Newcastle Disease Virus Induced Pathologies Severely Affect the Exocrine and Endocrine Functions of Pancreas in Chickens

Background: Newcastle disease virus (NDV) causes a highly contagious and devastating disease in poultry, Newcastle disease (ND), which is particularly characterized by extensive pathologies in the digestive, respiratory and nervous systems. ND cause heavy economic losses to the world poultry industry by decreasing growth rate, decrease egg productions, mortality and morbidity. Although, signicant advances have been made in the vaccine development, but outbreaks are reported in vaccinated birds leading decrease growth rate. Methods: In this study, we report the damage caused by the NDV infection in the pancreas of vaccinated as well specic pathogen free chickens. Results: The histopathological examination of the pancreas showed sever damage in the form of partial depletion of zymogen granules, acinar cell vacuolization, necrosis, and apoptosis, congestion in the large and small vessels, sloughing of epithelial cells of pancreatic duct, and mild perivascular edema. Increased plasma levels of corticosterone, somatostatin, were observed in NDV infected chicks at 3 and 5-day post infection (DPI). Slight decrease in the plasma concentrations of the insulin were noticed at 5 DPI. Signicant changes were not observed in the plasma levels of glucagon. Furthermore, NDV infection has decreases the activity and mRNA expression of amylase, lipase, and trypsin from the pancreas. Conclusion: Taken together, our ndings highlight that NDV induces extensive tissue damage in pancreas, decrease the activity and expression of pancreatic enzymes and increase plasma corticosterone and somatostatin. haemagglutinin-neuraminidase (HN), (M), (P), (L). these involved in the AOAV-1 induce pathologies in poultry. and 5 days post infection (DPI) in EDTA-coated tubes, and immediately transferred to the laboratory, maintaining the cold chain. To obtain the plasma, blood samples were centrifuged (2000 × g) for 10 min at 4 º C and stored at − 80 º C until further analysis. A total of four chickens per experimental treatment were sacriced for the collection of pancreatic tissue at 1, 3, and 5 DPI. All the tissue samples were immediately put in liquid nitrogen and stored at -80 º C until further analysis. assay the manufacturer’s instructions Diagnostics GmbH, Mannheim, Germany). Tissue sections were deparanized and rehydrated by the graded levels of xylene and ethanol. Tissue sections were treated with proteinase K and incubated at 37 º C for 25 min. Activity of the endogenous peroxidase activity was blocked with hydrogen peroxide. The sections were incubated at 37 º C with the terminal TdT nucleotide mixture for 2 hours. Nuclei were counterstained with 3 µg/ml 4′, 6′-diamidine-2-phenylindole (DAPI) and visualized by using uorescence microscopy. Then slides were digitalized by using Pannoramic SCAN (3DHISTECH Ltd., Hungary), and studied by using CaseViewer 2.2 (3DHISTECH Ltd., Hungary). Five different high power elds (HPF) were selected to count the number of apoptotic cells using Image-Pro Plus software (version 6.0 for Windows).


Background
Newcastle disease (ND) is highly infectious and one of the most distressing viral diseases of poultry, leading to the heavy economic losses to the world poultry industry [1]. ND is caused by the virulent strains of Avian orthoavulavirus 1 (AOAV-1), also known as ND virus (NDV) [2]. AOAV-1 belongs to the to the order Mononegavirales, family Paramyxoviridae, and genus orthoavulavirus, and contains non-segmented, negative-sense, and single stranded RNA genome of 15 kb length [3]. The proteins encoded by the AOAV-1 genome are fusion (F), haemagglutinin-neuraminidase (HN), matrix (M), phosphoprotein (P), and large polymerase (L). From these proteins, F and HN are mainly involved in the AOAV-1 induce pathologies in poultry.
It is well known that ND decreases the growth rate and egg production for long period in infected chickens [16]. However, AOAV-1 induced pathological changes on the digestion and absorption of nutrients remain elusive. For the digestion of lipids, proteins, carbohydrates, and nucleic acids; different enzymes are produced by the digestive system including salivary glands, intestine, liver, and pancreas [17]. Pancreatic enzymes such as trypsinogen, chymotrypsinogen, elastase and procarboxypeptidases for the digestion of proteins; pancreatic amylase for the digestion of carbohydrates; phospholipase, lipase, and cholesterol esterase for the digestion of lipids; and two type of nucleolytic enzymes namely ribonuclease and deoxyribonuclease [17][18][19][20]. The endocrine pancreatic tissue have clusters of cells known as islets of Langerhans, which produce different hormones including insulin, glucagon, somatostatin, and avian pancreatic polypeptide (APP) [21]. These hormones regulate the glucose homeostasis, glucose and amino acid transport, lipogenesis, glycogenolysis, hypoglycerolemia, regulate the level of plasma free fatty acid, regulate the expression of lipogenic enzymes, intestinal motility, and gall bladder secretions [21].
Previous studies have shown the involvement of pancreas in NDV infection and histopathological lesions were reported in chickens [12,14,22,23], ducks [2,24], geese [25], and turkeys [26]. However, the endocrine and exocrine functions of the pancreas are not clear in NDV infected chickens. Therefore, present study was planned to investigate the pathophysiology of pancreatic functions in NDV infected speci c-pathogen-free (SPF) and vaccinated and then NDV challenged chickens.

Chickens and Experimental design
Speci c pathogen free embryonated eggs were purchased from the Merial Vital Laboratory Animal Technology Company (Beijing, China) and were incubated at the laboratory facility of SHVRI, CAAS. The hatched-out SPF chicks were reared, vaccinated, and challenged in positive pressure isolators in high containment facility. All the birds were provided with ad libitum access to feed and water throughout the experiment. At the age of 9 weeks, chickens were randomly divided into three groups having 20 chicken each.
Birds in the group 3 (VAC + CHA) were vaccinated at the age of 9 weeks with 100 µl of LaSota at the dose rate of 10 3 embryo infective dose (EID 50 ) via intranasal and eye drop routs, prepared in phosphate buffered saline (PBS). After 2 week of vaccination of VAC + CHA group, all the birds in group 2 (CHA), and VAC + CHA group were challenged with ZJ1 suspended in ice cold PBS have a titer of 10 6.5 EID 50 per bird via right eye and choanal slit in 100 µl volume. Dose of the challenged and vaccinated viruses were selected on the basis of previous experiment of Cornax et al. [29]. Birds in the group 1 (CON) were given the same dose of PBS, via the same routs, as a mock infection.

Sampling
Four birds per group were randomly selected for collection of blood and tissue samples. Blood samples were collected from the jugular vein at 1, 3, and 5 days post infection (DPI) in EDTA-coated tubes, and immediately transferred to the laboratory, maintaining the cold chain. To obtain the plasma, blood samples were centrifuged (2000 × g) for 10 min at 4 º C and stored at − 80 º C until further analysis. A total of four chickens per experimental treatment were sacri ced for the collection of pancreatic tissue at 1, 3, and 5 DPI. All the tissue samples were immediately put in liquid nitrogen and stored at -80 º C until further analysis.

Plasma hormones
Plasma levels of the somatostatin, insulin and glucagon were determined by using chicken speci c commercial assay kits [Nanjing Jiancheng Bioengineering Institute (HO92-SS; H183 GC; H203 Insulin)]. All samples were analyzed within single assay to avoid interassay variations. The absorbance of the microplates was determined using the Epoch microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) at the optical density value of 450 nm. The plasma levels of corticosterone (CORT) were measured using a chicken speci c commercial ELISA kit (CSB-E11991C; Cusabio Biotech. Co., Ltd., Wuhan, China). The standard curve was made by using the CurveExpert 1.4. The limits of detection for the insulin and glucagon were 0.5mIU/ml and 5 ng/L, respectively. The inter and intra assay coe cient of variation was less than 10% and 12% respectively.

Digestive enzyme activities in pancreas
To determine the activity of the pancreatic enzymes, a small portion (about 1 mg) of the pancreatic tissue was homogenized in 9 volumes (W/V) of ice-cold phosphate buffer saline (PBS), using a Tissuelyser-24 (Jingxin Technology, Shanghai, China) at 20 Hz for 2 minutes, and instantly centrifuged at 3000 × g at 4 °C for 10 minutes. The supernatant was collected for the determination of digestive enzyme activities. To determine the activity of trypsin (A080), lipase (A054) and amylase (C016) by calorimetric methods, commercial kits were used (Nanjing Jiancheng Bioengineering Institute, China). Pre-experimental standardizations were performed for each of the targeted enzyme to determine the optimum dilution for the measurement of enzymes. Protein concentration of the supernatants were determined by using the kits from Beyotime, (Beyotime, Nanjing, China; P0010).
Total RNA extraction and quantitative real-time PCR The quantitative real time PCR (qRT-PCR) was performed to measure the mRNA expressions of pancreatic digestive enzymes including amylase, lipase, and trypsin genes. The chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. The primers used in the present study were synthesized by Sangon Biotech Co., Ltd (Shanghai, China) and are described in the Table 1. Total RNA of the pancreas samples were extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA pellet was dissolved in RNase-free water. Total RNA was quanti ed using a NanoDrop spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA) and purity was assessed by measuring the absorbance ratio at 260 to 280 nm. Reverse transcription was performed using HiScript II (Catalogue # R233; Vazyme Biotech Co., Ltd., China), according to the manufacturer's instructions. Expression of target genes were measured by performing the qRT-PCR on CFX96 Touch Real-Time PCR Detection System (BioRad, USA), using SYBR Premix (Dongsheng, Biotech, China), and data were normalized with GAPDH. The qRT-PCR was performed with a nal volume of 20 µl. The PCR cycles are as follows: 94 º C for 3 minutes, followed by 40 cycles of 95 º C for 15 s, 60 º C for 15 s and 72 º C for 20 s. All the ampli cations generated expected amplicons with single, sharp fusion curves. All experiments were carried out in triplicate. The changes of mRNAs were presented as fold expression and calculated using 2 −ΔΔCT method [30].

Viral loads in the pancreas
The virus copy numbers were detected from the pancreas as described previously [9]. Brie y, the ZJ1 strain was grown in 10-day-old SPF embryonated eggs and allantoic uid was collected after 60 h of infection. The TRIzol (Invitrogen, Carlsbad, CA, USA) reagent was used to extract the viral RNA from the allantoic uid as per the manufacturer's instructions. The ZJ1 M gene was ampli ed using the primers mentioned in the Table 1, and con rmand the size (1095 bp) of the fragment by electrophoresis. The HiPure Gel Pure DNA Mini Kit (AnGen Biotech, Guangzhou, China) was used to purify the PCR product and cloned into a plasmid vector to construct a standard curve. The pancreatic tissue from the chickens was homogenized and RNA was extracted as mentioned above. About 1 µg RNA extracted from the pancreas samples was reverse transcribed to cDNA with HiScript II (Catalogue # R233; Vazyme Biotech Co., Ltd., China), and quantitative PCR was performed with SYBR Premix (Dongsheng, Biotech, China). Virus copy numbers were calculated using the standard curve [9].

Histopathology
Samples of the Pancreatic tissue were collected from the middle region and xed in 10% neutral buffered formalin, embedded in para n. and 5 µm thick sections were obtained. The sections were stained with hematoxylin and eosin (H&E) following the standard procedures [31]. The slides were digitalized by using Pannoramic SCAN (3DHISTECH Ltd., Hungary) and histological lesions were studied by using CaseViewer 2.2 (3DHISTECH Ltd., Hungary) [32].

TUNEL assay
For the detection of apoptotic cells, 5 mm thick tissue sections of the pancreas were subjected to the transferase dUTP nick-end labeling (TUNEL) assay according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany). Tissue sections were depara nized and rehydrated by the graded levels of xylene and ethanol. Tissue sections were treated with proteinase K and incubated at 37 º C for 25 min. Activity of the endogenous peroxidase activity was blocked with hydrogen peroxide. The sections were incubated at 37 º C with the terminal TdT nucleotide mixture for 2 hours. Nuclei were counterstained with 3 µg/ml 4′, 6′-diamidine-2-phenylindole (DAPI) and visualized by using uorescence microscopy. Then slides were digitalized by using Pannoramic SCAN (3DHISTECH Ltd., Hungary), and studied by using CaseViewer 2.2 (3DHISTECH Ltd., Hungary). Five different high power elds (HPF) were selected to count the number of apoptotic cells using Image-Pro Plus software (version 6.0 for Windows).

Statistical analysis
The data were analyzed by two-way ANOVA test, with challenge and time points as the main effects [33], and Graph Pad Prism 6.0 software (GraphPad Software, Inc., CA, USA) was used to generate the graphs. The graphical results were expressed as mean ± standard deviation (M ± SD). Results with P < 0.05 were considered as statistically signi cant. When a signi cant main effects was observed, TUKEY test was used to compare the differences among groups.

Viral load in the pancreas
The transcriptional level of the NDV M gene was determined to examine the viral load in the pancreas using the qRT-PCR assay at 3 and 5 DPI. The viral copy numbers per µl are shown in the Fig. 1. There was non-signi cant difference in the viral load of VAC + NDV and CHA groups at 3 DPI and viral copy numbers were signi cantly less at 3DPI compared to 5 DPI in infected groups. Maximum viral load observed in the CHA group at 5 DPI and was signi cantly higher compared to VAC + NDV group. The negative control group remained negative for NDV.

Histopathology
There were not signi cant changes observed in the control group. At certain locations, presence of intranuclear eosinophilic material could be nuclear edema or nuclear invagination or incidental nding. In CHA group, at 1 DPI, pancreas was intact, multifocal lymphoid nodules in the proximity of branches of pancreatic ducts, and occasionally, hyperplasia in the endocrine pancreas was seen. Rarely, cells in the acinar tissue had eosinophilic intranuclear material. This was only observed in the endocrine tissues. There was mild perivascular edema. Rare multifocal apoptotic cells (rounded, hypereosinophilic with pyknotic nuclei) were noticed. At 3 DPI, in CHA group, eosinophilic intranuclear material was noticed in endocrine pancreas. Multifocal to diffusely, there was partial depletion of zymogen granules form the exocrine pancreas. Acinar cells had occasional single to multiple vacuoles in the apical portion of the cell. Multifocally, there was mild perivascular edema and occasionally individualization of acinar cells. Few acinar cells undergo single cell necrosis/apoptosis and other cells had mild cytoplasmic vacuolation. No signi cant changes were observed in the endocrine pancreas. Occasionally, there was lymphocytic in ltration in the exocrine tissue. Rarely, there were eosinophilic intranuclear inclusion bodies in the endocrine tissue (Fig. 2, E). At 5 DPI, in CHA group, exocrine cells had a complete loss of zymogen granules, rounded off, and separated from neighboring cells. The exocrine and endocrine tissues had lost the original structure, and cells were individualized. There was sever congestion in the large and small vessels. The epithelial cells of pancreatic duct were sloughed and lost nuclear detail as well. There was mild perivascular edema. Occasionally single cell necrosis (Fig. 2, H). There was cytoplasmic vacuolation of acinar cells in the exocrine tissue and partial depletion of zymogen granules.
In VAC + CHA group, at 1 DPI pancreatic tissue shad, multifocal, partial to complete depletion of zymogen granules, cytoplasmic vacuolation (degeneration) and sometimes necrosis of individual acinar cells of pancreas. At 3 DPI, the acinar cells undergo a spectrum of changes that range from degeneration, with loss of zymogen granules, cytoplasmic vacuolation and swelling of the cells, to single cell necrosis, with cells becoming individualized, and shrunken with pyknotic nuclei. There were multifocal, randomly distributed lymphoid nodules. Whereas at 5 DPI, partial or complete depletion of zymogen granules were observed. Frequently, cytoplasmic vacuolation with eosinophilic hyaline inclusion in acinar cells of pancreas were seen throughout the exocrine part. There were multifocal lymphoid nodules throughout exocrine pancreas. There was marked increase in the cytoplasmic vacuolation and single cell necrosis of acinar cells and due to partial depletion of zymogen granules, acinar cells looked ductless. There is mild to moderate interstitial edema in exocrine portion of pancreas and separation of acinar cells from neighboring cells. One focal area of lymphocytic in ltration in the peripancreatic duct branch, perivascular edema was observed. Persistently, scattered lymphocytes were observed throughout the exocrine pancreas. Most of the observed lesion were perivascular in this slide (Fig. 2, I). Individualized round acinar cells with dark pyknotic nuclei showed sever single cell necrosis throughout the exocrine tissue. There was distortion of the acinar cells and separation from the neighboring cells. Diffusely, the areas of cytoplasmic vacuolation of acinar cells are seen.

Apoptosis in the pancreas
To estimate NDV induced apoptosis in the chicken pancreatic tissue, TUNEL positive cells were counted (Fig. 3). The apoptotic cells had green stained nuclei. As presented in the Fig. 3A, the number of apoptotic positive cells in the pancreas was similar between the CON and NDV infected groups at 1 DPI. In the NDV challenged groups, the number of apoptotic cells were signi cantly increased compared to CON at 3DPI. The increase in the number of apoptotic cells was less evident in the VAC + NDV group compared to CHA, but, still it was signi cantly higher than the CON (Fig. 3, B) at 5 DPI.

Plasma concentrations of hormones
The plasma concentrations of CORT were signi cantly elevated (P < 0.05) in the CHA group compared to CON at 3 DPI. Signi cant difference was not found in the concentrations of CORT in CON and VAC + CHA (Fig. 4, A). However, at 5 DPI, NDV challenge had signi cantly increased the plasma CORT level in CHA and VAC + CHA groups compared to the CON group.
Plasma concentrations of the glucagon and insulin are represented in the Fig. 4 (B, C respectively). Statistically, non-signi cant effect of NDV challenge on glucagon was observed at 3 and 5 DPI, but numerically, slight decrease in the plasma levels of NDV infected birds were noticed compare to CON group. The plasma levels of insulin remained unaffected at 3 DPI, but signi cant decrease was observed in CHA group compared to control at 5 DPI (Fig. 4, C).
Plasma concentration of the somatostatin is presented in the (Fig. 4, D). The plasma concentrations of the somatostatin were signi cantly increased (P < 0.05) in CHA (from 261114c) group but, this increase was not signi cant in CON and VAC + CHA groups, at 3 DPI. Birds in the CHA group tends to have higher concentration (P < 0.05) of somatostatin at 5 DPI compared to CON and VAC + CHA groups.

Activity and expression of pancreatic enzymes
The results for amylase activity and expression are presented in Fig. 5 (A, B). At 3 DPI, chickens in the CHA group showed decreased activity of amylase (p < 0.05), compared to the CON group. However, amylase activity in the pancreas of the VAC + CHA group of chickens was similar to that observed in the CON group, at 3DPI. More drastic decrease in the activity and expression of amylase in the pancreas of CHA groups of chickens were observed at 5 DPI compared to CON and VAC + CHA group. Furthermore, activity of amylase in the VAC + CHA was signi cantly less than control and, more than CHA group. The expression of amylase at 5 DPI from the CHA and VAC + CHA groups were signi cantly (p < 0.05) less than the CON group.
There was non-signi cant effect of NDV challenge in SPF and vaccinated chickens on the activity of lipase at 3 DPI (Fig. 5, C). NDV infection in SPF chickens resulted in decrease activity of lipase compared to chickens in the CON and VAC + CHA groups. Lipase mRNA expression in the pancreas had signi cantly decreased in NDV infected birds (p < 0.05), compared to CON at 3, and 5 DPI (Fig. 5, D).
The NDV challenge signi cantly affected the activity and expression of pancreatic trypsin (Fig. 5, E, F). Activity of trypsin was signi cantly decreased in NDV challenge groups compared to CON, and this decrease was more obvious at 5 DPI. The mRNA expression of trypsin was signi cantly higher in the pancreas of CON birds (p < 0.05), compared to CHA and VAC + CHA groups.

Discussion
The present study comprehensively reported the effect of NDV infection on the pancreas in vaccinated as well as SPF chickens. We persuasively demonstrated the replication of NDV in the pancreas of chicken in vaccinated and SPF chickens. In the study, chickens were vaccinated with 10 3 EID50 of the Lasota and challenged with 10 6.5 EID 50 of the ZJ1 after 2 week of vaccination because, at this dose of vaccine and challenge, pathogenic virus can replicate, can cause conjunctivitis and mild depression {Cornax, 2012 #1671}. Histological studies of the chickens in the current experiment advocate the severe damage caused by the NDV in the pancreas. The NDV infection had caused the partial depletion to complete loss of zymogen granules form the exocrine pancreas, cytoplasmic vacuolation and necrosis of acinar cells cell, and eosinophilic intranuclear material was noticed in endocrine pancreas, but these were less evident at 3 DPI compared to 5 DPI. Histopathological results of the present study are in agreement with the previous studies [12,14,[22][23][24].
The NDV infection in vaccinated as well as SPF chicken resulted in signi cant decrease in the activities and expression of pancreatic enzymes. These decreased activities and expression of amylase, lipase and trypsin may be related to extensive damage caused by the NDV to exocrine portion of pancreas in the infected birds. These decreased activities of the pancreatic enzymes may be one of the insults to decrease the digestion and weight gain in NDV infected vaccinated birds. Decrease activities of the digestive enzyme may be related to poor production performance after NDV outbreaks in commercial poultry. Our hypothesis was that chickens having less vaccine dose can suffer with pancreatitis which may leads to decrease production of digestive enzyme.
It is well studied, that hypothalamic-pituitary-adrenal axis is involved in acute stress and regulate the in ammatory response to an infectious challenge. Stress is strongly correlated with the exacerbation of many viral infections [34,35]. In this study increased plasma levels of corticosterone were found in NDV infected chicken. Increased levels of corticosterone have been related to decrease growth rate, decreased antibody response, decreased number of lymphocytes, and reduced size of lymphoid organs [36].
Corticosterone decrease the insulin sensitivity in chicken [37]. In this study slight decrease in the plasma level of insulin and glucagon was noted at 5 DPI in NDV infected chicken. It may be due to less structural and morphological changes in the endocrine pancreas after NDV infection, as noted in the histopathology results. Avian pancreas has higher levels of glutathione, which exhibits protective effects on the beta cells [38]. This increased level of glutathione may be related to less pathogenicity in the endocrine pancreas because, decreased levels of glutathione was observed in the plasma and intestines of NDV infected chickens [6,39].
The somatostatin, also known as growth hormone-inhibiting hormone, is a 14 amino acid peptide that regulates the endocrine system. Somatostatin is produced by the delta cells (δ-cells or D cells) [40]. Previous studies had suggested the production of SS from the endocrine cells in the proventriculus, small intestine [41], pancreas [42] and expression in the testis [43]. There are two forms of chicken somatostatin, somatostatin-14 and somatostatin-28, which are encoded by the same gene (PSS1) but another variant exist which is expressed in the brain and pancreatic islets [21,44]. The somatostatin inhibits the production of many hormones including insulin, glucagon, growth hormone, gastrin, vasoactive intestinal peptide, and thyroid-stimulating hormone [45]. In the present study, increase plasm levels of somatostatin were found in NDV challenged birds. Increased somatostatin mRNA expression has been described after stimulation with cytokines in murine macrophage [46]. Similarly, increased plasma concentration of somatostatin in endotoxin injected sheep [47], septic pigs [48], simian immunode ciency virus infected macaque, then infected with Mycobacterium Avium [49], increased expression in rabies virus infected brain [50] and secreted by human adipocytes after stimulation with activated macrophages, LPS, and IL-1β [51]. The increased plasma concentrations of somatostatin may be involved to counter the in ammation caused by infection. The SS has been described as anti-in ammatory and immunosuppressive and its analogues have been used as potential treatments for in ammation [52,53].

Conclusions
In summary, present study suggest that the pancreas could be a potential target of NDV infection. Evidently, exocrine pancreas is more severely affected by NDV compared to endocrine pancreas. The NDV infection leads to decrease in production and activity of digestive enzyme from the pancreas, even in the vaccinated chickens. Similarly, NDV infection also induce the increased levels of corticosterone and somatostatin, and decreased the production of insulin as evident from the plasma levels. These pathologies of the pancreas may be the leading cause of decreased production performance of NDV infected vaccinated birds.  The number of apoptotic positive cells in the pancreas was similar between the CON and NDV infected groups at 1 DPI.

Abbreviations
Page 15/16 Figure 4 Plasma concentration of the somatostatin