Detection of infectious laryngotracheitis virus (Gallid herpesvirus-1) from clinically infected chickens in Egypt by different diagnostic methods

Document Type : Full paper (Original article)


1 Department of Virology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt

2 Department of Pathology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt

3 Department of Parasitology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt


Infectious laryngotracheitis (ILT) disease is an acute highly contagious viral disease leading to massive economic losses to the national poultry industry. This study aimed to identify the most accurate and rapid diagnostic methods to rescue layer poultry farms from intense outbreaks in Egypt. Fifty pathological specimens were collected and subjected to virus isolation (VI), histopathology, direct fluorescent antibody technique (FAT) and polymerase chain reaction (PCR). Egg inoculation revealed stunted growth and white pock lesions on chorioallantoic membranes (CAM) in 23 samples. Isolation and propagation of infectious laryngotracheitis virus (ILTV) in cell culture showed syncytia formation 5 days post infection in 20 inoculated samples. PCR resulted in successful amplification of a 647 bp fragment of the thymidine kinase (TK) gene in 25 field samples. Histopathological examination of inoculated CAM showed intranuclear inclusion bodies with infiltration of inflammatory cells. Direct FAT showed intra-cytoplasmic apple green reactions in 18 examined tracheal tissues. PCR has been shown to be more sensitive, accurate and rapid than VI, FAT and histopathological examination.


Abbas, F and Andreasen, JR (1996). Comparison of diagnostic tests for infectious laryngotracheitis. Avian Dis., 40: 290-295.
Abbas, F; Andreasen, J; Becker, R; Ahmed, M; Awan, MA; Wadood, A and Sonn, A (2010). Production of monoclonal antibodies against the challenge strain of infectious laryngotracheitis virus of chickens and their use in an indirect immunofluorescent diagnostic test. Pak. Vet. J., 30: 163-166.
Abdo, W; Magouz, A; El-Khayat, F and Kamal, T (2017). Acute outbreak of co-infection of fowl pox and infectious laryngotracheitis viruses in chicken in Egypt. Pak. Vet. J., 37: 321-325.
Ahmed, A; Khan, TA; Kanwal, Y; Raza, M; Akram, SF; Rehmani, NA and Kazmi, SU (2009). Molecular identification of agents causing respiratory infections in chickens from southern region of Pakistan from October 2007 to February 2008. Int. J. Agric. Biol., 11: 325-328.
Ali, A; Mansour, S; Mohamed, M; Ali, H and Shahin, A (2014). Molecular characterization of thymidine kinase and glycoprotein G genes from a possible vaccine induced infectious laryngotracheitis outbreak in Egypt. Pak. Vet. J., 34: 381-385.
Al Shekaili, T (2015). Epidemiological studies on avian influenza and other respiratory viruses in backyard poultry in Oman. Ph.D. Theses. University of Liverpool. PP: 15-17.
Bagust, TJ; Jones, RC and Guy, JS (2000). Avian infectious laryngotracheitis. Rev. Sci. Tech., 19: 483-492.
Cattoli, G; Drago, A; Maniero, S; Toffan, A; Bertoli, E; Fassina, S; Terregino, C; Robbi, C; Vicenzoni, G and Capua, I (2004). Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds. Avian Pathol., 33: 432-437.
Chacón, JL and Ferreira, AJP (2008). Development and validation of nested-PCR for the diagnosis of clinical infectious laryngotracheitis. J. Virol. Methods. 151: 188-193.
Craig, MI; Rojas, MF; van der Ploeg, CA; Olivera, V; Vagnozzi, AE; Perez, AM and König, GA (2017). Molecular characterization and cluster analysis of field isolates of avian infectious laryngotracheitis virus from Argentina. Front. Vet. Sci., 4: 212.
Crespo, R; Woolcock, PR; Chin, RP; Shivaprasad, HL and Garcia, M (2007). Comparison of diagnostics techniques in an outbreak of infectious laryngotracheitis from meat chickens. Avian Dis., 51: 858-862.
Cobo, F (2012). Application of molecular diagnostic techniques for viral testing. Open Virol. J., 6: 104-114.
Coppo, M; Hartley, C and Devlin, J (2013). Immune responses to infectious laryngotracheitis virus. DCI. 41: 454-462.
El Zowalaty, M; Yogesh, P; Abd El-Latif, H; El Sayed, M and Goyal, S (2011). Selective isolation of avian influenza virus (AIV) from cloacal samples containing AIV and Newcastle disease virus. J. Vet. Diag. Invest., 23: 330-332.
Fuchs, W; Veits, J; Helferich, D; Granzow, H; Teifke, JP and Mettenleiter, TC (2007). Molecular biology of avian infectious laryngotracheitis virus. Vet. Res., 38: 261-279.
Guy, JS and Bagust, TJ (2003). Laringotracheitis. In: Saif, YM; Barnes, HJ; Fadly, AM; Glisson, JR; McDougald, LR and Swayne, DE (Eds.), Diseases of poultry. (11th Edn.), Ames, Iowa, IowaStateUniversity Press. PP: 121-134.
Guy, JS and Garcia, M (2008). Laryngotracheitis. In: Saif, YM; Fadly, AM; Glisson, JR; McDougald, LR; Nolan, LK and Swayne, DE (Eds.), Diseases of poultry. (12th Edn.), Ames, Blackwell Publishing. PP: 137-152.
Hassan, K; Shany, S; Al-Hussien, A; Dahshan, M; El-Sawah, A and El-Kady, M (2016). Prevalence of avian respiratory viruses in broiler flocks in Egypt. Poult. Sci., 95: 1271-1280.
Helferich, D; Veits, J; Mettenleiter, TC and Fuchs, W (2007). Identification of transcripts and products of the UL31, UL37, UL46, UL47, UL48, UL49, and US4 gene homologues of avian infectious laryngotracheitis virus. J. Gen. Virol., 88: 719-731.
Hidalgo, H (2003). Infectious laryngotracheitis: a review. Braz. J. Poult. Sci., 5: 157-168.
Humberd, J; Garcia, M; Riblet, S; Resureccion, R and Brown, T (2002). Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction. Avian Dis., 46: 64-74.
Islam, MS; Khan, MSR; Islam, MA; Hassan, J; Affroze, S and Islam, MA (2010). Isolation and characterization of infectious laryngotracheitis virus in layer chickens. Bangl. J. Vet. Med., 8: 123-130.
Kaboudi, K; Nciri, J; Amara, A; Laarbi, I; Moalla, N; Bouzouaia, M and Ghram, A (2016). Histopathological and molecular diagnosis of infectious laryngotracheitis in Tunisia - First report. IJLR. 6: 34-45.
Kantor, AB; Moore, WA; Meehan, S and Parks, DR (2016). A quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. Curr. Protoc. Cytom., 77: 1-23.
Kim, HR; Oem, JK; Bae, YC; Kang, MS; Lee, HS and Kwon, YK (2013). Application of real-time reverse transcription polymerase chain reaction to the detection the matrix, H5 and H7 genes of avian influenza viruses in field samples from South Korea. Virol. J., 10: 85.
Kimberly, R; García, M; Spatz, S and Tablante, N (2014). Molecular epidemiology of infectious laryngotracheitis: a review. Avian Pathol., 43: 2.
Kirkpatrick, NC; Mahmoudia, A; Colson, CA; Devlin, JM and Noormohammadi, AH (2006). Relationship between mortality, clinical signs and tracheal pathology in infectious laryngotracheitis. Avian Pathol., 35: 449-453.
Koski, D (2015). The complexity of infectious laryngotracheitis virus: a focus on vaccination and challenge viruses. Theses and Dissertations. 14550. IowaStateUniversity. PP: 4-5.
Lee, SW; Markham, PF; Markham, JF; Petermann, I; Noormohammadi, AH; Browning, GF; Ficorilli, NP; Hartley, CA and Devlin, JM (2011). First complete genome sequence of infectious laryngotracheitis virus. BMC Genomics. 12: 197.
Leenaars, M and Hendriksen, CF (2005). Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR. 46: 269-279.
Loncoman, CA; Hartley, CA; Coppo, MJ; Vaz, PK; Diaz-MeÂndez, A and Browning, GF (2017). Development and application of a TaqMan single nucleotide poly-morphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host. PLoS ONE. 12: e0174590.
Madbouly, HM; Sagheer, MAB; Ata, NSS; Kutkat, MA and Zaher, KS (2005). Identification and purification of a local isolate of ILTV. J. Vet. Sci., 39: 11-20.
Noroozian, H and Vasfi-Marandi, M (2007). Detection of avian influenza virus of H9 subtype in the feces of experimentally infected chickens by RT-PCR. Arch. Razi Inst., 62: 181-189.
Office International des Epizooties (2004). Avian infectious laryngotracheitis. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Birds). 5th Edn., Paris: World Organisation for Animal Health. PP: 889-895.
OIE (2014). Office of International Epizootics Terrestrial Manual chapter 2.3.3. Avian infectious laryngotracheitis 1-11.
Ojkic, D; Swinton, J; Vallieres, M; Martin, E; Shapiro, J; Sanei, B and Binnington, B (2006). Characterization of field isolates of infectious laryngotracheitis virus from Ontario. Avian Pathol., 35: 286-292.
Oldoni, I; Rodríguez-Avila, A; Riblet, S and García, M (2008). Characterization of infectious laryngotracheitis virus (ILTV) isolates from commercial poultry by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Avian Dis., 52: 59-63.
Ou, SC and Giambrone, JJ (2012). Infectious laryngotracheitis virus in chickens. World J. Virol., 1: 142-149.
Pang, Y; Wang, H; Girshick, T; Xie, Z and Khan, MI (2002). Development and application of a multiplex polymerase chain reaction for avian respiratory agents. Avian Dis., 46: 691-699.
Parra, S; Nuñez, L and Ferreira, A (2016). Epidemiology of avian infectious laryngotracheitis with special focus to South America: an update. Rev. Bras. Ciência Avíc., 18: 551-562.
Preis, IS; Fiúza, AT; Silva, CC; Braga, JF; Couto, RM; Martins, ND and Ecco, R (2014). Pathological, immunohistochemical, and molecular findings in commercial laying hens and in backyard chickens naturally infected with the infectious laryngotracheitis virus. Rev. Bras. Cienc. Avic., 16: 359-366.
Rashid, S; Naeem, K; Ahmed, Z; Saddique, N; Abbas, M and Malik, S (2009). Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens. Poult. Sci. J., 88: 2526-2531.
Sary, K; Chénier, S; Gagnon, K; Shivaprasad, H; Sylvestre, D and Boulianne, M (2017). Esophagitis and pharyngitis associated with avian infectious laryngotracheitis in backyard chickens: two cases. Avian Dis., 61: 255-260.
Shehata, A; Halami, M; Sultan, H; Abdelrazik, A and Vahlenkamp, T (2013). Chicken embryo origin-like strains are responsible for infectious laryngotracheitis virus outbreaks in Egyptian cross-bred broiler chickens. Virus Genes. 46: 423-430.
Spackman, E; Senne, DA; Myers, TJ; Bulaga, LL; Garber, LP and Perdue, ML (2002). Development of real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H9 haemagglutinin subtypes. J. Clin. Microbiol., 40: 3256-3260.
Taha, Z; Allawe, A; Kadhum, M and Abbas, A (2017). Isolation and sequencing of field isolates of infectious laryngeotracheitis virus in Iraq. J. Entomol. Zool. Stud., 5: 882-886.
Tao, Q; Wang, X; Bao, H; Wu, J; Shi, L; Li, Y; Qiao, C; Yakovlevich, SA; Mikhaylovna, PN and Chen, H (2009).
Detection and differentiation of four poultry diseases using asymmetric reverse transcription polymerase chain reaction in combination with oligonucleotide microarrays. J. Vet. Diag. Invest., 21: 623-632.
Villarroel, A (2015). Practical clinical epidemiology for the veterinarian. 1st Edn., Ames, Iowa: John Wiley & Sons Inc., PP: 100-108.
Williams, R; Savage, CE and Jones, RC (1994). A comparison of direct electron microscopy, virus isolation and a DNA amplification method for the detection of avian infectious laryngotracheitis virus in field material. Avian Pathol., 23: 709-720.
Zaher, KS and Syame, SM (2009). Diagnosis of infectious laryngotracheitis virus using routine and advanced techniques under local Egyptian field conditions. WJZ. 4: 57-62.
Zhao, Y; Kong, C; Cui, X; Cui, H; Shi, X; Zhang, X; Hu, S; Hao, L and Wang, Y (2013). Detection of infectious laryngotracheitis virus by real-time PCR in naturally and experimentally infected chickens. PLoS ONE. 8: e67598.