The study determined the occurrence and distribution of transmissible viral proventriculitis (TVP) and its correlation with infectious bursal disease (IBD) in broiler chickens from 78 flocks in 5 complexes in North Carolina. On initial gross evaluation of proventriculi from all complexes, flocks were diagnosed with TVP, but microscopic examination confirmed TVP in only 3 flocks from one complex. A subsequent study of this complex found 9 of 15 flocks were TVP-positive by histopathology, with 5 initially positive and 4 more on re-sampling. All initially positive flocks also tested positive for an adenovirus-like virus (AdLV) using immunofluorescence assays. Only 3 f

1. LRH: R. Monleon et al.
RRH: TVP in NC Broilers
Occurrence and Distribution of Transmissible Viral Proventriculitis in
North Carolina Broiler Chickens
R. MonleonA
, M. AlfonsoA
, J. RaduB
, J. S. GuyA
, and H. J. BarnesA,C
A
Poultry Health Management, Department of Population Health and Pathobiology,
College of Veterinary Medicine, North Carolina State University,
4700 Hillsborough St., Raleigh NC 27606
B
Schering-Plough Animal Health Corp.,
1095 Morris Ave, Union NJ 07083
For submission to: Avian Diseases
C
Corresponding Author. Dr. H. John Barnes, Poultry Health Management, Department
of Population Health & Pathobiology, College of Veterinary Medicine, North Carolina
State University, 4700 Hillsborough Street, Raleigh, NC 27606.

2. 2
SUMMARY. The occurrence and distribution of transmissible viral
proventriculitis (TVP) and the correlation between TVP and infectious bursal disease
(IBD) was determined in chickens from 78 flocks in 5 broiler complexes located in
different geographical regions of North Carolina. On gross evaluation of proventriculi,
all complexes were initially diagnosed with TVP-positive (TVP+) flocks. However,
microscopic examination confirmed 3 TVP+ flocks in only one complex. In a
subsequent second study of the TVP+ complex, nine out of 15 flocks were identified as
TVP+; five on initial sampling and four more on re-sampling 2 weeks later or just prior
to processing. Proventriculi were additionally evaluated with an indirect fluorescent
antibody (IFA) test for an adenovirus-like virus (AdLV). All five TVP+ flocks in the
initial sampling had microscopic lesions of TVP and also tested positive for AdLV. Only
3 flocks were positive for both TVP and IBD.
RESUMEN. La ocurrencia y la distribucion de proventriculitis viral transmissible
(TVP), y la correlacion entre TVP y la enfermedad infecciosa de la bolsa (IBD) fue
determinada en 78 parvadas de pollos de 5 complejos de broilers localizados en
diferentes areas geograficas de Carolina del Norte. Al inicio macroscopicamente los
proventriculos, en todos los complejos fueron positivos a TVP (TVP+), sin embargo la
evaluacion microscopica confirmo 3 parvadas TVP+, y en un solo complejo. En un
estudio posterior en el complejo TVP+, nueve de quince parvadas fueron identificadas
como TVP+; cinco subsiguientes a la primera toma de muestras, y cuatro mas en una
retoma dos semanas despues o antes del procesado. Los provenctriculos fueron
adicionalmente evaluados con un test indirecto de fluorescencia de anticuerpo (IFA)
contra un virus similar a adenovirus (AdLV). Las cinco parvadas comprobadas en la toma
de muestra inicial tuvieron lesiones microscopicas y fueron positivas a AdLV. Solo 3
parvadas fueron positivas para ambos TVP e IBD.

3. 3
Keywords: broiler, proventriculitis, transmissible viral proventriculitis, TVP, North
Carolina, adenovirus-like virus
Abbreviations: AdLV = adenovirus-like virus; BHE = bursae histopathology evaluation;
BHE+ = BHE-positive; IBD = infectious bursal disease; IBDV = infectious bursal
disease virus; IBV = infectious bronchitis virus; IFA = indirect fluorescent antibody;
PFTE = proventriculi fresh tissue evaluation; PHE = proventriculi histopathology
evaluation; PXTE = proventriculi fixed tissue evaluation; TVP = transmissible viral
proventriculitis; TVP+ = TVP-positive; SPF = specific pathogen free

4. 4
Transmissible viral proventriculitis (TVP) was described in 1996 as a disease of
broiler chickens characterized by lymphocytic proventriculitis and necrosis primarily
involving the deep mucosal glands (6). The infectious transmissible nature of TVP has
been confirmed with experimental reproduction of the disease using proventricular
homogenates from affected chickens (2, 4, 8, 9, 20, 21). Demonstration of virus within
lesions and reproduction of TVP with filtrates of affected proventriculi provide evidence
for a virus as the primary cause of the disease (2, 9).
In the initial description of TVP, based on naturally infected birds, virus particles
were identified within nuclei and vacuolated spaces in the cytoplasm of affected
proventricular epithelial cells in lesional areas. They were considered the likely cause of
TVP, but the specific identity of the virus could not be determined (6). In subsequent
studies, similar, though slightly larger virus particles were identified in experimentally
infected chickens (9, 21). Recently, virus particles similar to those described in the
initial description of the disease (6) were identified and partially characterized as an
adenovirus-like virus (AdLV) (8). A strain of AdLV (R11/3) reproduced TVP in specific
pathogen free (SPF) chickens following oral inoculation at two weeks of age (8). Other
infectious agents have also been identified in chickens with TVP, including avian
reovirus (1, 11, 12, 18), adenovirus (10, 12, 21), corona virus (IBV) (15, 25), and
birnavirus (IBDV) (2, 5, 9, 17, 24). However, the role of IBDV in TVP remains
controversial. Recent studies suggest that although the virus is present in proventriculi of
affected birds, it may not be involved in actually causing the disease (4, 19, 21).

5. 5
Several diagnostic methods for TVP identification have been used to determine
the most accurate way to identify the disease. Histopathology is considered among the
best diagnostic methods as TVP lesions are characteristic of the disease. Microscopic
lesions of TVP occur only in the proventriculus and are characterized by necrosis of
glandular epithelial cells, lymphocytic infiltration of the lamina propria of mucosa and
proventricular glands, hyperplasia of duct epithelium, and replacement of lost glandular
epithelium by duct epithelium. Lesions are most prominent in the central areas of
mucosal glands where the intermediate epithelium between the glandular and ductal
epithelia is located (8). Gross examination of fresh / fixed proventriculi has been used in
the past as a method to determine lesion score (2, 9). In addition, electron microscopy
can be used to diagnose TVP: intranuclear or intracytoplasmic virus particles are found in
affected proventricular epithelial cells (6, 8, 9, 21). Indirect fluorescent antibody (IFA)
test provided satisfactory results in identifying the probable cause of TVP: AdLV R11/3
in frozen tissues (8). RT-PCR and PCR have successfully identified IBDV and IBV in
affected chickens (5, 21). DNA in situ hybridization was attempted to identify
polyomavirus and adenovirus in replicate proventriculus sections, but failed (6).
TVP has been identified in the USA (2, 6, 8, 9, 21), Australia (23), and the
Netherlands (7, 10). Recently, a series of histology TVP cases confirmed by
histopathology in Panama (3), and suspected cases in Spain (22) suggest that the disease
is more widely spread than previously supposed.
Limited information has been published about occurrence and distribution of TVP
in the US Poultry Industry. During a survey of broilers in Georgia (USA) with
proventricular lesions between 1992 and 1995, compatible light microscopy TVP was the

6. 6
most commonly observed lesion in 109/220 birds (49.5%) (6). No further information is
available regarding occurrence and distribution of this disease in a commercial
environment.
The importance of TVP is still currently debated, despite TVP-infected chickens
being considered more costly to produce (7, 13) and that proventriculitis has been
associated with a wide range of negative effects on production and processing parameters
(2, 8). No published study has established the economic significance of TVP.
In addition, no survey has previously been conducted to determine the occurrence
and distribution of TVP in a specific geographical area. Evaluation of fresh and fixed
tissue, including lesion scoring have been conducted (2, 8), however correlation with
histopathology / IFA testing has not been yet determined. Likewise, association of TVP
with IBDV (2, 5, 9), is controversial (4, 19, 21) and requires further examination. Our
study was done to answer some of these specific questions related to TVP. We
determined the occurrence and distribution of TVP in a representative geographical area
of North Carolina, compared different diagnostic techniques to determine correlations
among them, and evaluated the presence of IBD lesions associated with TVP.
MATERIALS AND METHODS
Case materials and experimental design. Between November, 2004 and
March, 2005, proventriculi and bursae were collected from 530 broilers in 78 flocks from
four different integrators in five different complexes (A-E) located in various areas of
high poultry density in North Carolina (Figure 1). Six studies were conducted (1-6); two
at the same complex (studies 2, 3). The number of flocks sampled in each study varied
from 9 to 16, with an age range of 6 to 56 days (Table 1). Flocks from each complex

7. 7
were evaluated to determine the prevalence of TVP and IBD. All methods of evaluation
were not used in all studies. Samples of proventriculi and bursae were fixed in 10%
neutral buffered formalin and proventriculi were transferred to 70% ethanol after 72 hrs.
Tissues were processed for histopathology using conventional paraffin embedding and
staining with hematoxylin and eosin. After identification by histopathology of TVP
lesions in chickens from flocks in study 2, a second study (study 3) was conducted,
consisting of two parts. Fifteen flocks were examined initially, followed by a second
evaluation either two weeks later or just prior to processing.
Proventriculi fresh tissue evaluation (PFTE). Fresh proventricular tissue
samples were evaluated in study 5 (16 flocks). A scale was developed to score the
lesions (0=no lesions to 3=severe lesions). A lesion criterion was based on observation
of thickened and distended glands containing viscous white material in the wall, and a
lumen with thickened and rugose mucosal appearance and indistinct papillary orifices (7).
Proventriculi fixed tissues evaluation (PXTE). Fixed proventricular tissues
were collected and evaluated in studies 1, 2, 4, and 5 (49 flocks). A scale was developed
to score the lesions (0=no lesions to 3=severe lesions). A lesion criterion was based on
observation of thickening of the wall and checkered appearance with subserosal
disseminated gray-white polygonal foci (7).
Proventriculi histopathology evaluation (PHE). Proventricular tissues were
sampled from all flocks. A scale was developed to score the lesions (0=no lesions to
3=severe lesions). The lesion criteria were based on observation of glandular epithelial
necrosis, duct epithelial hyperplasia, replacement of glandular epithelium with ductal
epithelium, and increased interstitial lymphoid tissue in glands (8). A set of four pieces

8. 8
of tissue that scored 0 to 3 respectively in fresh evaluation was collected separately for
histopathology evaluation.
Indirect fluorescent antibody test (IFA). Pieces of proventriculi used were
snap-frozen on dry ice in OCT tissue embedding medium (Fisher Science Education,
Hanover Park, IL) and stored at –80C until sectioned on a cryostat. An IFA test specific
for AdLV (R11/3) (8) was performed on 15 samples in the first part of study 3.
Independent testing was done simultaneously, and correlated with histopathology results.
Bursal histopathology evaluation (BHE). Bursae tissues were sampled from 77
flocks of the 78 flocks in the study. No samples were collected during the second part of
study 3. A scale was developed to score the lesions (0=no lesions to 3=severe lesions).
Lesion criteria included degeneration or necrosis of lymphocytes in the medullar area of
the bursa, heterophil replacement of lymphocytes, hemorrhage, edema, cystic cavities in
medullary areas of follicles, and fibroplasia in interfollicular connective tissue (14).
Statistical analysis. Statistical analysis was performed using McNemar’s chi-
square test (Microsoft® Office Excel for Windows XP) (16) to determine significance (p
= < 0.05) between PFTE and PHE, PXTE and PHE, IFA and PHE, and BHE and. PHE.
RESULTS
Proventricular fresh tissue evaluation. Seven flocks out of 16 (43.8 %) were
scored as positive in the single fresh proventriculi evaluation study (study 5) (Table 2).
The age of positive flocks was 21, 24, 29, 35, 52, 52 and 59 days.
Proventricular fixed tissue evaluation. Twenty-six flocks out of 49 (53.1 %)
from the four studies evaluated using fixed tissues (1, 2, 4 and 5) scored positive (Table
2). Seven flocks out of 9 were positive in study 1 (78%) at ages 18, 25, 29, 33, 36, 40

9. 9
and 50 days. Six flocks out of 12 were positive in study 2 (50%), at ages 18, 34, 42, 44,
48, and 51 days. Five flocks out of 12 were positive in study 4 (42%), at ages 28, 35, 42,
42, and 48 days. Eight flocks out of 16 were positive in study 5 (50%), at ages 15, 28, 29,
35, 35, 52, 52 and 52 days.
Proventricular histopathology evaluation. Three of 63 flocks (4.76%) from
complex B only (20% of complexes) were scored positive in the initial five studies (1, 2,
4, 5, and 6) (Table 2). All three positive flocks were found in study 2. Ages of the
positive flocks were 18, 34, and 48 days. In part one of study 3, five flocks out of fifteen
(33.3 %) were positive. Ages of the positive flocks were 17, 18, 21, 27 and 28 days. In
the second part of study 3, the same fifteen flocks from part one were re-sampled, and
nine flocks out of fifteen (60%) were positive. The additional positive flocks were
identified in samples obtained within one week of market age (58-days).
Indirect fluorescent antibody test. Five of fifteen (33.3%) flocks were positive
in the IFA test performed in the first part of study 3 (Table 2). These five flocks
corresponded with same flocks that had independently been diagnosed as TVP-positive
(TVP+) by histopathology.
Bursal histopathology evaluation. All of the complexes had positive flocks
based on bursal histopathology (Table 2). In total, 38 out of 77 (49.36%) flocks were
positive in the initial five complexes (1, 2, 4, 5, and 6) and the first part of study 3. The
age range of the positive flocks was 18 - 52 days.
Statistical analysis. Statistical results showed significant differences between
PFTE and PHE, PXTE and PHE, and PHE and BHE. No significance was found
between PHE and IFA.

10. 10
DISCUSSION
One of our main objectives was to determine if TVP could be diagnosed on the
basis of gross lesions. Our study indicated that when using direct evaluation (PFTE /
PXTE) of the proventriculi for the diagnosis of TVP, the results significantly disagree
with histopathology. Fresh tissue evaluation and fixed tissue evaluation resulted in a
number of false positives that in our opinion ruled out the possibility of using direct
evaluation as a definitive tool for TVP diagnosis
Occurrence of TVP within complexes as diagnosed by histopathology was one
out of five (20%). These data were lower than our expectations at the beginning of these
studies, especially considering industry concerns. The only previous reference
highlighted 109 cases (1.7%) with TVP-compatible lesions from a study of 6437 cases
over a period of 4 years (6). Nevertheless, data in that study were not tabulated by
complex, hence it is not possible to compare our results with it. In study 2, the number of
positive flocks was only 3 (25%), and 4 months later in study 3 it increased to 5 (33%)
and 9 (60%), suggesting a possible increase in distribution of the disease within the
complex. Caution should be observed regarding the increase in prevalence in study 3
from 5 to 9 positive flocks. The variability in this complex may be attributed to a real
increase in prevalence or to an insufficient number of samples chosen in each flock.
Comparison of histopathology with IFA brought an interesting result. The 5
positive in histopathology were positive to IFA against the AdLV R11/3 antigen. This
corroborates Guy et al. (8) studies, providing evidence that an AdLV antigen was present
in TVP-affected proventriculi. Therefore AdLV may be considered a likely cause of

11. 11
TVP. Additionally, histopathology can be used as a simple, economic, and definitive
method of diagnosing TVP.
Controversy regarding the role of IBDV in TVP has been a long-standing issue:
currently published data links IBDV as a major causative agent of TVP (2, 5, 9) in
contrast to naturally-occurring TVP in the absence of IBDV referenced by Pantin-
Jackwood (19) and other studies (21). Results in our study demonstrate that TVP can
significantly (p = < 0.05) occur in the absence of IBDV (22/48=46%), suggesting that
IBDV is likely not the primary cause of TVP. These results are consistent with our
hypothesis that AdLV is the primary cause of TVP and that IBDV likely plays a
secondary role that may facilitate the establishment of the disease.
In the future, it would be informative to conduct additional studies of TVP in a
larger population and in different areas to better understand its national occurrence and
distribution. As data related to processing was contradictory in previous studies (2),
additional research regarding production and processing parameters should provide a
correct and accurate evaluation of industry loss incurred due to TVP disease. Direct
(fresh, fixed) diagnosis of the disease may require additional review, however our data
showed that it should not be used as a primary diagnostic tool. Additional diagnostic
techniques (i.e.: DNA in situ hybridization) should be investigated and correlated with
actual proven methods (i.e.: histopathology). Finally, the fundamental role of IBDV in
TVP needs further clarification. The isolation of IBDV from TVP-affected chickens and
subsequent reproduction of TVP disease in SPF chickens suggests a possible key role of
IBDV in the development of TVP disease.

12. 12
ACKNOWLEDGMENTS
The research reported in this paper was supported in part by a grant from the US
Poultry and Egg Association and funds provided by the North Carolina State University
College of Veterinary Medicine. We would like to thank Mr. T. Cimino of Merial-Select
and Mr. J. Ledbetter of Schering-Plough for their help with this project. The Author
would also like to thank Drs. O. J. Fletcher, D. H. Ley, and Miss. C. Germain for editorial
support.
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