Slov Vet Res 2015; 52 (1): 15-21 UDC 636.5.09:578.834:57.083
Original Scientific Article
ultrastructural study of the trachea in experimentally infected broilers with ibv serotype 4/91
Saeed Seifi1 *, Zahra Boroomand2
1Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, 2Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran
Corresponding author, E-mail: saeedseifi57@gmail.com
Summary: Avian infectious bronchitis virus (IBV) is a globally distributed Coronavirus that causes tremendous economic loss in the poultry industry. The primary target cells of the virus are located in the respiratory tract. Strain-dependent, IBV may spread to other epithelia and cause nephritis or drop in egg production. Subcellular changes in broilers trachea induced by infectious bronchitis virusserotype 4/91 were examined bytransmission electronmicroscopy(TEM). Seventy 1-day-old commercial broiler chicks were divided randomly into two groups (control and experimental), and at the age of 21 days, all birds in the experimental group were challenged intranasally with the virus. Four birds from challenge group and two birds from the control group were euthanized at 1, 2, and 4 days post inoculation (PI) and tracheae were isolated after necropsy and examined with TEM. Mild tracheal rales, coughing and gasping were seen in the experimental group. As gross lesions, hyperemia and edema in tracheal mucosa were observed. IBV infection resulted in hypertrophy of goblet cells, their rupture, and the formation of excess mucus. At the level of the ciliated cells, complete deciliation of the tracheal surface was observed. Swelling and increase in the amount of endoplasmic reticulum was seen in infected birds.
Key words: transmission electron microscope; infectious bronchitis virus; serotype 4/91; trachea; broilers
Introduction
Avian infectious bronchitis virus (IBV), an enveloped, positive-sense single stranded RNA virus, is a member of the Coronaviridae family of the order Nidovirales that causes highly contagious disease of poultry, which poses a major threat to the poultry industry. The avian coronavirus IBV has a unique morphology. It is an envelope virus that is spherical to pleomorphic with evenly dispersed spike-like projections on the
Received: 14 February 2014
Accepted for publication: 24 December 2014
surface of the virion. Clinical cases of infectious bronchitis (IB) are associated with respiratory, reproductive, digestive and renal infections in domestic poultry and in various other avian species (1). Although effective vaccines are available and utilized routinely in commercial poultry production, the virus tends to mutate frequently (2). Little or no cross-protection occurs between different serotypes of infectious bronchitis virus (3), therefore, continuous determination of the epidemic serotype and production of new generations of vaccines are crucial for controlling IB in each geographic region or country. The IBV
16
S. Seifi, Z. Boroomand
serotype 4/91 (also named 793/B and CR88), was first reported in Britain in the early 1990s (4, 5), and is one of the most common IBV serotypes worldwide (3). In Iran, the presence of Massachusetts serotype as the major circulating IBV was confirmed in 1994 (6). By isolation and serologic identification of some field strains, VASFI MARANDI and BOZORGMEHRI FARD (7), suggested the presence of IBV variant(s) in Iran. Serotype 4/91 was isolated in Iran by MOMAYEZ et al. (8) and a recent study revealed that it has been the dominant IBV serotype throughout 1994-2004 in Iran (9). In the present study, ultrastructural changes in the trachea due to infection with IBV serotype 4/91 were investigated.
Materials and methods
Virus preparation
IBV serotype 4/91 isolated from broiler flocks in Iran (10) was used in this study. Virus propagation was performed in 10-day-old embryonated chicken eggs. Eggs were obtained from a respiratory disease-free flock. The embryo lethal dose (ELD50) of infected allantoic fluid was calculated according to the REED and MUENCH'S (11) formula. In the present study, allantoic fluid containing 1065 ELD50/0.1ml of the virus was used to induce the disease.
Experimental Design
This study was performed after receiving approval from the Animal Ethics Committee of the Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran. Also, it was conducted with respect to the International Guidelines for research involving animals (Directive 2010/63/EU). Seventy, 1-day-old commercial broiler chicks (Ross breed) were divided randomly into two groups (fifty chicks in the experimental and twenty chicks in the control group). Experimental and control groups were housed in separate experimental rooms. Food and water were supplied ad libitum. No vaccine was used in either of the two groups. Prior to virus challenge, all birds were found free from IBV antibodies (Flock Check IBV ELISA test kit, IDEXX Laboratories Inc., Westbrook,
ME). Tracheal swabs were taken from chicks for detection of other respiratory pathogens such as avian influenza virus (H9N2), Newcastle disease virus and Mycoplasma spp. by molecular assay before and after IBV inoculation (12) and the results were negative. At the age of 21 days, all birds in the experimental group were challenged intranasally with 0.2 ml of allantoic fluid virus suspension (titre 1065 EID50 per 0.1ml). Control birds were sham inoculated with an equal volume of normal saline. Four birds from the challenge group and two birds from the control group were euthanized with chloroform at 1, 2, and 4 days post inoculation (PI) and tracheae were isolated after necropsy.
Electron microscopy
Tracheal specimens were fixed in 2.5% gluteraldehyde-2% formaldehyde, washed in sodium cacodylate buffer solution (pH 7.4), post-fixed in 1% OsO4, dehydrated in ascending concentration of ethanol, and embedded in medium Spurr's resin. Tracheal sections (1 ^m) stained with toluidine blue were examined for trimming of the blocks to areas of interest. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined in a Philips CM10 transmission electron microscope (13).
Results
Clinical signs and gross lesions
No clinical signs and gross lesions were observed in control group. In experimental group mild tracheal rales, coughing and gasping were seen at 24 hours PI. Severity of the signs was less after 5 days PI. Slight hyperemia and edema in tracheal mucosa were noticed in the euthanized birds. No mortality was observed in either group.
Electron Microscopy
In chicks from the control group the tracheal surface was lined by a pseudostratified columnar epithelium. The surfaces of control group tracheas were covered with abundant cilia and some goblet cells. The following changes were seen in chicks of experimental group at different days
Ultrastructural study of the trachea in experimentally infected broilers with ibv serotype 4/91
17
post inoculation. In day 1 PI, virus particles were localized in the tracheal epithelial cells (Figure 1). In day 2 PI, limited infiltration of heterophils and lymphocytes to the tracheal epithelium, inflammation and congestion, hypertrophy of goblet cells, mild edema and partial loss of cilia were seen. In day 4 PI, deciliation of the tracheal epithelium was increased and complete deciliation was seen at some sections. Other changes in this time were: infiltration of plasma cells, and
appearance of vacuoles containing lymphocytes and heterophiles (Figure 2), edema in sub mucosal layer, severe degeneration of the epithelial cells and deformation of the mucous gland. Viral particles were observed outside surface cytoplasmic membranes and occasionally within cytoplasmic vesicles (Figure 3). Swelling and increase in the amount of endoplasmic reticulum were seen at 4 PI (Figure 4).
Figure 1: Viral particles in epithelial cells of chicken trachea at day 1 after infection. x 28500
Figure 2: Trachea at 4 days post infection. Electron-dense particles in hypertrophied Goblet cells. Partial deciliation of the tracheal epithelium. x 1550
18
S. Seifi, Z. Boroomand
Figure 3: Viral particles are seen in the Golgi apparatus and in electron-dense areas at day 4 after infection. x 21000
Figure 4: Heterophile degranulation at day 4 after infection. x 2950 (ER: Endoplasmic reticulum, HD: Heterophile degranulation)
Ultrastructural study of the trachea in experimentally infected broilers with ibv serotype 4/91
19
Discussion
The progression of tracheal lesions induced by inoculation of commercial broiler chicks with infectious bronchitis virus serotype 4/91 was examined by tracking subcellular changes using transmission electron microscopy. Serotype 4/91 of IBV was first isolated in 1985 in France, and then spread to many countries in Europe, Japan, Saudi Arabia, Thailand and Mexico (5, 14). Despite the regular vaccinations on chicken farms, mostly with Massachusetts (Mass) strains, IB still exerts a severe negative impact on the poultry industry in Iran. Cross-protection between different serotypes of IBV is variable; hence vaccination failure may be due to low homology (26%) between the 4/91 and Massachusetts serotype vaccines such as the H120 strain (15, 16). Therefore, it is very important to understand the effects of this virus on broilers.
Replication of IBV occurs in the ciliated epithelium and mucous cells within 24 hr after intratracheal or aerosol inoculation, and viral particles are confined to small vacuoles of cytoplasm (17, 18). Severity of the tracheal lesions are varied due to different inoculation routes, strains and inoculum sizes of IBV and age of chicken infected (17, 19, 20). In our experiment, tracheal epithelial changes such as hyperemia and edema were similar to those described previously (21, 22).
According to the results, complete deciliation was seen at 4 PI, which is in agreement with results of UPPAL and CHU (23), NAKAMURA et al. (20), and ABD EL RAHMAN et al. (24). Loss of cilia was observed at day 2 PI and was complete at day 4 PI. The increase in the damage to ciliated cells destruction between 1 and 4 days PI reflects the replacement of the highly differentiated, pseudostratified epithelium containing ciliated cells by a simple squamous to cuboidal epithelium without cilia. Replacement by cuboidal cells is best interpreted as the regeneration phase during which the basal cells at the periphery of a lesion slide across the underlying fibroblastic connective tissue to close the lesion. An early strong activation of the goblet cells was observed in inoculated chicks which most likely reveal the initial IBV infection of these cells. The subsequent decrease in the goblet cells activation score can be attributed to the goblet cells exhaustion and elimination due to virus replication as severe ultrastructural decay and rupture of these cells was observed
after the initial activation. Cilia are responsible for propelling the entrapped particles (bacteria, virus, dust etc.) for disposal. Reduced ciliary motility or disrupted ciliated epithelium could be expected to adversely affect the resistance of birds to microorganisms that normally enter their bodies via the respiratory system (25). On the other hand, this study confirmed that IBV initially infects the upper respiratory tract, where it is restricted to the ciliated and mucus-secreting cells (26).
In Iran, the most prominent lesion in respiratory disease infected flocks is severe exudation in trachea, which leads to tubular cast formation in the tracheal bifurcation and extending to the lower bronchi (27). This is followed by destruction of the cilia by factors such as infectious bronchitis virus.
In normal conditions in which the goblet cells are not activated, the mucus layer that covers the tracheal epithelium is largely washed away during the reparative steps. After virus inoculation goblet cells were ruptured and deformed. This finding is in line with results of MAST et al. (28).
In conclusion, although no mortality was seen in infected group in experimental condition, IBV serotype 4/91 can increase the susceptibility to the respiratory disorders by tracheal hyperemia and deciliation in field status.
Acknowledgements
The authors would like to thank Mrs. S. Shobeikeh for her technical help.
References
1.	Cavanagh D. Coronaviruses in poultry and other birds. Avian Pathol 2005; 34: 439-48.
2.	Wang L, Junker D, Gollisson EW. Evidence of natural recombination within the S1 gene of infectious bronchitis virus. Virology 1993; 192: 710-6.
3.	Cavanagh D. Coronavirus avian infectious bronchitis virus. Vet Res 2007; 38: 281-97.
4.	Adzhar A, Gough RE, Haydon D, Britton P, Shaw K, Cavanagh D. Molecular analysis of the 793/B serotype of infectious bronchitis virus in Great Britain Avian Pathol 1997; 26: 625-40.
5.	Cavanagh D, Mawditt K, Britton P, Naylor CJ. Longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broiler us-
20
S. Seifi, Z. Boroomand
ing type-specific polymerase chain reactions. Avian Pathol 1999; 28: 593-105.
6.	Aghakhan SM, Afshar N, Fereidouni SRN, Marunesi C, Khodashenas M. Studies on avian viral infectious in Iran. Arch Razi Inst 1994; 44/45: 1-10.
7.	Vasfi Marandi M, Bozorgmehri Fard MH. Isolation and identification of infectious bronchitis viruses in chicken I Iran. In: Proceedings of the 21st Worlds Poultry Congress. Motreal, Canada, 2000.
8.	Momayez R, Pourbakhsh SA, Khodashenas M, Banani M. Isolation and identification of infectious bronchitis virus from commercial chickens. Arch Razi Inst 2002; 53: 1-9.
9.	Shoushtari AH, Toroghi R, Momayez R, Pourbakhsh SA.793/B type, the predominant circulating type of avian infectious bronchitis viruses 1999-2004 in Iran: a retrospective study. Arch Razi Inst 2008; 63: 1-5.
10.	Seifi S, Asasi K, Mohammadi A. Natural co-infection caused by avian influenza H9 subtype and infectious bronchitis viruses in broiler chicken farms. Vet Arh 2010; 80: 269-81.
11.	Reed LR, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938; 27: 493-7.
12.	Roussan DA, Haddad R, Khawaldeh G. Molecular survey of avian respiratory pathogens in commercial broiler chicken flocks with respiratory diseases in Jordan. Poult Sci 2008; 87: 444-8.
13.	Reynols ES. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 1963; 17: 208-12.
14.	Cook JKA, Orbell SJ, Woods MA, Huggins MB. A survey of the presence of a new infectious bronchitis virus designated 4/91(793/B). Vet Rec 1996; 138: 178-80.
15.	Cavanagh D. Severe acute respiratory syndrome vaccine development: experience of vaccination against avian infectious bronchitis corona-virus. Avian Pathol 2003; 32: 567-82.
16.	Cavanagh D, Ellis MM, Cook JKA. Relationship between sequence variation in the S1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo. Avian Pathol 1997; 26: 63-74.
17.	Ducatelle R, Meulemans G, Coussement W, Hoorens J. Aetio-pathology of the fowl trachea
early after inoculation with H52 infectious bronchitis virus. Zbl Vet Med B 1984; 31: 151-60.
18.	Purcell DA, Clarke JK. The replication of infectious bronchitis virus in fowl trachea. Arch Gesamte Virusforsch 1972; 39: 248-56.
19.	Fulton RM, Reed WM, Thacker HL. Cellular response of the respiratory tract of chickens to infection with Massachusetts 41 and Australian T infectious bronchitis viruses. Avian Dis 1993; 37: 951-60.
20.	Nakamura K, Cook JKA, Otsuki K, Hug-gins MB, Frazier JA. Comparative study of respiratory lesions in two chicken lines of different susceptibility infected with infectious bronchitis virus: histology, ultrastructure and immunohis-tochemistry. Avian Pathol 1991; 20: 241-57.
21.	Kotani T, Shiraishi Y, Tsukamoto Y, et al. Epithelial cell kinetics in the inflammatory process of chicken trachea infected with infectious bronchitis virus. J Vet Med Sci 2000; 62: 129-34.
22.	Mahdavi S, Tavasoly A, Pourbakhsh A, Momayez R, Shamseddini M. The immunohisto-chemistry study of lesions due to avian infectious bronchitis (serotype 4/91) on different tissues in specific pathogen free chicks. J Vet Res 2007; 62(4): 97-101.
23.	Uppal PK, Chu HP. An electron-microscope study of the trachea of the fowl infected with avian infectious bronchitis virus. J Med Microbiol 1970; 3: 643-7.
24.	Abdel Rahman S, El- Kenawy AA, Neumann U, Herrler G, Winter C. Comparative analysis of the sialic acid binding activity and the tropism for the respiratory epithelium of four different strains of avian infectious bronchitis virus. Avian Pathol 2009; 38: 41-5.
25.	Fedde MR. Relationship of structure and function of the avian respiratory system to disease susceptibility. Poult Sci 1998; 77: 1130-8.
26.	Dhinakar Raj G, Jones RC. Infectious bronchitis virus: immunopathogenesis of infection in the chicken. Avian Pathol 1997; 26: 677-706.
27.	Nili H, Asasi K. Avian influenza (H9N2) outbreak in Iran. Avian Dis 2003; 47: 828-31.
28.	Mast J, Nanbru C, van den Berg T, Meulemans G. Ultrastructural changes of the tracheal epithelium after vaccination of day-old chickens with the La Sota strain of Newcastle disease virus. Vet Pathol 2005; 42: 559-65.
Ultrastructural study of the trachea in experimentally infected broilers with ibv serotype 4/91
21
ULTRASTRUKTURNA RAZISKAVA SAPNIKA V POSKUSNO OKUŽENIH BROJLERJEIH Z IBV SEROTIPA 4/91
S. Seifi, Z. Boroomand
Povzetek: Aviarni virus kužnega bronhitisa (IBV) je globalno razširjen korona virus, ki povzroča ogromno gospodarsko škodo v perutninski proizvodnji. Primarne tarčne celice virusa so v dihalnem traktu. IBV se lahko razširi tudi na druga epitelijska tkiva in povzroči npr. vnetje ledvic ali padec proizvodnje jajc.
S transmisijsko elektronsko mikroskopijo (TEM) smo pregledali znotrajcelične spremembe sapnika brojlerjev po okužbi z virusom kužnega bronhitisa serotipa 4/91. Sedemdeset enodnevnih brojlerskih piščancev je bilo naključno razdeljenih v dve skupini (kontrolna in poskusna). Pri starosti 21 dni so bili vsi piščanci v poskusni skupini okuženi z virusom preko nosu. Štiri piščance iz poskusne skupine in po dva iz kontrolne smo usmrtili v 3 časovnih točkah po okužbi, natančneje po enem dnevu, po dveh in po štirih dneh. Ob obdukciji smo izolirali sapnike in jih pregledali s TEM. V poskusni skupini smo opazili blago obliko hropenja, kašelj in zasoplost. Močneje sta bila izražena hiperemija in edem sluznice sapnika. Okužba z IBV je povzročila hipertrofijo celic goblet, njihovo pokanje in prekomerno tvorbo sluzi. Na ravni ciliarnih celic smo opazili popolno odsotnost migetalk na površini sapnika. Pri okuženih piščancih smo opazili tudi otekel in povečan endoplazemski retikulum.
Ključne besede: transmisijski elektronski mikroskop; virus kužnega bronhitisa; serotip 4/91; sapnik; brojlerji