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

BSchering-Plough Animal Health Corp.,

1095 Morris Ave, Union NJ 07083

For submission to: Avian Diseases

CCorresponding 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

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

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

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

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

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

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

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

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

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

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

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.

REFERENCES

1. Apple R.O., J. K. Skeeles, G. E. Houghten, J. N. Beasley, and K. S. Kim.

Investigation of a chronic feed-passage problem on a broiler farm in northwest Arkansas.

Avian Dis. 35:422-425. 1991.

2. Bayyari, G. R., W. E. Huff, J. M. Balog, N. C. Rath, and J. N. Beasley.

Experimental reproduction of proventriculitis using homogenates of proventricular

tissues. Poult. Sci. 74:1799-1809. 1995.

3. Calderon, E. Personal communication. 2005.

4. Dormitorio, T. V., J. J. Giambrone, and F. J. Hoerr. Experimental reproduction of

transmissible proventriculitis in broilers. Proc. International Poultry Scientific Forum, Jan

24-25, Atlanta, GA. pp. 23. 2005.

5. Dormitorio, T. V., J. J. Giambrone, F. J. Hoerr, T. F. Kelly, and S. B. Lockaby.

Effect of vaccination against IBDV on incidence and severity of proventriculitis in SPF

leghorns. Proc. International Poultry Scientific Forum, Jan. 15-16, Atlanta, GA. pp. 40.

2001.

13

6. Goodwin, M. A., S. Hafner, D. I. Bounous, K. S. Latimer, E. C. Player, F. D.

Niagro, R. P. Campagnoli, and J. Brown. Viral proventriculitis in chickens. Avian Pathol.

25:369-379. 1996.

7. Goodwin, M. A., and S. Hafner. Viral proventriculitis. In: Diseases of Poultry,

11th ed. H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, Y. M. Saif, and D.

E. Swayne, eds. Iowa State University Press, Ames, IA. pp. 383-388. 2003.

8. Guy, J. S., H. J. Barnes, L. Smith, R. Owen, and F. J. Fuller. Partial

characterization of an adenovirus-like virus isolated from broiler chickens with

transmissible viral proventriculitis. Avian Dis. 49:344-351. 2005.

9. Huff, G. R., Q. Zheng, L. A. Newberry, W. E. Huff, J. M. Balog, N. C. Rath, K.

S. Kim, E. M. Martin, S. C. Goeke, and J. K. Skeeles. Viral and bacterial agents

associated with experimental transmission of infectious proventriculitis of broiler

chickens. Avian Dis. 45:828-843. 2001.

10. Kouwenhoven, B., F. G. Davelaar, and J. Van Walsum. Infectious

proventriculitis causing runting in broilers. Avian Pathol. 7:183-187. 1978.

11. Kouwenhoven, B., M. Vertommen, and E. Goren. Investigations into the role of

reovirus in the malabsortion syndrome. Avian Pathol. 17:879-892. 1988.

12. Lenz, S. D., F. J. Hoerr, A. C. Ellis, M. A. Toivio-Kinnucan, and M. Yu.

Gastrointestinal pathogenicity of adenovirus and reovirus isolated from broiler chickens

in Alabama. J. Vet Diagn. Invest. 10:145-151. 1998.

13. Leonard, J., and S. Schmittle. Proventriculitis, proventriculosis distinctions are

important. Feedstuffs, January 2, 67(1):12. 1995.

14

14. Lukert, P. D., and Y. M. Saif. In: Diseases of Poultry, 11th ed. H. J. Barnes, J. R.

Glisson, A. M. Fadly, L. R. McDougald, Y. M. Saif, and D. E. Swayne. Iowa State

University Press, Ames, IA. pp. 161-179. 2003.

15. Ma, F. L., X. L. Fu, G. Z. Chen; S. Y. Jia; R. M. Chen; W. S. Liu; H. Xuan. The

isolation, identification and experimental vaccination of avian infectious bronchitis virus

causing proventriculitis. Chinese J. Vet. Sci. 24:225-227. 2004.

16. Microsoft® Office Excel Software for Windows XP. Microsoft, Inc., Redmond,

WA, 2003.

17. Newberry, L. A. Determination of the role of standard and variant strains of

infectious bursal disease virus in the induction of viral proventriculitis and enteric disease

in chicken. Ph.D. Dissertation. University of Arkansas, Fayetteville, AR. 1996.

18. Page, R. K., O. J. Fletcher, G. N. Rowland, D. Gaundry, and P. Villegas.

Malabsorption syndrome in broiler chickens. Avian Dis. 26:618-626. 1982.

19. Pantin-Jackwood, M. J., and T. P. Brown. Infectious bursal disease virus and

proventriculitis in broiler chickens. Avian Dis. 47:681-690. 2003.

20. Pantin-Jackwood, M. J., T. P Brown, Y. Kim, and G. R. Huff. Proventriculitis in

broiler chickens: Effects of immunosuppression. Avian Dis. 48:300-316. 2004

21. Pantin-Jackwood, M. J., T. P. Brown, and G. R. Huff. Reproduction of

Proventriculitis in Commercial and Specific-Pathogen-Free Broiler Chickens. Avian Dis.

49:352-360. 2005.

22. Pizarro, M. Personal communication. 2005.

15

23. Reece, R. Transmissible (infectious?) proventriculitis of Australian broiler

chickens. Proc. Australian Vet. Poultry Assoc.. Conf., Goldcoast, Queensland, Australia.

pp. 35-38. 2002.

24. Shapiro, F., and I. Nir. Stunting syndrome in broilers: physical, physiological, and

behavioral aspects. Poult. Sci. 74:33-44. 1995.

25. Yu, L, Y. Jiang, S. Low, Z. Wang, S.J. Nam, W. Liu, and J. Kwang.

Characterization of three Infectious Bronchitis Virus isolated from China associated with

proventriculitis in vaccinated chickens. Avian Dis. 45:416-424. 2001.