Chicken proventricular necrosis virus related transmissible viral proventriculitis in broiler chickens in Poland

Transmissible viral proventriculitis (TVP) is an infectious disease reported in all production types of chickens. TVP is manifested in decreased body weight gains, wide weight diversity of birds in the ock and poor feed conversion. Histopathological examination seems to be the most reliable method for conrming the disease. Although TVP etiology has not been explicitly dened, a novel virus identied as a member of the Birnaviridae family, named chicken proventricular necrosis virus (CPNV) has been isolated from clinical cases of TVP and it is now considered as a potential factor of a disease. The study was undertaken in order to reproduce the disease under laboratory conditions and to evaluate the etiology of rst described Polish case of TVP.


Conclusions.
We have demonstrated that CPNV was involved in the development of the disease. We did not record the presence of IBDV in the TVP or control birds, despite our recording a strong seroconversion against IBDV in the birds from the TVP group. CPNV belongs to the same family as IBDV, which allows us to assume serological cross-reactivity between them. This possibility of CPNV infections affecting IBDV antibody levels detected by commonly available ELISAs should be taken into account under poultry eld conditions and diagnosis. The role of FAdV in the development of TVP needs further evaluation.

Background
Transmissible viral proventriculitis (TVP) is an infectious disease reported in the production of all types of chickens and having signi cant impact on the poultry industry. The typical pathological lesions observed in the course of TVP affect the proventriculus and are described as proventricular enlargement, thickening of its walls and spotty discoloration in the cross-section. In isolated cases, small hemorrhagic changes are observed in the proventricular mucosa [1].
The rst reports on TVP date back to 1978 and come from the Netherlands [2], when Kouwenhoven et al. reported a case of proventriculitis in commercial chicken broilers and proved that TVP was induced by an infectious factor. Since then, TVP cases have been identi ed and reported in the USA, Australia, China, South Korea, Spain, France, the UK, and Poland among other countries [3][4][5][6][7][8][9][10].
The etiology of TVP has not been explicitly de ned so far. Studies on TVP etiopathology imply the involvement of infectious bursal disease viruses (IBDV) of the Birnaviridae family, IBDV-like viruses, infectious bronchitis viruses (IB) of the Coronaviridae family, reoviruses (REO), picornaviruses, fowl adenoviruses (FAdV), adeno-like viruses, or mixed infections in the development of typical pathological lesions [3-6, 8, 10-15]. Recently a novel virus identi ed as a member of the Birnaviridae family has been isolated from clinical cases of TVP. Preliminary studies con rmed that this virus differed signi cantly from the Avibirnavirus genus IBDV. This virus was named chicken proventricular necrosis virus (CPNV), and an RT-PCR method has been developed which enables its detection [13]. Unfortunately, cases of TVP which were negative for CPNV as well as cases positive for CPNV presence without typical TVP changes has also been reported [12], which makes the etiology of TVP unresolved and further research are still necessary.
Clinically, TVP is manifested mainly in broiler chickens by decreased body weight gains, wide weight diversity of birds in the ock and an increased feed conversion ratio [1]. The disease, which usually affects up to 50% of the birds in the ock, can signi cantly reduce the cost-effectiveness of production.
Considering its problematic etiology, TVP diagnosis is di cult. Histopathological examination seems to be the most reliable method for con rming the disease. The histopathological lesions observed in the case of TVP exclusively affect the proventriculus and are manifested in a triad of lesions related to the necrosis of glandular epithelial cells (even up to 80% of cells in the proventricular mucosa), a strong lymphatic in ltration in the lamina propria of the mucosa and among the proventriculus glands, hypertrophy of the epithelial cells of the excretory ducts of proventricular glands with successive replacement of the epithelial glandular cells with hypertrophied cells of the excretory ducts. The severity of these lesions can vary depending on the duration of the disease [1,16].
Given the recent cases of TVP recorded in Poland [9], a laboratory-conditions study was undertaken that attempted to reproduce the clinical course of TVP in broiler chickens by inoculating them with a homogenate of proventriculi from a con rmed TVP eld case. The research was also aimed at identifying the etiological factor in domestic cases of TVP.

Body weight
The mean body weight of the control and TVP-infected birds is summarized in Table 1. Over a 14-day span after the infection, the birds from the control group gained 1.58 kg on average, while the birds from the TVP group gained 1.1 kg. In other words, after the infection, the body weight gain in the TVP group was signi cantly less and lower by 30.38% than in the control group.

Gross Lesions
No lesions typical of TVP were recorded in any of the groups at the time of TVP infection on the 24th day of life. In the TVP group at 14 days after the infection, enlargement of the proventriculus was noted in 6 out of the 8 birds examined, thickening of the proventricular wall was also observed in 6 out of 8, and discoloration was registered in 5 ( Fig. 1). No TVP lesions were recorded in the control group at this time.
The relevant data are summarized in Table 2. No other anatomopathological lesions were observed in any of the birds from either the control or TVP groups. In this group gross lesions associated with TVP, which were characterized as enlargement of the proventriculus (enlargement), thickening of the proventriculi wall (thickening) and discoloration recorded in the proventriculi wall cross-section (discoloration) were recorded at 14 dpi. No changes were observed in the proventriculi of Control birds.
* signi cant difference in the number of proventriculi with gross lesion in TVP group in comparison to the Control group at the same dpi.
** signi cant difference in the number of proventriculi with gross lesion within the group (TVP or Control) at 14 dpi in comparison to 0 dpi.

Histopathological Lesions
Histopathology revealed no TVP lesions in the proventriculi of birds from either group on the day of infection. In the TVP group at 14 dpi, necrosis, hyperplasia, and in ltration ( Fig. 2) were recorded in 7 out of the 8 proventriculi examined. No lesions were recorded in the control group at this time. The results of the histopathological examination are provided in Table 3. In this group histopathological lesions associated with TVP, which were characterized as necrosis of glandular epithelium (necrosis), hypertrophy and hyperplasia of ductal epithelium, replacement of glandular epithelium by hyperplastic ductal epithelium (hyperplasia) and multifocal to severe in ltration of lymphoid cells (in ltration) were recorded at 14 dpi. No histopathological changes were observed in Control birds.
* signi cant difference in the number of proventriculi with histopathological lesion in TVP group in comparison to the Control group at the same dpi.
** signi cant difference in the number of proventriculi with histopathologica lesion within the group (TVP or Control) at 14 dpi in comparison to 0 dpi.

Serological Results
The results of the serological examination are summarized in Table 4. In the TVP group, a signi cant increase in the anti-FAdV and anti-IBDV antibodies level was recorded 14 dpi in comparison to their level on the day of infection. At the same time, levels of these antibodies were signi cantly higher in the TVP group than in the Control group 14 dpi. Table 4 Mean IB, REO, FAdV and IBDV antibody levels ± SD in Control and TVP group at different dpi.

Molecular Biology
The results of molecular studies are shown in Table 5. All samples collected at 0 and 14 dpi in the control group were negative in all assays. In the TVP group at 0 dpi, all samples were also negative. In this group at 14 dpi, the samples of proventriculi (100% of samples tested) were positive for CPNV, while those of spleens and livers were positive for FAdV.

Discussion
Clinically, TVP is manifested by lower body weight gains, wide weight diversity of birds in the ock and an increased feed conversion ratio [1]. Goodwin et al. [4] reported that suboptimal body weight gains could result from pepsinogen-and hydrochloric acid-producing cell destruction and that, in severe cases of TVP, 80% of these cells are destroyed due to necrosis caused by infection. In the course of TVP no increased mortality is observed in the ock, but the number of culled birds increases signi cantly. In our study no increased mortality was manifest but poorer body weight gain was recorded in the TVP group.
Additionally, in this group both the anatomical and histopathological changes typical of TVP were observed. Given the fact that such lesions were not recorded in the control group and that no other lesions apart from those related to TVP were observed in any of the groups examined, we may conclude that we have succeeded in the reproduction of TVP under experimental conditions. Our study showed that over the 14-day period post infection the body weight gains of birds were lower by over 30% than those of the control birds.
To date, the etiological agent causing TVP has not been clearly established. However, the infectious nature of the disease has been emphasized many times, which is re ected in our work. Studies on TVP etiopathology imply the coaction of infectious bursal disease viruses, infectious bronchitis viruses, reoviruses, picornaviruses, adenoviruses, adeno-like viruses, IBDV-like viruses, or mixed infections in the development of characteristic lesions [3-6, 8, 10-15]. Recently, CPNV was suggested as a possible TVP causative agent [6]. With what is currently known about CPNV, it seems that the rst description of this virus comes from 1996 when Goodwin et al. [4] observed hexagonal virus particles in the nuclei and cytoplasm of proventricular cells in a case of TVP [4]. However, a more detailed description along with the taxonomy of CPNV was provided later by Guy et al. [13]. Chicken proventricular necrosis virus is a twentywalled, non-enveloped virus with a diameter of approximately 75 nm. As genetic material, it has doublestranded RNA, organized into two segments (c. 3.8 and 3.4 kbp). Within the putative viral protein-1encoding gene (VP), CPNV has a motif characteristic of Birnaviridae encoding the RNA-dependent RNA polymerase. However, based on the phylogenetic studies of the VP1 gene, it has been established that this virus is different from other known members of this family of viruses. It is interesting that clinical TVP was reproduced with the use of isolated CPNV [7].
Recent research from the UK also suggests the involvement of CPNV in the development of TVP in chickens. In two studies, the authors reported that CPNV was detected in 22 and 47% of con rmed clinical cases of TVP [11,12]. It remains unknown, however, if all cases of this disease are of the same infectious origin. There are cases of TVP in which CPNV cannot be identi ed, as well as disease cases in which CPNV can be identi ed but which do not meet the diagnostic criteria of TVP [7,11,12]. In addition, it is suggested that the pathogenesis of TVP could be more complex and may result from other viral, bacterial or fungal co-infections [1].
Due to seroconversion against FAdV and IBDV being observed in the studied TVP group, we performed a further molecular analysis to con rm the presence of these viruses as well as the CPNV. At 14 dpi, we con rmed the presence of CPNV in 100% of the proventriculus samples collected from birds from the TVP group, which indicates the involvement of this virus in TVP development. We also con rmed the presence of FAdV in the TVP-infected birds. It is di cult to clearly state the nature of FAdV's involvement in TVP development in our study. In our opinion, two hypotheses are likely. The rst is that CPNV infection contributed to the activation of a latent FAdV infection in the experimental birds and the second is that these viruses were present in the infectious material. However, the fact that FAdV was not detected in the proventriculi of TVP-infected birds but only in spleen and liver samples supports the hypothesis of latent infection. The explanation of these relationships requires further research. Once again, these results highlight the complex nature of TVP etiology.

Conclusions
In this experiment we were able to reproduce the clinical TVP in broiler chickens under laboratory conditions with the use of homogenates of proventriculus from con rmed, rst described Polish case of TVP. We have demonstrated that CPNV was involved in the development of the disease. To our surprise, we did not record the presence of IBDV in the TVP or control birds, despite our recording a strong seroconversion against IBDV in the birds from the TVP group. The putative etiological agent CPNV belongs to the same Birnaviridae family as IBDV, which allows us to assume serological cross-reactivity between them. This should be taken into account during serological evaluation under eld conditions, as the possibility also should of CPNV infections affecting IBDV antibody levels detected by commonly available ELISAs. On the other hand, the possibility of using IBDV ELISA kits for TVP diagnosis might be considered. Undoubtedly, these issues, as well as the contribution of FAdV in the development of TVP require further research.

Birds
Commercial Ross 308 broiler chicks of both sexes, purchased from one hatchery and one hatch, were used in the experiment. The trial was conducted in isolated pens of the Pavilion of Experimental Poultry Infections, at the Department of Avian Diseases, University of Warmia and Mazury in Olsztyn, which are maintained at a biosafety PCL 3 conditions. Water and feed were given to birds ad libitum. Rearing conditions were consistent with Aviagen (Aviagen, USA) recommendations.

Experiment Layout
The experiment was carried out with 24 commercial broiler chickens. The birds were reared until day 24 of life, after which they were divided into two groups (n = 12). One served as the control, and the other was infected with a proventriculus homogenate originating from a con rmed eld TVP case. Before the infection (0 days post infection -dpi), 4 birds selected randomly from each group were euthanized and subjected to autopsy examination to check for macroscopic lesions typical of TVP. Afterwards, proventriculi were collected for histopathological examination, and samples of them and of other internal organs (intestines, spleen, liver, and bursa of Fabricius) were taken for molecular analysis. For histopathological examination, the proventriculus samples were xed in a 10% formaldehyde solution. Samples for molecular analysis were frozen at − 20 °C until they were analyzed. The remaining birds from both groups were weighed and blood samples were collected for serological analysis. Blood was centrifuged at 1 500 × g for 15 minutes and the serum obtained was frozen at − 20 °C until it was analyzed. After infection, the birds were reared for the next 14 days. At 14 dpi, blood was sampled from all birds from both groups for serological analysis. Next, the birds were weighed and euthanized. Pathological lesions in the proventriculus were investigated and recorded during the anatomopathological examination, after which samples of this organ were collected for histopathological and molecular examinations. Production results were presented as the mean body weight (kg) +/− standard deviation (SD).
For euthanasia, chickens were placed in a chamber with Carbogen (95% O2 + 5% CO2). After 1 min, Carbogen was slowly replaced by 100% CO 2 . This method of euthanasia is responsible for stress reduction in the birds.

Proventriculi Homogenate And Infection
Proventriculi from the rst Polish case of TVP [9] were used for the experimental infection. After homogenization, proventriculus samples were processed through three freeze-thaw cycles. After centrifugation (2 000 × g, 15 minutes), the supernatant was stored and used for the infection. Broiler chickens from the TVP group were infected with 5 mL of the supernatant per bird. The supernatant was given to birds per os, directly with a probe to the crop. At the same time, the birds from the control group were given PBS in the same way.

Serological Analysis
A commercial kit of ELISA Ab Tests (IDEXX Laboratories, USA) was used to determine the titer of anti-IBV, anti-REO, anti-FAdV, and anti-IBDV speci c IgY in broiler serum. Particular stages of the tests were performed with an Eppendorf epMotion 5075 LH automatic pipetting station (Eppendorf, Germany), a BioTek ELx405 automatic multi-well plate washer (BioTek, USA), and a BioTek ELx800 plate reader. Sample to positive (S/P) ratio, plus/minus standard deviation (SD) were computed for each group in each sampling period.

Gross Lesion Evaluation
The birds were investigated for the presence of lesions typical of TVP in the proventriculus during an anatomopathological examination. Enlargement of the proventriculus coupled with thickening of its walls and their spotty discoloration in the cross-section were recorded. Results were collated as the number of birds with recorded pathological lesions relative to the total number of birds examined.

Histopathology
During necropsy, samples of the central part of the proventricular wall were embedded in 10% formalin (pH 7.4) and processed for histopathological examination. After passing the samples through intermediate liquids (increasing concentrations of alcohol and xylene) they were embedded in para n blocks. Sections of the examined samples 4 µm thick were stained with hematoxylin-eosin and microscope samples were scanned with a Pannoramic MIDI scanner (3DHISTECH, Budapest, Hungary). Samples were considered TVP positive if the following histopathological lesions were registered during examination: necrosis of glandular epithelium (necrosis), hypertrophy and hyperplasia of ductal epithelium, replacement of glandular epithelium by hyperplastic ductal epithelium (hyperplasia) and multifocal-to-severe in ltration of lymphoid cells (in ltration). For PCR analysis, ve 4 um sections from each para n block (each bird) were put into xylene and prepared for PCR analysis.

CPNV Identi cation
Para n xed sections of the proventriculi were used for CPNV identi cation. One mL of xylene was added to each sample comprising ve para n-xed proventriculus sections (each 5 µm thick) and these were incubated for 5 minutes at 50 °C in order to remove para n residues. Further RNA isolation steps were performed with an Isolate II

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare no competing interests.
Funding Publication costs nancially supported by Minister of Science and Higher Education in the range of the program entitled "Regional lnitiative of Excellence" for the years 2019-2022, Project No.  Gross lesions. Proventriculi of TVP (left) and Control (right) broiler chicken examined at 14 dpi. Typical pathological lesions observed Proventriculi enlargement, thickening of its walls and spotty discoloration in the cross-section were recorded in 6 out of 8 TVP infected birds, which are the three characteristic gross lesions related to transmissible viral proventriculitis.