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Proceeding of the ACVP/ASVCP

Concurrent Annual Meetings

Dec.3-7, 2011
Nashville, Tennessee, USA

Next Meeting : December 1-5, 2012 - Seattle, WA, USA

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Histomorphometry of Bone Marrow and Other Tissues in Diseases of

Broiler Chickens
Floyd D. Wilson, BS, DVM
Clinical Professor of Pathology
Department of Pathobiology and Population Medicine,
MVRDL & PRDL, CVM, MSU
fwilson@mvrdl.msstate.edu

Abstract:
Quantification of bone marrow cellular elements present in the mid-shaft femur of broiler
chickens and heavy fowl demonstrated age-associated changes in normal chicken populations.
In addition, often extensive elevations in bone marrow cellularity were present in birds
manifesting with evidence of septicemia-toxemia and with both severe localized or systemic
inflammatory conditions. Dramatic elevations occurring at high incidence in bone marrow
cellularity were documented for some, but not other viral diseases. However, quantification of
the marrow also demonstrated clear numerical differences between severe bone marrow
hyperplasia occurring in response to infrectious disease and those present in myeloid leukosis.
Simple morphometric also was utilized to document quntiative changes in the glomeruli in
broiler manirfesting with inclusion body hepatitis associated glomerulonephritis, for the
demonstration of age associated changes in the myocardium of the heart and a variety of
morphometric methods were applied for the comparison normal bursas to those manifesting
with microscopic pathologic changes.

Hematopoietic Studies:
Background:
While dramatic changes in the bone marrow of poultry manifested with infectious and neoplastic
diseases are often seen by diagnostic poultry histopathologists, only a limited amount of
quantitative information on avian bone marrow in normal and abnormal conditions is available in
the scientific literature and potential bone marrow changes in many common diseases in poultry
have not been investigated (1-6). Thus, an effort was made to develop simple quantitative
methods for the evaluation of bone marrow in broiler and other classes of commercial poultry.
The quantitative method was developed and applied for the establishment of normal bone
marrow status [including defining age-associated changes] and also for documentation bone
marrow abnormalities occurring with septic-toxemic disease conditions, bacterial and viral
diseases and also avian neoplastic conditions involving the bone marrow. In addition, simple
morphometric methods that have been developed for the measurement of microscopic
parameters in the kidney, heart and bursa will also be discussed.

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Bone Marrow Histomorphometric Methods:

Bone marrow is collected from the mid-shaft femur region and a plug of marrow is then
subjected to routine histological processing following formalin fixation. H &E stained 5 µm thick
sections were used for all evaluations. The bone marrow sections were evaluated at 400-x
magnification using a 1 mm2 ruled ocular grid or reticule The number of both mature heterophils
and large primitive or blastic cells present/ total grid area were enumerated for a total of 10
fields for each samples. Mitotic figures and adipocytes were similarly enumerated for select
studies.

Species, Age & Site Considerations:

It is important to recognize some fundamental differences between avail and mammalian bone
marrow as illustrated in figure 1. Unlike mammals, bone marrow in birds does not contain
megakaryocytes.and erythropoiesis normally occurs within the sinusoids rather in the
surrounding interstitial tissues. Avian peripheral blood erythrocytes are oval-shaped and
nucleated; unlike the typically round and non-nucleated erythrocytes found in mammals.
However, similar to mammals, bone marrow within central long bones undergoes transition to a
largely fatty composition with aging. For example, our studies on mid-shaft femoral bone
marrow demonstrated substantial reduction in hematopoietic elements occurring between day
one and forty days of age. However, regions of active hematopoiesis persist within the
metaphyseal regions. Lymphoid aggregates are present which tend to increase with aging in
response to repeated antigenic stimulation (1).

Quantitative Studies on Bone Marrow Age-Associated Changes:

Critical to the elucidation of quantitative pathological changes occurring in the bone marrow of
poultry is documentation of normal composition including age-associated alterations. To this aim
we conducted studies on quantification of mid-shaft femoral bone marrow in broiler chickens of
various ages. The results are shown in figure 2 and demonstrate a marked and progressive
reduction in the concentration of mature and primitive granulocytes occurred between day one
and forty. For example the average number of mature heterophils/ grid field decreased from
around 170 on day one of age to a value of near 50 by forty days of age. The average number
of primitive granulocytes went from 14 to around 1over the same time period.

Bone Marrow Changes in Poultry Condemned for Localized

Inflammatory Process (IP or Cellulitis) and Septcemia-Toxemia (Sep-
Tox) Catagories.

Using the quantitative bone marrow method the cellularity of marrows obtained from grossly
normal appearing aged heavy fowl and market age broiler chickens were compared to those
manifesting with local infections or evidence of septicemia-toxemia [Sep-Tox]. These pilot
studies utilized heavy fowl condemned during processing and field cases of broiler chickens
(10-12). The purpose of these studies was determine if “systemic effects” are present in the
bone marrow in birds with “local” inflammatory lesions, to test a simple histiomorphometirc

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model for quantitation of such potential effects in the bone marrow and to document bone
marrow changes in septicemia /toxemia in poultry (conditions with arguable public health
significance (7,8,9). Examples of normal and hyperplastic bone marrows are shown in figure 3.
The experimental design included the following groups:

Negative Control: Normal Birds Condemned for Mutilation or Contamination.

Positive Control - Septicemia/Toxemia.

Experimental Group - Localized Inflammatory Process [IP or Cellulitis].

The results for quantification of marrows in heavy fowl and broilers demonstrated dramatic and
highly significant elevations in the number of both primitive and mature granulocytes for birds
manifesting with severe local and systemic inflammatory diseases. While values for normal
birds remained around 50/ grid field, average values for septic birds ranged from over 150 for
heavy fowl and around 250 for the field broilers exhibiting sep-tox changes (Figure 4). Bone
marrow heterophil values for cellulitis (IP) birds were typically intermediate between the normal
and septic birds.

Figure 1: Comparison Mammalian & Poultry Marrow

Feline Marrow FIP Hyperplasia 1000-x IBH Chicken Hyperplastic BM 1000-x

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Figure 2: Mature Heterophils Age Changes

250

200
Cells/ Field

150

100

50

0
15 Days 23 Days 29 Days 35-44 Days Market Age

Figure 3: Normal & Hyperplastic Market-

Age Broiler Bone Marrows@ 400-x

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Histomorphometric Findings in the Spleen and Bone Marrow of

“Small Birds” and Other Carcass Cohorts Condemned at Slaughter
Under the Sep-Tox Disposition Category.
The pilot studies in heavy fowl and broilers utilized sep-tox birds manifesting with gross changes
of definitive septicemia or toxemia [small size, muscle wasting, erythemia, cyanosis,
dehydration and/or hemorrhages]. The previous studies documented bone marrow changes in
chickens manifesting with severe localized bacterial infections or generalized septic conditions.

However, additional bone marrow studies were done on broiler chickens that were actually
condemned during routine slaughter inspection for the sep-tox disposition category [as opposed
to being selected from field cases]. The purpose of these studies was the scientific
documentation of the pathological spectrum comprising the “septicemia/toxemia” [sep/tox]
disposition category employed in poultry slaughter inspection (13). A total of 87 condemned and
19 grossly normal carcasses were evaluated in the study. 45% of condemned carcasses were
judged to be small and they exhibited a much higher incidence of “sep-tox” inclusive parameters
[muscle wasting, skin discoloration and dehydration] than in the large condemned carcasses.
Conversely, a higher incidence of conditions typically more reflective of localized infectious
disease [air sac, cellulitis and liver mottling-cholangitis] was present in the large relative to small
bird cohorts:

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The studies were facilitated by employing the simple histomorphometric model for the
quantification of bone marrow and some morphometric measurements on the spleen were also
performed. Comparisons between findings for small and large bird condemned cohorts were
also done.

Spleen Histomorphometrics: Measurement of % Histiocytic Area:

Spleen follicular areas were enlarged in most condemned carcasses and the bone marrow
cellularity increased in a subset of the carcasses. The spleen follicles demonstrated expansion
of cells with histiocytic morphology with a relative depletion of lymphocytes. Histomorphometric
studies were initiated to document the follicular histiocytic areas. Using the NIH Image J
morphometric software program, the total area of interest was first measured using the
rectangle tool (2). Using the polygonal tool, the area occupied by histiocytes within all individual
follicles present within the area of interest were measured, the individual follicular areas
calculated, the sum of all histiocytic area was detemined and the % follicular histiocytic area
relative to the total area of interest was determined. The results are shown in figure 5 and
demonstrate a clear increase in histiocytic follicular area for the sep-tox group relative to
controls.

The % incidence of follicular histiocytic areas present above two defined thresholds (greater
than 30% and above 35%) were also determined for control and sep-tox condemned carcasses
and the results are also presented in figure 5. In addition, an increased incidence for follicular
histiocytic area expansion was observed in the small compared to the large carcass cohorts that
was particularly apparent at the >35% follicular area threshold.2w

Figure 5: Spleen Morphometric % Follicular

Histiocytic Area [Average % Histiocytic Area & %
Incidence ]
45%

40% Ave % Histiocytic Area

35% % incidence >30%

Score

30% % Incidence > 35%

25%

20%
Septic
15%
Control Condemn
10%
1 2

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Bone Marrow Histiomorphometrics:

The bone marrow morphometric results demonstrated a moderate elevation in the number of
absolute heterophils in septic birds compared to normal, but the elevations were of a lesser
magnitude than that seen for selected severe sep-tox birds in the broiler field studies. However,
much greater difference between control and sep-tox birds was evidenced for the % incidences
of granulocytic hyperplasia based on the defined thresholds of >75 cells/ grid area for bone
marrow mature heterophils and > 4 cells/ grid area for primitive cells (figure 6)..

The incidence of heterophil hyperplasia went from 12% in controls up to 31% in the sep-
tox condemned group. In addition, while a zero incidence of primitive cell hyperplasia
was seen for control bone marrows, 19% of the sep-tox group exhibited substantial
elevations.

Figure 6: Bone Marrow Histomorphometrics

35%
Mature Heterophis
30%
Sep-Tox
Primitve Cells
% Incidence

25%

20%
Normal
15%

10%

5%

0%
1 Group 2

E. Coli Intravenous Injection Bone Marrow Study:

A small study was also done to directly document the influence of experimental E. coli sepsis on
the bone marrow (12).

For this purpose market age broilers received intravenous injections of approximately 107 E. coli
organisms and non-injected birds served as controls. Groups of birds [3 birds /group] sacrificed
on post-injection days 1, 3 and 6 and bone marrow was evaluated using our routine
histomorphomeric method. Gross pathology demonstrated the production of dramatic

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hepatosplenomegay [140 and 180 percent increase in liver and spleen weights respectively] in
injected relative to control]. The results for bone marrow mature heterophils are presented in
figures 7. While considerable variation was seen at some time points, the results demonstrated
elevations in both primitive and mature granulocytes occurred in response to E. coli injection.

Figure 7: E. coli Induced Kinetic Marrow

Mature Heterophils
200
180
160 Control
140
Injected
Cells/ Field

120
100
80
60
40
20
0
1 3 6
Post Injection Day

Bone Marrow Changes with Specific Bacterial Disease:

Outbreak of Onithobacterium rhinotracheale Meningoencephalitis in

Commercial Broiler Chickens:
An outbreak of elevated mortality associated with clinical neurological signs including torticollis
and incoordination was observed in 32-day-old mixed-sex, Ross-Ross cross commercial broiler
chickens. Half of the submitted birds also grossly demonstrated unilateral or bilateral hypopyon.
Onithobacterium rhinotracheale was recovered from 60% of the brains & 33% of the eyes
cultured (14).

Microscopic pathology included variable meningoencephalitis,enophthalmitis and bone marrow

granulocytic hyperplasia. The average for mature bone marrow heterophils approached 200/
grid field, while the average for primitive granulocytes was over 8/ grid area. The results for
bone marrow counts for the ORT birds are presented and compared to other results for marrow
hyperplastic and neoplastic conditions in figure 8.

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To our knowledge this case represented the first detailed pathological documentation of an
outbreak of Onithobacterium rhinotrachialis with neurological signs in Mississippi poultry and
associated with a high incidence of previously unreported or unusual lesions of endophthalmitis
and bone marrow granulocytic hyperplasia.

Bone Marrow Findings in Viral Diseases:

West Nile Infection in a Wild Turkey:

One of the first avian viral diseases for which bone marrow was evaluated at our laboratory was
a case of natural west nile virus occurring in a wild turkey. While bone marrow counts were not
performed, the bone marrow demonstrate marked granulocytic hyperplasia by routine histology
(15). The results for immunohistochemistry demonstrate strong staining for west nile virus
antigen by primitive forms of marrow granulocytes (15).

Morphomeric Studies on Kidney and Bone Marrow in Broiler Chickens

with Inclusion Body Hepatitis:
Routine histological evaluation of tissues obtained from an outbreak of inclusion body hepatitis
in Mississippi poultry demonstrated a high incidence of glomerulonephopathy and bone marrow
hyperplasia (16). Grossly, these birds manifested with classical hepatic pathology that was also
accompanied by renal enlargement. Characteristic intranuclear adenoviral inclusion bodies were
usually demonstrated in the livers of most birds, and fowl adenovirus was recovered from livers
of many of the groups that were also positive with PCR testing 916).

Birds with hepatitis exhibited a high incidence of membranoproliferative glomerulonephritis and

bone marrow obtained from many also demonstrated a dramatic hypercellularity of the femoral
bone marrow owing to marked elevations in both primitive and mature forms of granulocytic
elements (figure 8).

Glomerular size was determined using the ImageJ NIH software program and the total cell
counts of corresponding glomeruli were also determined using an ocular grid. The average
glomerular area values for normal glomeruli in the sub-capsular cortical and central kidney
regions respectively were of 1,794 µm2 and 5,304 µm2. In contrast, glomerular measurements
for kidneys exhibiting glomerulonephritis by routine histopathology, demonstrated average
values for the two regions of 4,727 µm2 and 11,063 µm2. The average glomerular cell counts for
the two regions in normal’s were 44 and 107 cells/glomeruli, while averages obtained for birds
with glomerulonephritis were 90 and 193 cells/glomeruli.

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The results for bone marrow cellularity in part are shown in figure 8 and demonstrated a
dramatic elevation in both primitive and mature granulocyte populations. We have subsequently
observed similar elevations in marrow cellularity in other cases of inclusion body hepatitis, but
also one case manifesting with normal marrow cellularity.

To our knowledge, the studies represent the first observation of either a high incidence of
glomerulonephritis or severe bone marrow granulocytic hyperplasia occurring in broiler chickens
with IBH.

Infectious Laryngotracheitis:
Bone marrow was also examined from broilers manifesting having PCR documented cases of
infectious laryngotracheitis [ILT] and compared to marrows of birds from the same flocks not
exhibiting clinical signs of ILT. The results or bone marrow quantitation are presented in figure 8
and compared to marrow results previously obtained for birds having inclusion body hepatitis.
No detectible changes were appreciated in the marrow in ILT-positive birds relative to controls
in contrast to the dramatic hyperplasia that we have seen in many cases of inclusion body
hepatitis.

Avian Myeloblastosis:
Histomorphomeric studies were also performed on bone marrow samples taken from market
chickens manifesting with avian myeloblastosis. The results for myeloblastosis are compared to
our results for normal and hyperplastic bone marrows in figure 8 and demonstrate profound
difference when compared to severely hyperplastic marrows seen with various viral and
bacterial diseases.

New Approaches for Measurement of Bone Marrow

Histopathology:
A small pilot study was done to test the utility of other histomorphometric methods for the
quantitation of marrow cellularity. One method utilized the imageJ software program to
determine the optical density [pixal density] of selected marrows from normal, hyperplastic and
myelod leukosis bone marows. This approach is somewhat similar to that reported for the
documentation of bone marrow cellularity in birds manifesting with chicken anemia viral disease
(4). The results for bone marrow optical density obtained for normal, hyperplastic and myeloid
leukosis in the feasibility study are shown in figure 9.

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Bone Marrow in Protozoan Disease:

Toxoplasmosis in a backyard Guinea fowl with bone marrow
Involvement:
An adult backyard Guinea fowl was submitted to the poultry diagnostic laboratory that
microscopically demonstrated multifocal necrosis and inflammation of the spleen, lung and heart
associated with the variable occurrence of protozoa cysts and tachyzoites compatible with
toxoplasmosis (17). The diagnosis was confirmed with immunohistochenmistry and PCR
testing. Although not counted, the bone marrow exhibited marked multifocal necrosis. edema
and fibrin exudation with marked intra-sinusoidal erythroid and lesser granulocytic hyperplasia
and also intra-lesional protozoan cysts.

\\

Other Organ Morphometric Studies:

Other histomorphometric method studies that were done included employment of nuclear
density determinations for the evaluation of cadiomyopathies and pancreatic hyperplasia as-
well-as optical density and microscopic geometric evaluations of the bursa.

Histomorphometric Studies on Age-Associated Changes in the

Myocardium of Meat-Type Chickens:

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We were interested in determining if quantitative histomorphometrics could be used for

documentation of possible microscopic cardiomyopathy in poultry manifesting with clinical
cyanosis or cardiac diseases

Changes in both gross dimensions and microscopic profiles of hearts in various

cardiomyopathies have been reported for poultry, and abnormal echocardiograms have been
observed with ascites syndrome in broilers and roosters with cyanosis. Preliminary studies were
done to establish normal age-associated changes in the myocardium using simple
histomorphometric evaluations.

The heart studies were based on studies using this approach for the documentation of
quantitative hyperplastic changes occurring in pancreatic acinar cells in rats fed soy trypsin
inhibitor (18).

The nuclear density of myocardial myofibers was determined for various age groups including
aged birds appearing clinically normal or exhibiting transitory cyanosis (19). The numbers of
nuclei per grid field at 400-x magnification for a total of ten fields was determined for each
sample. The preliminary results for myocardial age-associated changes in nuclear density are
presented in figure 11.

Figure 11: Changes in Nuclear Density with Age
200
180
160 147 142
Nuclei /Field @400-x

140
120 106
100
80 68 67
60
40
20
0
1 to 10 11 to 20 21 to 40 200 to 300 > 300
Age Group Days

Other Heart Histomorphometric Measurements:

A variety of other histomorphometric measurements were also investigated on select heart

samples that included myocardial fiber nuclear width, nuclear area and cell width. Select
samples of hearts from birds having low and high nuclear densities were utilized and
measurements made from a published micrograph for J virus associated cardiomyopathies

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were also performed (20). In addition, studies have also been done comparing results for
nuclear density quantitation to results obtained using a particle counting method [automated
nuclear density measurements using the ImageJ particle analysis]. For this purpose the NIH
imageJ software program was utilized for measurements. Following calibration with a
micrometer; heart sections from having documented high or low nuclear densities were
measured. A positive control for cardiomyopathy was provided by a published micrograph of J-
virus associated cardiomyopathy (20). Ten 400-x fields were evaluated for each heart. The
results for comparisons of the various methods in the pilot study are presented in table 1. While
considerable differences were observed between actual counts and estimates using particle
analysis, the averages for the two methods were similar and suggest that refinement of the
particle analysis method by adjustments of various thresholds might provide for a crude
automated method for estimating changes occurring in the myocardium with cardiac diseases.

Table 1: Comparison of Manual Cell Counts with Automated

Counts Using ImageJ Software Morphometric Program

Sample Actual Cell Count ImageJ "Particle” Number

1 115 109
2 169 142
3 171 183
4 132 133
5 105 99
6 233 348
7 134 142
8 130 140
9 141 153
10 191 332
11 226 176
12 173 192
Average ± SDev = 160 ± 41 Average ± SDev = 179 ± 80

Correlation Coefficient = 0.78

Comparison with Published Cardiomyopathy Images:

One useful applications of cardiac histomorphometric is that it allows for comparisons with
published photomicrographs of poultry exhibiting documented cardiomyopathies. We compared
our results for hearts demonstrating either low or high myocardial densities to a published image
of cadiomyopathy in broilers associated with experimental congenital avian viral leukosis
infections (20). The results are shown in table 2 and demonstrate a dramatic reduction in
myocardial nuclear density [and conversely enlargement of myocardial fiber size] in leukosis-
associated cardiomyopathy; even when compared to our low nuclear density bird.

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Table 2: Comparison of "Experimental” to “Publication”

Values:

Micrograph Sample Nuclei/ 400-x Field

High Nuclear Density 136

Low Nuclear Density 34
Publication
a. Direct Count 49
b. *Corrected Count 17

*Correction for Magnification = Actual or Direct Count x 0.34

The following conclusions were made from the myocardial histomorphometric study:

1. Histomorphometric studies document a progressive reduction in myocardial nuclear

density with increasing age

2. A spectrum of histomorphometric changes in various parameters were appreciated

within the aged roosters [nuclear density, nuclear size and myofiber width] some of
which might indicate possible early preclinical cardiomyopathy.

3. The various quantitative cardiomorphometric methods can be utilized to evaluate

published photomicrographs from birds with documented cardiomyopathies

Bursa Morphometrics (Comparison of Microscopic Methods

for Bursa Histopathology Evaluation):
A large number of studies have demonstrated the utility of using routine histopathological scoring
and morphometric procedure in documenting microscopic pathology of the bursa (21-24). We
were interested in the development of such methods in our laboratory and in the comparison of
these methods in view of the large numbers of bursa samples that were provided.

The microscopic pathology of 600 bursa samples from four broiler farms was histologically
evaluated and subsets of the samples were also evaluated using quantitative histomorphometric
methods [simple generalized subjective histopathology scoring, additive detailed histological

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scoring and quantitative measurement of optical density and other morphometric parameters].
In the first method, all bursas were given a subjective severity score for overall histopathology
using a range of 0 to 5 plus [0 = Absent; 1 = Minimal; 2 = Mild; 3 = Moderate; 4 = Marked, and 5
= Severe]. The detailed cumulative subjective histopathology scoring method involved first
assigning individual scores to multiple subcategories of microscopic pathology and than
generating a generalized histopathology score by summation of the component scores. The
following parameters were scored: lymphoid depletion; surface scalloping, heterophilic
inflammatory infiltration, interstitial inflammatory cell infiltration, edema, fibrosis, and cystic
changes. The third method employed was to measure the optical density of gray scale
photomicrographs of the individual bursas and measurement of cortex to medullary ratios using
actual measurments of microscopic areas or radominized measurement of the cortical width.

All morphometric evaluations employed the ImageJ software program provided by NIH
[http://imagej.nih.gov/ij/download.html]. One morphometric method employed was the
measurement of optical density using gray scale photomicrographs standardized using a Kodak
optical density cailbration image. In addition, individual bursas and measurement of cortex to
medullary ratios using actual measurements of microscopic areas or radominized measurement
of the cortical width were performed.

The results demonstrated that within the limits of methods we employed for measurement of
optical density, there appeared to be no major difference for overall OD between the groups.
This is not to say that other procedures for measuring OD might not be more sensitive.
However, there were observable differences in OD between the least and most severely
affected bursa subgroups that tended to correlate to histopathology severity scores and the
geometric measurements obtained for the subgroups.

Values for the most severely affected and least affected bursas for the various methods are
compared in figure 12. While the magnitude of differences were similar and extensive for both
the cumulative pathology and % cortex area [31% and 37% respectively], the percentage
difference between least and most severe groups for optical density was dissimilar and of a
much less magnitude. In addition while the group differences were highly significant for % cortex
area [≤ 8.12E-06], they were of lower significance [≥ 0.04] for optical density.

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References:
1. Avian Histopathology, 3rd Edition, Chapter 1. Hemic System, , Oscar J. Fletcher
and Tahseen Abdul-Aziz Eds., American Association of Avian Pathologists Omni
Press, Madison WI, pp. 2-10, 2008.
2. All morphometric evaluations employed the ImageJ software program provided by
NIH [http://imagej.nih.gov/ij/download.html].
3. Tumor Diagnosis Manual, The Differential Diagnosis of Lymphoid and Myeloid
Tumors in the Chicken, 1st Edition, Richard L. Witter, Isabel M. Gimeno, Arun R.
Pandirai and Aly M. Fadly Eds., American Association of Avian Pathologists,
Omnipress, Jacksonville, Florida, 2010.
4. Luz Garcia, Victor Bermudez, Mariela Brett, et al. Quantitative analytical
technique applied to histopathology of birds infected experimentally by the virus of
chicken anemia virus. Microscopy Proceedings Diagnostic Pathology, 3 (Suppl 1):
S21doi, 2008.

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5. Lesions in Bone and Bone Marrow in Myeloid Leukosis Occurring Naturally in

Adult Broiler Breeders, Nakamura, K. Ogiso, Tsukamotoa, K et al. Avian Diseases
44: 215-221, 2000.
6. Lysis of Myelocytes in Chickens Infected with Infectious Bursal Disease Virus. M.
Inoue, A. Fujita, and K. Maeda, Vet Pathol 36: 146, 1999.
7. HACCP Inspection Model Project: Young Chicken Inspection:, Federal Register:
November 2, 2000 (Volume 65, Number 213, Notices, Pages 65828-65829.
8. Fisher, M.E., Trampel, D.W., Griffith, R.W. Postmortem Detection of Acute
Septicemia in Broilers. Avian Dis. 42(3): 452-461, 1998.
9. Gomis SM, Goodhope R, Kumor. Isolation of Escherichia coli from cellulitis and
other lesions of the same bird in broilers at slaughter. L, Caddy et al Avian Dis
43(3):491-6, 1999.
10. F.D, Wilson, FD, T. Cumming, S. Hubbard, D. Magee and L. Pace. Morphometric
Studies on Inflammatory Diseases in Poultry: Presented at the AAVLD Annual
Meeting, Greensboro, NC, Oct 2004.
11. Wilson, FD, Cummings, T, Hubbard, S and Ewing, M. Detection of Bone Marrow Effects
Produced by “Sep/Tox” and “IP” Disposition Conditions in Poultry Using a Simple
Histomorphometric Method, Presented at the American Association of Avian Pathologists
Meeting, AVMA/AAAP Annual Meeting, Philadelphia, Pennsylvania, July 24-28, 2004.
12. Floyd D. Wilson, Timothy, S. Cummings and Mark A. Burleson Histopathological
Effects of Intravenous Injections of E. coli on the Bone Marrow in Broilers
Including Quantitative Histomorphometric Changes Presented at the AVMA,
AAAP Meeting, July 15-19, Honolulu, Hawaii, 2006.
13. Floyd Wilson, Fred Hoerr, Tim Cummings, and Marty Ewing. Routine Histological &
Histomorphometric Finding in the Spleen and Bone Marrow of “Small Birds” and Other
Carcass Cohorts Condemned at Slaughter Under the Sep-Tox DispositionCategory.
Presented at the American Association of Avian Pathologists, Annual Meeting,
Washington, DC, July 14-18, 2007.
14. Floyd Wilson, Shuping Zhang,, Sue Ann Hubbard and Danny Magee Unusual
Pathology Manifested with an Outbreak of Ornithobacterium rhinotracheale
Associated Meningoencephalitis in Commercial Broiler Chickens. Presented at
the American Association of Avian Pathologists, , July 11 - 14, Seattle,
Washington , 2009.
15. Z. Zhang, F. Wilson, R. Read, L. Pace, S. Zhang. Detection and Characterization of
Naturaly Acquired West Nile Virus Infection in a Female Wild Turkey J Vet Diagn Invest
18: 204-208, 2006.
16. Floyd D. Wilson, William R. Maslin, Robert W. Wills, Philip A. Stayer, Carlos
Gabriel Senties-Cue and Danny L. Magee. High Incidence of Glomerulonephritis
Associated with Inclusion Body Hepatitis in Meat-type Chickens: Routine
Histopathology and Histomorphometric Studies. Avian Diseases 54(3):975-980,
2010].
17. Kelly Jones, Floyd Wilson, and Scott Fitzgerald and Matte Kiupel. A Natural
Outbreak of Toxoplasmosis in a Backyard Flock of Guinea Fowl in Mississippi.
Presented at the American Association of Avian Pathologists Meeting, July 16-19,
St. Louis Missouri, 2011.
18. J.C.Smith, , F. D. Wilson, P.V. Allen and D.L. Berry, Hyperplasia of the Rat
Pancreas Produced by Short-Term Dietary Administration of Soya Derived Protein

62nd Annual Meeting of the American College of Veterinary Pathologists and

46th Annual Meeting of the American Society for Veterinary Clinical Pathology
2011 - Nashville, Tennessee, USA
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and Soybean Trypsin Inhibitor.

J. Applied Toxicology, 32: 346-350, 1989.
19. Floyd Wilson, Kelli Jones, Danny Magee & Lanny Pace Histomorphometric &
Histologic Studies on Age-Associated Changes in the Myocardium and Arteries of
Meat-Type Chickens. . Presented at American Association of Avian Pathologists,
August 2-4, Atlanta, Georgia, 2010.
20. N.L. Stedman and T.P. Brown Cadiomyopathy in Broiler Chickens Congenitally
Infected with Avian Leukosis Virus. Vet. Pathol. 39:161=164, 2002
21. Floyd D. Wilson, Alejandro Banda and Ivan Alvarado
Comparison of Microscopic Methods for Bursa Histopathology Evaluation.
Presented at the American Association of Avian Pathologists Meeting, July 16-19,
St. Louis Missouri, 2011.
22. M. Nishizawa, M., Paulillo,A.C., Bernardino A et all Evaluation of
anatomopathological, serological, immunological responses and protection in
broilers vaccinated with live infectious bursal disease vaccines. Arq. Inst. Biol.,
São Paulo 74: 219-226, 2007
23. Henry C W, Brewer R N and Edgar S A. (1980). Studies on infectious bursal
disease in chickens. 2.Scoring microscopic lesions in the bursa of Fabricius,
thymus, spleen and kidney in gnotobiotic and battery reared White Leghorns
experimentally infected with infectious bursal disease virus. Poultry Science
59: 1006-1017, 1980.
24. Krishnamohan Reddy et al. Bursal Lesion Scoring for the Assessment of Severity
of Infectious Disease.http://www.tanuvas.tn.nic.in/tnjvas/vol4(1)/25-28.pdf

62nd Annual Meeting of the American College of Veterinary Pathologists and

46th Annual Meeting of the American Society for Veterinary Clinical Pathology
2011 - Nashville, Tennessee, USA