteria would induce a better specializa-

tion of the immunologic response.14,32 Animals vaccinated

against paratuberculosis develop granulomas similar to those

seen in the adult sheep and have been shown to be more resis-

tant to Map infection,21 further supporting this hypothesis. In

our study, the ewes came from a flock considered free of this

disease, although the absolute lack of mycobacteria in the

environment cannot be ensured since distribution of Map is

widespread.16 Moreover, some of the ewes used in this study

were especially old (between 9 and 11 years old), increasing

the probability of previous contacts with different mycobac-

teria, although no differences were seen in the type and inten-

sity of the lesions in comparison with the rest of the ewes.

Differences in the amount and functionality of the intestinal

Table 1. Number (%) of Animals Positive to Map Detection by Culture and by Nested PCR (n-PCR)a

Sites

HD Lambs (n ¼ 8) LD Lambs (n ¼ 8) HD Ewes (n ¼ 13) LD Ewes (n ¼ 9)

Culture n-PCR Culture n-PCR Culture n-PCR Culture n-PCR

ICV 4 (50.0) 8 (100.0) 0 4 (50.0) 0 7 (53.9) 0 3 (33.3)dIL 2 (25.0) 7 (87.5) 0 1 (12.5) 0 4 (30.8) 0 1 (11.1)mJPP 0 6 (75.0) 0 3 (37.5) 0 6 (46.2) 0 2 (22.2)MLN 0 7 (87.5) 0 4 (50.0) 0 7 (53.9) 0 4 (44.4)

All the animals, regardless of the time of sacrifice, are included. dIL, distal ileum; ICV, ileocecal valve; Map, Mycobacterium avium subsp paratuberculosis; mJPP,medium jejunal Peyer patch; MLN, mesenteric lymph node; PCR, polymerase chain reaction.aThe culture technique was carried out from fresh tissue samples and the n-PCR was performed from paraffin-embedded tissues. High-dose (HD) and low-dose(LD) infected animals are analyzed in this table.

Delgado et al 7

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lymphoid tissue between lambs and adults27,37 also could have

played a role in the higher resistance of adult ewes to Map

infection.

This study also emphasizes the importance of the intestinal

lymphoid tissue in the establishment of paratuberculosis infec-

tion since most of the granulomatous lesions were detected in

this area, as has been reported in previous studies using young

animals.21,23,24,33 Furthermore, in this study, this finding has

been observed in both lambs and ewes, leading to the conclu-

sion that the Peyer patches would be the primary portal of entry

for Map infection, regardless of the age and their degree of

development. The functional differences in the intestinal lym-

phoid tissue between young and adult sheep have not been

deeply investigated, but some reports27,37 describe that ileal

lymphoid tissue suffers a marked involution as it ages, and the

remaining follicles show increased numbers of T and B cells in

the follicle and prominent intrafollicular T-cell areas, predomi-

nantly harboring CD4þ lymphocytes. Thus, although further

investigation on this topic would be needed, it is likely that

these differences in the lymphoid tissue between young

animals and adults could have played a role in the different

evolution of the lesions.

Regarding the location and intensity of granulomatous

lesions, the granuloma count showed individual variability.

The considerable between-animal variation in pathology has

already been reported as a typical feature of natural and experi-

mental paratuberculosis.4,24,31 In our study, the ICV lymphoid

tissue was more frequently affected in the ewes, whereas the

lesions predominated in the medial JPP in the lambs (Fig. 9).

These 2 areas have already been reported to be some of the

most frequently affected in previous studies in small rumi-

nants.10,17,33,41 Some morphological and lymphocyte distribu-

tion similarities have been reported between ICV and JPP,

compared with IPP,11,41 suggesting that they could have had

an influence on the higher presence of lesions in the ICV and

the JPP than in the IPP. On the other hand, whereas the ICV

is equally affected in all the animals, lesions appear more fre-

quently in the mJPP in the lambs but not in the ewes. This could

indicate a different functional pattern between both lymphoid

tissues depending on the age of the animal, as it has been

already seen to occur in pigs.26 These possible functional dif-

ferences between the 2 sites could explain the fact that lesions

were more easily detected in the ICV than in the JPP despite the

similar level of Map DNA detection by nested PCR in both

locations. According to our findings, apart from the ICV, the

sensitivity of the histological diagnosis of paratuberculosis

would be more efficient if samples from the JPP were also

taken, especially from the medial zone.

The predominance of initial lesions in the ileocecal lymph

nodes or caudal mesenteric lymph node is an already known

fact in bovine19,25 or deer.2,7 However, in our study, the lymph

node involvement was less severe than in the intestinal lym-

phoid tissue. In the lambs, the granulomas were only found

in the caudal mesenteric lymph node when granulomas were

seen in the lamina propria (multifocal b lesions). These find-

ings are in agreement with other studies in ovine, in which the

initial lesions were reported to be established first in the intest-

inal lymphoid tissue and would not be detected in the related

lymph nodes until the infection had progressed to more

advanced stages.17,21,33

Only 1 ewe (9 years old at the moment of infection) showed

multifocal lesions, with some granulomas located in the LP,

suggesting that the infection could have progressed to more

advanced forms. Although it cannot be completely ruled out

that similar lesions could have been missed in the other ewes,

it is tempting to speculate that the immune response in this par-

ticular sheep, due to individual factors, could not have been

able to control the progression of Map, with the subsequent for-

mation of a higher amount of granulomas and their spread to

the LP. It is worth highlighting that the peripheral immune

response of this animal did not differ from its counterparts in

the group.

No granulomatous lesions consistent with paratuberculosis

were observed in any of the ewes and lambs infected with a low

dose of Map. Nor was any Map colony isolated from the tissues

or feces of these animals, although Map DNA was identified by

nested PCR. Besides, the peripheral immune response, evalu-

ated by the specific interferon (IFN)–g production, was similar

to that from the control group.13 These results indicate that the

establishment of an infection and the formation of granuloma-

tous lesions is directly dependent on the infectious dose. This

hypothesis has already been suggested by other authors, who

established a correlation between the amount of the dose

received and the severity of the infection induced, in

ovine,5,30,31 bovine,39 or red deer.29 Reddacliff and Whitting-

ton36 reported that doses below 104 CFU were not high enough

to induce a histologically and bacteriologically detectable

infection in Merino sheep, although other authors have found

evidence of infection using a dose of 103 CFU.5,31 These dis-

crepancies could be caused by the different strains of Map used

in these studies, since variation on the pathogenicity depending

on different Map strains has already been reported.42

The detection of nucleic acids of Map by nested PCR in

lambs and ewes from the LD-infected group was an interesting

finding. Map DNA was not detected by this technique in the

control animals, so it seems likely that the mycobacteria had

invaded the tissue of the animals after the experimental admin-

istration of the lowest dose but were not able, at least until 210

dpi, to trigger an immune response or cause lesions detectable

by the methodology used here. In our study, if the length of the

experimental infection had been prolonged, the ewes and lambs

from the LD-challenged group might have ended up develop-

ing a cell-mediated immune response detectable by IFN-gproduction and even some lesions. Finally, the possibility can-

not be ruled out that, in this group, an initial infection might

have been established, but the affected animals could have been

able to overcome it and recover before 110 to 120 dpi, although

this alternative seems unlikely.

AFB and Map antigens were only clearly identified by ZN and

IHC in the intestine from the HD-infected lambs with multifocal

lesions, characterized by the presence of granulomas in the LP.

The low sensitivity of both techniques in focal paratuberculosis

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lesions, located in the lymphoid tissue, has already been reported

in previous studies and is mainly explained because of the low

amount of AFB in these types of lesions.10,17,19,22,33 It should also

be remarked that the ZN technique can only stain those AFB with

an intact cell wall and thus is unable to detect the bacillus present

in its cell wall–deficient form or as spheroplasts.9 However, this

possibility seems unlikely as the IHC labeling, which is assumed

to be able to detect degraded or previously digested forms of the

bacilli,8,12,22 did not identify any Map antigen in the focal lesions

from this study.

This study has determined that adult sheep, older than

2 years, can get infected by experimental exposure of Map,

as do 1-month-old lambs. In both cases, the infection leads to

the establishment of lesions in the intestine. However, in the

ewes, the lesions appear almost exclusively in the intestinal

lymphoid tissue and are characterized to be focal and well

demarcated, whereas in the lambs, they progress into more

advanced forms, spreading into areas of mucosa not related

to the Peyer patches and to the regional lymph nodes. Only the

oral inoculation of high levels of Map was able to induce

visible lesions in the tissues both in lambs and in ewes. Thus,

the dose of Map inoculum administered to the animals in

experimental conditions has an influence on the establishment

of the infection.

Acknowledgements

We thank J. Reyero and G. Belver for technical assistance.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to

the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for

the research, authorship, and/or publication of this article: This work

was supported by grant AGL2008-05820-C02 of the Spanish Ministry

of Science and Innovation and by grant FP6-2004-FOOD-3B from the

ParaTBTools project (European Union). L. Delgado was recipient of a

predoctoral fellowship from the Spanish Ministry of Education. J.

Benavides is supported by CSIC through the JAE-Doc program,

financed in part by the European Social Fund (ESF).

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