Association of severity of enteric granulomatous inflammation with disseminated Mycobacterium avium subspecies paratuberculosis infection and antemortem test results for paratuberculosis in dairy cows.
ABSTRACT Disseminated infection (DI) of Mycobacterium avium subspecies paratuberculosis (MAP) in cattle may impair cow health, potentiate spread of disease, and is a potential food-safety risk. The objectives of this study were to determine the association between severity of histologic enteric lesions and the occurrence of DI, clinical signs, and positive fecal culture and serum ELISA results. Bacteriologic fecal culture and serum ELISA were performed on 40 dairy cows from MAP-infected herds. Cows were classified as having DI if MAP was isolated from any of 11 extra-intestinal tissues collected postmortem. A grade of 0-3, corresponding to the severity of histologically evident granulomatous inflammation was determined for sections of ileum, jejunum, mesenteric lymph node, and ileocolic lymph node. An overall intestinal inflammation (OII) grade of 0-3 was assigned to each cow. The proportion of cows with DI increased with tissue-specific lesion grade and OII grade. All cows with grade 3 inflammation in any single tissue had DI, however, some cows with DI had grade 1 inflammation or no lesions. In general, there was a positive association between OII grade and clinical signs, gross enteric lesions, and positive ELISA and fecal culture results. However, 12% of OII grade 0 cows had clinical signs (explained by other conditions recognized with necropsy), and the proportion of positive ELISA results was lower for OII grade 3 cows relative to grade 2 cows. Although MAP dissemination may occur early in the disease process, histopathology of intestinal tissues may be used to detect a substantial proportion of DI cows.
- SourceAvailable from: Kasra X Ramyar[Show abstract] [Hide abstract]
ABSTRACT: The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.Clinical and vaccine Immunology: CVI 05/2012; 19(7):1083-92. · 2.37 Impact Factor
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ABSTRACT: The infection biology of Mycobacterium avium subspecies paratuberculosis (MAP) has recently crystalized with added details surrounding intestinal invasion. The involvement of pathogen-derived effector proteins such as the major membrane protein, oxidoreductase and fibronectin attachment proteins have been uncovered. Mutations constructed in this pathogen have also shed light on genes needed for invasion. The host cell types that are susceptible to invasion have been defined along with their transcriptional response. Recent details have given a new appreciation for the dynamic interplay between the host and bacterium that occurs at the outset of infection. An initial look at the global expression pathways of the host has shown a circumvention of the cell communication pathway by MAP, which loosens the integrity of the tight junctions. We now know that MAP activates the epithelial layer and also actively recruits macrophages to the site of infection. These notable findings are summarized along with added mechanistic details of the early infection model. We conclude by proposing critical next steps to further elucidate the process of MAP invasion.Infection and immunity 08/2013; · 4.16 Impact Factor
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ABSTRACT: Paratuberculosis is a widespread disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). For long time the infection has been believed as confined to the intestine. Disseminated infection in ruminants with MAP has gained more interest in recent years. In this study the histological lesions, the distribution of MAP in intestinal and mammary tissues of seropositive sheep and the sensitivity of direct diagnostic tests are evaluated. Histopathological lesions were observed in the proximal jejunum, distal jejunum and ileum of 19 sheep. Types 3b and 3c lesions were observed. Sheep with subclinical infection had type 3c ileal lesions, while clinically infected animals had mainly type 3b ileal lesions (p < 0.05). Ziehl–Neelsen (ZN) staining was more sensitive for detecting acid-fast bacilli in type 3b intestinal lesions than in type 3c (p < 0.05). Immunohistochemistry (IHC) was more sensitive than ZN staining in detecting infected intestinal samples (p < 0.05). PCR was the most sensitive technique in detecting MAP in both intestinal and extra-intestinal samples (p < 0.05). MAP was mainly observed in mammary tissues of sheep with type 3b intestinal lesions, although these findings were not statistically significant (p > 0.05). The number of MAP in the intestine might influence the clinical course and the local histopathological lesions, but probably further factors contribute to the discrimination between local and general infection.Small Ruminant Research 06/2012; 105:295-299. · 1.10 Impact Factor
Association of severity of enteric granulomatous inflammation
with disseminated Mycobacterium avium subspecies
paratuberculosis infection and antemortem test
results for paratuberculosis in dairy cows§
M.M. Dennisa,b,*, M.C. Antognolib, F.B. Garryc, H.L. Hirstc,
J.E. Lombardc,d, D.H. Goulda, M.D. Salmanb
aVeterinary Diagnostic Laboratory, College of Veterinary Medicine and Biomedical Sciences,
Colorado State University, Fort Collins, CO 80523-1681, USA
bAnimal Population Health Institute, Department of Clinical Sciences, College of Veterinary Medicine and
Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1681, USA
cIntegrated Livestock Management, Department of Clinical Sciences, College of Veterinary Medicine and
Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1681, USA
dUnited States Department of Agriculture, Animal and Plant Health Inspection Services, Veterinary Services,
Centers for Epidemiology and Animal Health, Fort Collins, CO 80526, USA
Received 1 January 2008; received in revised form 26 February 2008; accepted 28 February 2008
Disseminated infection (DI) of Mycobacterium avium subspecies paratuberculosis (MAP) in cattle may impair cow health,
potentiate spread of disease, and is a potential food-safety risk. The objectives of this study were to determine the association
between severity of histologic enteric lesions and the occurrence of DI, clinical signs, and positive fecal culture and serum
ELISA results. Bacteriologic fecal culture and serum ELISAwere performed on 40 dairy cows from MAP-infected herds. Cows
were classified as having DI if MAP was isolated from any of 11 extra-intestinal tissues collected postmortem. A grade of 0–3,
corresponding to the severity of histologically evident granulomatous inflammation was determined for sections of ileum,
jejunum, mesenteric lymph node, and ileocolic lymph node. An overall intestinal inflammation (OII) grade of 0–3 was assigned
to each cow. The proportion of cows with DI increased with tissue-specific lesion grade and OII grade. All cows with grade 3
inflammation in any single tissue had DI, however, some cows with DI had grade 1 inflammation or no lesions. In general, there
was a positive association between OII grade and clinical signs, gross enteric lesions, and positive ELISA and fecal culture
Available online at www.sciencedirect.com
Veterinary Microbiology 131 (2008) 154–163
§Some data existing in this manuscript were included in an abstract presented here: Dennis, M.M., Antognoli, M.C., Garry, F.B., Hirst, H.L.,
Lombard, J.E., Gould, D.H., Salman, M.D., 2007. Association of disseminated Mycobacterium avium subspecies paratuberculosis infection
with severity of granulomatous enteritis and mesenteric lymphadentis in cattle. In: Nielsen, S.S. (Ed.), Proceedings of the Ninth International
Colloquium on Paratuberculosis, Tsukuba, Japan, pp. 72–73.
* Corresponding author at: Faculty of Veterinary Science, University of Sydney, 425 Werombi Road, Camden, NSW 2570, Australia.
E-mail address: firstname.lastname@example.org (M.M. Dennis).
0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
results. However, 12% of OII grade 0 cows had clinical signs (explained by other conditions recognized with necropsy), and the
occur early in the disease process, histopathology of intestinal tissues may be used to detect a substantial proportion of DI cows.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Diagnosis; Disseminated infection; Fecal culture; Histology; Mycobacterium avium subspecies paratuberculosis; Serology
Paratuberculosis is an enteric disease of ruminants
caused by Mycobacterium avium subspecies para-
tuberculosis (MAP). Infected cattle develop granulo-
matous enteritis and mesenteric lymphadenitis and
may show diarrhea, reduced milk production, and
weight loss. The production losses associated with
paratuberculosis are a substantial economic burden to
cattle industries (Ott et al., 1999).
In a significant proportion of cattle infected with
MAP, the organism can be isolated from tissues other
than small intestine and associated lymph nodes, a
condition termed disseminated infection (DI) (Antog-
noli et al., 2008; Ayele et al., 2004; Brady et al., 2008;
Hines et al., 1987; Koenig et al., 1993; Pavlik et al.,
2000; Sweeney et al., 1992). Whether organism
dissemination potentiates development of clinical
illness is unknown, but it is possible that cattle with
DI may represent a food safety risk or may provide the
basis for alternative modes of transmission (other than
through fecal shedding). The mechanism of organism
dissemination in cattle with DI has not been
determined but is thought to occur through periods
of sporadic bacteremia (Hines et al., 1987; Koenig
et al., 1993). Since DI is prevalent among cattle with
clinical signs, or cattle shedding large numbers of
organism in feces, some have considered the
occurrence of DI to be a feature of late-stage infection
(Huda and Jensen, 2003; Pavlik et al., 2000; Whitlock
and Buergelt, 1996). However, others have reported
that many cattle with DI do not have clinical
paratuberculosis (Antognoli et al., 2008; Brady
et al., 2008). Thus, further study is needed to
understand the pathogenesis of DI.
In bovine paratuberculosis, the persistence of MAP
in enteric tissues and the extent of the associated
host’s immune response are thought to be key factors
for progression of disease (Radostits et al., 1994).
Histological evaluation of enteric tissues is a conven-
tional method to gauge MAP persistence and the
severity of a host’s cell-mediated immune response.
However, the association between histological lesion
severity and the occurrence of DI has not yet been
defined. Furthermore, there is a shortage of datawhich
describe the relationship between histological lesion
severity and other aspects of MAP infection status,
including clinical signs of disease, fecal shedding of
organism, and humoral immune response.
The objectives of the present study were to clarify
the relationship between enteric pathology and
dissemination of MAP, manifestation of clinical signs,
secondary objective was to determine whether the
severity of histological lesions of small intestine and
mesenteric lymph nodes could predict DI status of
Forty mature dairy cows, culled from 4 MAP-
infected herds, were enrolled in this study as
previously described (Antognoli et al., 2008). Briefly,
the study population was intended to represent non-
infected cattle and cattle within various stages of
infection. The majority of the cows were selected
based on prior positive serum ELISA results and
others were selected based on poor physical condition
at time of enrollment. Six cows with negative
historical ELISA results and normal physical appear-
ance were also included.
Amount of body fat coverage was used to assign a
through 5 (Ferguson et al., 1994). Cows were
classified as having clinical signs consistent with
paratuberculosis if their BCS was less than 2.5 and
diarrhea was present.
M.M. Dennis et al./Veterinary Microbiology 131 (2008) 154–163155
Blood and fecal samples were collected from all
cows prior to euthanasia and tested as previously
described (Antognoli et al., 2008). Serologic testing
was performed with a commercial ELISA for
antibodies against MAP according to the manufac-
turer’s instructions (Herdcheck, IDEXX Laboratories
Inc., Westbrook, ME, USA). Results for the ELISA
were calculated and reported as S:P ratios using the
following equation: (OD of sample ? OD of negative
control)/(OD of the positive control ? OD of negative
control). Test results were classified as positive if the
S:P ratio was ?0.25 or negative if the S:P ratio was
<0.25 as per manufacturer’s recommendations. Con-
ventional culture of feces and tissues on Herrold egg
yolk medium was conducted at the Colorado State
University Veterinary DiagnosticLaboratory. Bacterial
morphology, mycobactin dependence, and time of
growth were characteristics used for organism con-
firmation. Cattle were humanely euthanized using a
captive bolt followed by intravenous injection of
potassium chloride. Ileum, jejunum, ileocecal lymph
node, mesenteric lymph node, hepatic lymph node,
liver, kidney, lung, supramammary lymph node, retro-
pharyngeal lymph node, prescapular lymph node,
popliteal lymph node, myocardium, longissimus coli
muscle, and extensor carpi radialis muscle were
sampled aseptically for bacteriologic culture.
2.2. Characterization of cattle
Each cow’s infection status was determined using
results from bacteriologic culture of tissue and feces.
Cows were classified as having DI if MAP was
isolated from tissues other than small intestine,
mesenteric lymph node, and ileocecal lymph node.
Cattle were categorized as having infection limited to
the gastrointestinal tract (GI) if isolation of MAP was
limited to feces, small intestine, mesenteric lymph
node, and ileocecal lymph node. Cattle were classified
as non-infected (NI) if MAP was not isolated from
feces or tissues.
Ileum, jejunum, ileocecal lymph node, mesenteric
lymph node, and a liver sample (meant to simulate a
biopsy) were sampled for histological examination.
Sampled portions of small intestine were taken from
areas showing gross lesions consistent with para-
tuberculosis, including thickened mucosa and enlar-
gement of serosal lymphatic vessels. If gross lesions
were absent, samples were taken from the proximal
region of bowel segment. The mesenteric lymph node
with largest diameter was sampled; if gross intestinal
lesions were present, the mesenteric lymph node was
taken from mesentery adjacent to affected jejunum.
Tissues were immersed in 10% neutral buffered
formalin. Full thickness portions of small intestine
were trimmed transversely along the longitudinal axis
offormalin-fixed bowel. Carewas taken to include the
anti-mesenteric aspect of bowel and to produce
sections of equivalent size. Lymph nodes were
sagitally partitioned in the area where diameter was
greatest. Tissues were embedded in paraffin and were
routinely processed. Five micrometer thick sections
were prepared and stained with hematoxylin and eosin
and Ziehl–Neelsen acid fast stain.
Histological examination was completed by a
single pathologist (MMD) who had no knowledge of
clinical findings and reasons for which cows were
enrolled in the study. Sections were examined for the
presence of granulomatous inflammation and for acid
fast bacilli (AFB). A grading system was used to
categorize the severity of granulomatous inflamma-
tion. Points were assigned to each tissue according to
quantity and microanatomical distribution of epithe-
lioid macrophages and Langhans-type multinucleate
giant cells (Table 1). The levels for leukocyte
distribution were designed to represent the character-
istics of lesions that have been documented to
manifest as this disease progresses (Buergelt et al.,
1978; Gilmour et al., 1965; Payne and Rankin, 1961).
For each tissue, tallied points were used to assign a
lesion grade from 0 to 3 (Table 2). Some lymph nodes
contained small nests of macrophages which con-
tained granular yellow-green refractile pigment.
macrophages and were excluded from the grading
An overall intestinal inflammation (OII) grade was
created to reflect the degree of granulomatous
inflammation detected in each cow. For each cow,
the OII grade was determined based on the sum of the
grades assigned to the 4 histopathologically evaluated
tissues. OII grade of 0 was assigned to cows with a
sum of 0, 1 (low severity) was assigned to those with a
M.M. Dennis et al./Veterinary Microbiology 131 (2008) 154–163 156
sum of 1–4, 2 (moderate) assigned to those with a sum
of 10–12. OII grade was only allotted to cows for
which all 4 tissues were available for histopathologic
evaluation (n = 38).
2.4. Statistical analysis
Fisher’s exact test was used to test for a difference
in the proportions of cows with gross lesions, clinical
signs, serum-ELISA results, fecal culture results, DI,
and tissues containing AFB between tissue-specific
lesion grade or OII grade groups. Stuart’s Tau-c was
used to compare the agreement of tissue-specific
lesion grade results between two tissues. A Kruskal–
Wallis test was used to compare median age and
median ELISA S:P ratios among OII grade groups. A
Mann–Whitney test was used to compare median S:P
ratios between two OII grade groups. Odds ratios with
95% confidence intervals (CI’s) were constructed to
compare the odds for DI among OII grade groups.
Sensitivity was calculated as the percentage of cows
with DI that had tissue-specific lesion grade or OII
grade of 2 or 3. Specificity was calculated as the
percentage of cows without DI (i.e. cows with NI or
GI) that had grade of 0 or 1. For sensitivity and
specificity, exact 95% CI’s were estimated using a
binomial distribution. For all analyses, p < 0.05 was
regarded as significant.
Twenty-eight (70%) cows of the study population
were found to be infected with MAP. Of these, 21
(75% of infected cows, 53% of study cows) had DI,
while 7 (25% of infected cows, 18% of study cows)
had GI. Granulomatous inflammation was detected in
a single non-infected cow; this cow had grade 1
inflammation in the mesenteric lymph node (thought
to represent a granulomatous reaction to a substance
other than MAP). All other cows with grade 1–3
inflammation in any tissue were infected, and all cows
with grade 3 inflammation and 83% cows with grade 2
inflammation in any tissue were characterized as
having DI (Table 3). However, 9 cows with DI had at
least one tissue with grade 1 or 0 inflammation.
Grade 1 inflammation consisted of scattered
individual Langhans-type multinucleate giant cells
and/or tiny clusters (<10 cells) of epithelioid
macrophages throughout the lamina propria or peyer’s
patches of intestinal sections, or in the paracortex of
M.M. Dennis et al./Veterinary Microbiology 131 (2008) 154–163 157
Lesion grade assigned to intestinal and lymph node tissues accord-
ing to total points (as described in Table 1)
Determination of tissue-specific lesion grade
Total pointsCorresponding severityLesion grade
Lesion grading system for small intestine and lymph node sections
Lesion grading system
Leukocyte distribution pattern
Small intestine Lymph nodes
Rare individual cells
Lamina propria or Peyer’s patches
Lamina propria and submucosa or
within/around lymphatic vessels
Paracortex and subcapsular
sinus or within/around
capsular lymphatic vessels
Expansion of subcapsular sinus3Many groupsLamina propria and submucosa,
and muscularis, serosa, or within/
around lymphatic vessels
Distortion and blunting of villi4Confluent/sheetsEffacement of cortex
Points were assigned for both total leukocytes and leukocyte distribution pattern by microanatomical location, then tallied. For each section,
tallied points were used to determine tissue-specific lesion grade (Table 2).
aPertaining specifically to epithelioid macrophages and/or Langhans-type multinucleate giant cells.
lymph node sections. Scant AFB were identified
within the cytoplasm of multinucleate giant cells of
these lesions with some difficulty. In all 5 cows with
OII grade of 1, inflammation was detected in only 1–3
of the 4tissues evaluated. Inflammation was evidentin
the ileum of 4 (80%) cows, ileocolic lymph node of 2
(40%) cows, mesenteric lymph node of 2 (40%) cows,
and jejunum of 1 (20%) cow.
Grade 2 inflammation consisted of groups of
epithelioid macrophages of varied size, uncommonly
admixed with multinucleate giant cells. For intestinal
tissues, these leukocytes were usually dispersed
throughout the submucosa in addition to the lamina
propria. In a few cows with grade 2 intestinal
inflammation, leukocytes were limited to within and
around serosal lymphatic vessels. In grade 2 lympha-
denitis, leukocytes were dispersed throughout para-
cortex and subcapsular sinus. Similarly, in a few cows
with grade 2 lymph node inflammation, leukocytes
were limited to within and around capsular lymphatic
vessels. AFB were identified in the tissues of most
cows with grade 2 lesions (Table 3), and were in
greater numbers than in tissues with grade 1 lesions.
Inflammation was identified in all histologically
evaluated tissues of cows with OII grade of 2. Mild
multi-focal granulomatous hepatitis was detected in
20% of OII grade 2 cattle (Table 4); no AFB were
identified in these lesions.
Grade 3 inflammation consisted of numerous
coalescing groups or confluent sheets of epithelioid
macrophages; multinucleate giant cells were rarely
identified. In grade 3 enteritis, leukocyte infiltration
always distorted, blunted, and fused villi. In addition,
groups of epithelioid macrophages were usually
transmurally distributed, found in submucosa, mus-
cularis, and/or serosa, and often around or within
lymphatics. For lymph node sections with grade 3
lesions, sheets of epithelioid macrophages usually
effaced cortex and/or expanded subcapsular sinus.
AFB were easily identified in sections with grade 3
inflammation and were in great numbers. The
proportion of lesions containing AFB increased with
grade (Table 3) (for each tissue type, p < 0.01).
Inflammation was identified in all histologically
evaluated tissues of cows with OII grade of 3. Mild
multi-focal granulomatous hepatitis was detected in
50% of OII grade 2 cattle (Table 4); no AFB were
identified in these lesions.
M.M. Dennis et al./Veterinary Microbiology 131 (2008) 154–163158
Number of cattle within each tissue-specific lesion grade group stratified by infection status and by presence or absence of intra-lesional AFB
Tissue-specific lesion grade
Values given in parentheses are in percentage. DI, Disseminated infection; G, infection confined to gastrointestinal tract; NI, not infected; AFB, acid fast bacilli; MLN, mesenteric
lymph node; ILN, ileocolic lymph node.
aFor two noninfected cows, ileum was the only tissue available for histopathologic evaluation.
Of 38 cows with paired mesenteric lymph node and
ileocolic lymph node samples, grades for each tissue
were concordant in 27 (71%) cows (t = 0.65). Grades
for paired ileum and jejunum samples were con-
cordantin33 (87%)cows (t = 0.77).Grades forpaired
ileum and ileocolic lymph node samples were
concordant in 25 (66%) cows (t = 0.62). Grades for
paired jejunum and mesenteric lymph node samples
were concordant in 28 (87%) cows (t = 0.68). Of 16
cows with AFB histologically recognized, 14 (88%)
had AFB recognized in all four tissues evaluated.
Median age did not vary among OII grade groups
(p = 0.1) (Table 4). Similar to tissue-specific lesion
grade results, all cows with OII grade of 3 had DI. The
proportion of DI cows increased with OII grade
(p < 0.01). Cows with OII grade of 2 or 3 were 40.0
(95% CI: 4.2–372.4) times more likely to have DI
relative to those with OII grade of 0 or 1. The
sensitivity and specificity of tissue-specific lesion
grade and OII grade of >1 for categorizing cows as
havingDI (relativeto bacteriologic tissue cultures) are
displayed in Table 5. No single tissue predominated in
its ability to accurately classify DI status of cattle. As
evidenced by overlapping CI’s, there were no
statistically significant differences in sensitivity or
specificity between each tissue evaluated.
The proportion of cows with clinical signs
(presence of diarrhea and BCS < 2.5) varied with
OII grade (p = 0.005). 56% of cows with OII grade of
2 or 3 had clinical signs. Of 11 cows with clinical
signs, 2 were without histologically detected enteric
inflammation; both were classified as NI. Severe
bronchopneumonia and lesions consistent with right-
sided heart failure were found on postmortem
examination of one cow. The other cow was found
to have severe eosinophilic enteritis.
M.M. Dennis et al./Veterinary Microbiology 131 (2008) 154–163159
Age, infection status, and test-results of cows in four tissue lesion severity groups
0 (N = 17) 1 (N = 5) 2 (N = 6) 3 (N = 10)
Values given in parentheses are in percentage. OII, Overall intestinal inflammation; DI, disseminated infection; GI, infection confined to
gastrointestinal tract; NI, not infected; LN, lymph node; AFB, acid fast bacilli.
aDI cows only (n = 21); MAP was not cultured from longissimus coli or extensor carpi radialis in any cow.
Theproportion ofcowswith grosslyevidententeric
lesions (including thickening of the mucosa of the
ileum or jejunum, enlargement of mesenteric lymph
nodes, and/or enlargement of small intestinal serosal
lymphatic vessels) varied with OII grade (p = 0.001).
All cows with OII grade of 2 or 3 had gross enteric
lesions. However, 29% of cows without histologically
have mild or moderate gross enteric lesions. Severe
gross lesions (consisting of severe thickening of
the mucosa of the ileum, notable enlargement of the
associated mesenteric LN, notable thickening of the
mucosa of the jejunum, and many thickened white-
colored serosal lymphatic vessels) were restricted to
six cows with DI that had an OII grade of 2 or 3.
The proportion of serum ELISA-positive cows
varied between OII grade groups (p < 0.001).
Furthermore, the median S:P ratio differed between
OII grade groups (p < 0.001) (Fig. 1). However, only
the differences in median S:P ratios between cows
with OII grade of 0 versus 2 and 0 versus 3 were
statistically significant (p < 0.005).
The proportion of cows that were fecal culture-
positiveincreasedwithOIIgrade(p < 0.05).Allcows
with an OII grade of 2 or 3 were fecal culture-positive.
However, of 22 fecal culture positive cows, 4 (18%)
had no detectable granulomatous inflammation in any
Thegrades forgranulomatous enteric inflammation
presented herein are believed to represent a spectrum
of lesion severity seen in cattle with natural
paratuberculosis. Mild level of inflammation (OII
grade 1) was not evenly distributed, whereas moderate
(OII grade 2) and severe (OII grade 3) levels of
inflammation involved all four tissues evaluated in
each affected cow. Typically, the severity of inflam-
mation within one tissue was equivalent or similar to
inflammation present in other tissues. These findings
suggest that lesions may have segmental distribution
early in the disease process, but usually progress
M.M. Dennis et al./Veterinary Microbiology 131 (2008) 154–163160
Fig. 1. Range of ELISA S:P ratio results for each overall intestinal inflammation (OII) grade group. Only the differences in median S:P ratios
between cows with OII grade of 0 vs. 2 and 0 vs. 3 were significantly significant (p < 0.005).
Sensitivity and specificity with 95% confidence intervals of each tissue-specific lesion score and overall intestinal inflammation (OII) grade of 2
or 3 for identifying cattle with DI
Accuracy of histopathologic grade for identifying cattle with DI
Se (95% CI)
Sp (95% CI)
76% (53, 92)
95% (74, 100)
71% (48, 89)
100% (81, 100)
67% (43, 85)
94% (71, 100)
62% (38, 82)
82% (57, 96)
71% (48, 89)
94.% (71, 100)
Se, Sensitivity; Sp, specificity; MLN, mesenteric lymph node; ILN, ileocolic lymph node; CI, confidence interval.
somewhat uniformly throughout the ileum, jejunum,
with the general belief that lesions first emerge in the
terminal small intestine (Radostits et al., 1994),
lesions were most consistently found in the ileum
of cows with mild enteric disease. Also in concurrence
with observation of others (Buergelt et al., 1978;
Hostetter et al., 2005), there were no histomorpho-
logical characteristics identified within lesions of the
study population to suggest that divergent types of
cell-mediated immune responses exist in cattle, as
described in ovine paratuberculosis (Perez et al.,
The proportion of cattle with DI increased with OII
grade. DI was nearly always present in cows with
severe enteric granulomatous inflammation (OII grade
of 3), but it was also present in most cows with
moderate lesions (OII grade of 2). However, 57% of
cattle with DI had no clinical signs, and some cattle
with DI had mild (OII grade of 1) or undetected
lesions. This is in concurrence with others who have
reported DI in asymptomatic cows (Ayele et al., 2004;
Brady et al., 2008; Pavlik et al., 2000; Sweeney et al.,
1992). Therefore, although advanced stage of infec-
tion seems an important determining factor for the
occurrence of DI, DI may also occur early in the
In the present study, two noninfected cows without
detected enteric inflammation had clinical signs
consistent with paratuberculosis (diarrhea and poor
body condition). However, postmortem examination
revealed conditions that could account for disease
signs in both cows. Others have reported poor
association between clinical signs and lesions of
paratuberculosis (Brady et al., 2008; Clarke, 1997).
The present study demonstrates that most cattle with
severe lesions have diarrhea and poor body condition,
but some cows with moderate tissue response to
infection will be asymptomatic. Additionally, some
non-infected cows with clinical signs suggestive of
paratuberculosis from MAP-infected herds have other
The present study found the proportion of cows
with ELISA-positive results to increase with OII
grades 0–2. Of 22 cows with OII grades of 0 or 1, only
4 (18%) had positive ELISA results. These findings
are in agreement with studies which have demon-
strated a higher proportion positive ELISA results in
populations of MAP-infected cattle with advanced
infection (as designated by heavy shedding of
organism in feces or the presence of clinical signs)
relative to those without (Milner et al., 1990; Sockett
et al., 1992; Sweeney et al., 1995). The proportion of
ELISA-positive results and median S:P ratios were
lower for cows with OII grade of 3 than those with OII
grade of 2. However, the magnitude of difference of
median S:P ratios was not statistically significant.
Serum ELISA’s have been demonstrated to have poor
repeatability over time (as opposed to repeat testing of
the same sample) (Barrington et al., 2003; Hirst et al.,
2002). This may be explained by gastrointestinal
protein loss, or anergy-associated reduction in anti-
body production, both which might occur in cattle
with severe lesions or advanced disease (Radostits
et al., 1994). Further studies using a larger sample size
would help decipher whether a true difference in
median S:P ratio exists between cows with moderate
and severe enteric pathology.
The proportion of cows that were fecal culture-
grade of 2 or 3 were fecal culture-positive. These
an important determinant factor for fecal shedding.
Three cows with OII grade of 1 were fecal culture-
negative. This may be explained by small quantity of
to uneven lesion distribution. MAP has been isolated
fromintestinal tissues offecalculture-negative cows in
sensitivity of fecal-culture when used to detect cows
with early-stage disease. Four cows with positive fecal
culture results were without detectable granulomatous
inflammation. Given the large number of enteric tissue
sections that may require histologic exam to detect
some infected cattle (Whitlock et al., 1996), it is
possible that examination of more than four sections
might have detected granulomatous inflammation in
orally exposed toMAP, may pass theorganism infeces
without establishing infection (Sweeney et al., 1992).
Tissue-specific lesion grades and OII grade were
of 2 or 3 designated a cow as having DI. Depending on
tissue evaluated, 61–76% of cows with DI were
correctly classified as having DI using this method
M.M. Dennis et al./Veterinary Microbiology 131 (2008) 154–163161
(sensitivity; Table 5). Conversely, the proportion of
non-DI cows that were incorrectly classified as having
DI ranged from 0 to 18% (1 ? specificity; Table 5).
Therefore, using lesion grade to predict DI appears to
be more accurate than using serum ELISA or fecal
culture results (Antognoli et al., 2008). Should a
causal relationship between MAP and human intest-
inal inflammatory conditions be established (Bull
et al., 2003; Collins et al., 2000; Hulten et al., 2001),
socioeconomic pressures may call for exclusion of
MAP-infected tissues from the human food supply.
Histological evaluation of intestinal tissues may be an
effective means to identify a large proportion of DI
cows. Since all cows with severe gross lesions (n = 6)
had DI and OII grade of 2 or 3, cows with severe gross
lesions identified at postmortem slaughter inspection,
could be condemned thereby reducing the number of
carcasses requiring histological examination.
In this study, conventional bacteriologic culture of
a select number of tissues was used as the criterion-
referenced standard for classifying DI status. Evalua-
tion of a larger number of tissues for MAP presence,
and use of other methods for the detection of MAP in
tissues, such as modified bacteriologic culture
techniques or PCR, may have detected a greater
number of study cows with DI. Following the uptake
of MAP by M-cells, subepithelial macrophages
transport MAP to the lymphatic system (Momotani
et al., 1988), and MAP-infected macrophages may
intermittently access systemic circulation (Koenig
et al., 1993). Because infected macrophages have
impaired intracellular killing mechanisms (Bendixen
et al., 1981), the distribution of macrophage-borne
MAP to extra-intestinal tissues through blood or
lymph may be a surprisingly common feature of
infection. In some cattle, DI may represent a period of
transient bacteremia, and perhaps all infected cattle
have DI at some point during infection.
The prevalence of DI is higher in subpopulations of
cattle with high to moderate-grade enteric granuloma-
tous inflammation relative to those with low-grade or
nondetected enteric inflammation. However, in some
cows, DI appears to occur at an early stage of infection
with no identifiable enteric lesions. There is an
association between enteric lesion severity and clinical
signs, gross lesions, fecal shedding, and humoral
useful means to screen slaughtered cattle for DI.
The authors thank Gail Thompson, Dennis Mad-
den, Jonathan Arzt, Davis Seelig, Shannon Swist,
Stuart Vandeventer, and Greg Wilkerson at the CSU
Veterinary Diagnostic Laboratory for technical assis-
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