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Byline: M. S. Imran, A. Aslam, M. Y. Tipu and T. Yaqub

Keywords: Newcastle disease virus, Infectious Bronchitis virus, Co-infection, Histopathology, Immunohistochemistry.


The respiratory diseases, caused by Newcastle disease viruses (NDVs) (Shabbir et al., 2013), Infectious bronchitis viruses (IBVs) (Rafique et al., 2018) and avian influenza viruses (AIVs) (Kausar et al., 2018), are frequent in commercial poultry in Pakistan. These viruses have abundant economic concerns due to their ability to cause infection either independently or in association with other viruses such as avian influenza virus, infectious bronchitis virus (Alexander and Senne, 2008; Roussan et al., 2008) or with other bacterial pathogens such as E.coli (Qamar-un-Nisa et al., 2018; Umar et al., 2018).

Newcastle disease virus (NDV) is a non-segmented, single stranded negative sense RNA virus which is classified within family Paramyxoviridae (Alexander and Senne, 2008). The whole genome (15198 bp) of the virus usually follows that "rule-of-six", 5'-NP-P-M-F-HN-L-3'. Based upon pathogenicity, NDVs are categorized into three pathotypes named as velogenic, mesogenic and lentogenic (avirulent) (Alexander and Senne, 2008). On the other hand, IBV is a single stranded

RNA virus that has positive-sense genome surrounded by an envelope of a lipid coat (Deming and Baric, 2008). The whole genome (27.6 kb nucleotide) encodes different structural and functional proteins in an order of 5'-S-M-E-N-3' and, on the basis of molecular epidemiology, lineage VAR-II is continuously reported from chickens. It predominantly affects the respiratory tract (tracheal rales, gasping, sneezing and coughing), urinary tract (acute nephritis, stones development in the kidney microtubules and deposition of urates in the kidney) and the reproductive system (decline in egg production with inferior quality of eggs both internal and external) of the birds (Bande et al., 2016; Kiss et al., 2016). Both diseases are endemic in Pakistan and, apart from this fact, co-infection with NDV and IBV consequences may induce severe morbidity and mortality rates in commercial poultry due to synergistic relationship among both viruses for tissue tropism (Nili and Asasi, 2002).

However, scanty information regarding the alterations in histopathological changes during the course of co-infection caused by NDV and IBV is available in the country. Therefore, the current study was designed to investigate the infectious potential of each of the individual viruses as well as co-infection of NDV and IBV in broiler birds. These study outcomes are expected to further elucidate baseline information towards pathogenesis of both viruses either in alone or in case of co-infection in commercial poultry.


Management of birds and preparation of viruses: A total of one hundred and twenty (n = 210) day-old broiler chicks were procured from local well reputed ISO certified hatchery (AW breeder farms and hatcheries (pvt) Ltd., Kasur road, Lahore). Standard management conditions were provided to birds with ad libitum feeding and watering and to avoid any possibility of cross contamination. The birds received 16 hours of artificial light per day as per requirement of brooding of chicks. All birds were housed for three weeks for attaining maturity. Both NDV and IBV strains were isolated during outbreaks in broiler birds of poultry farms in Lahore (unpublished data) and found velogenic/pathogenic in nature. Virus isolation was performed using 9-d-old embryonated chicken eggs and confirmed by Haemagglutination Inhibition (HI) assay using NDV-and IBV-specific antisera as per described previously (OIE, 2012) and PCR assays (Shabbir et al., 2013).

The positive-harvested allantoic fluid of NDV and IBV were diluted in PBS containing gentamycin (250ug/mL) and penicillin (200U/mL) antibiotics to finally obtain a titer of 105.76 and 106.03 EID50, respectively. The EID50 of each of the virus was calculated independently as per method described previously (Reed and Muench, 1938).

Experimental design and clinical observation: All birds (n = 210) were divided into six different groups (35 birds per group) as group A (NDV-challenge), B (IBV-challenge), C (first NDV then IBV challenge after 24 hrs), D (first IBV then NDV challenge after 24 hrs), E (NDV and IBV challenge simultaneously) and F (negative control) before one week of start of experiment for acclimatization in environment. One day before, all birds were screened for presence of NDV, IBV and H9N2 viruses and specific antibodies using haemagglutination inhibition assay (HI) and reverse transcriptase polymerase chain reaction (RT-PCR) (Shabbir et al., 2013). Birds were monitored for clinical signs twice a day up to 6 dpi with special monitoring of respiratory and nervous disorders. A scoring system for evaluation of degree of severity of infection was used by following scales as no sign (0), slight or mild signs (1), moderate signs (2) and severe signs (3).

The mean score of clinical signs was measured as sum of clinical scores for each sign divided by the number of birds showing signs in each group as previously described (Jirjis et al., 2004).

Necropsy findings and histopathological examination: From each group, a total of 05 chickens each were randomly slaughtered at day 0 dpi and thereafter, 10 chickens from each group (A, B, C, D, E and F) were sacrificed at 2nd, 4th and 6th days post infection. The necropsy was performed during the post-mortem of birds. The presence of pathologic lesions of trachea, lung, proventriculus, kidney and caecal tonsils were observed and scored as follow no lesion (0), mild lesions (1), moderate lesions (2) and severe lesions (3). The sum of lesions score in one group was used for statistical comparison to investigate the severity of infection between groups. Approximately 5 mm3 tissue samples were collected from each aforementioned organ. For this purpose, tissue sections of corresponding organ preserved in 10% formalin were fixed on microscopic glass and stained with Hematoxylin and Eosin (HE) as per method described previously (Subtain et al., 2011).

The paraffin-embedded tissues were sectioned, mounted, stained with and examined under light microscope for histological changes under 100X magnification and 3X view field repetition. During histopathological examination, lesions or any pathological change was graded as follows no lesion (-), slight or mild lesions (+), moderate lesions (++) and marked or severe lesions (+++) as described previously (Jirjis et al., 2004).

Immunohistochemistry: The process was conducted as per commercially available IHC kit following manufacturer's instruction (ab64264 - Mouse and Rabbit Specific HRP/DAB (ABC) detection Kit, abcamA(r), Austria). Using 1.0% solution of Poly-L-lysine and/or trypsin in PBS for 20 minutes at 37AdegC, the tissue sections were deparaffinised from the microscopic slide and antigen was retrieved using 10X target retrieval solution at pH 6.0 inside a microwave in 100AdegC for 15 minutes followed by 20 minutes' incubation at room temperature. A 3.0% solution of H2O2 was employed to block the endogenous enzyme activity for peroxidase. The target tissue was incubated with the highly specific primary mouse monoclonal antibodies for 60 minutes in a humidifier chamber. The secondary antibodies (biotinylated anti-rabbit antibody) were labeled as peroxidase polymer conjugated to anti-mouse and anti-rabbit immunoglobulins and incubated for 30 minutes.

Following that, a solution containing peroxidase-conjugated streptavidin was added a nd tissue section was incubated for 10-15 minutes. Finally, counterstaining was done with a bath of Mayer's Hematoxylin for 2-3 min and sections were examined under phase contrast microscope using 400X magnification (Pantin et al., 2003). The tissue sections collected from group F (negative control) were incubated without specific primary antibodies.


Clinical presentation of experimentally infected commercial broiler chickens upon exposure of field isolates of ND and IB viruses: Upon experimental infection with NDV and IBV, varying degrees of clinical infections were observed in different groups (A, B, C, D and E). Overall, the birds of group E gained high score for the exhibition of clinical signs following group C, D, A and B (Fig. 1A). Additionally, the birds infected with IBV (group B) gained high score for the exhibition of respiratory signs as compared to those birds infected with NDV (group A). Whereas, the birds of group A gained high score for the presentation of nervous signs as compared to those birds infected with IBV (group B) (Fig. 1B). Birds of the infected groups C, D and E showed mild to high level of gasping signs at 2nd dpi. These signs were severe after 4th dpi.

Respiratory clinical signs were predominant in all of the inoculated groups and were intense and more severe until 6th dpi, with no clear differences in the pathogenicity caused by NDV or IBV strains. The severity of nervous signs was variable according to the groups such as high severe signs were observed in group E and gained maximum score. While, birds of the infected groups A, C and D showed moderate level of nervous signs at 3rd dpi. These signs were severe after 5th dpi. These clinical signs were predominant in all of the inoculated groups and were more severe until 6thdpi. No abnormal clinical signs either respiratory or nervous were observed in the negative control birds (group F) (Fig. 1A, B).

Gross pathological and histopathological findings: At necropsy most of the birds from group A-E showed variable intensity (mild, moderate and severe) of pathological lesions suggestive of NDV and IBV infection. Lesions on the parenchymatous organs/tissues particularly trachea, proventriculus, lungs, kidneys and caecal tonsils were recorded. Generally, haemorrhagic tracheitis in trachea, hyperaemia in lungs, enlargement of kidneys, haemorrhagic and swollen proventriculus and caecal tonsils were observed. There were observed the severe haemorrhagic tracheitis, mucosal congestion and catarrhal exudates in the trachea in all birds of group A, B, C, D and E. Many of the necropsies showed mild, moderate and severe congestion and haemorrhages in the trachea and lungs collected from the birds of group A and B. The Proventriculus was found swollen in many necropsies of birds of group A, C, D and E with the presence of haemorrhages in submucosa and mild hemorrhagic papillae.

The kidneys and caecal tonsils collected from the birds of group C and E showed mild to severe congestion and mild to moderate haemorrhages along with inflammation (Fig 2). At the histopathological examination, moderate to very severe pathogenesis of NDV and IBV infection was observed in trachea, lungs, proventriculus, kidneys and caecal tonsils collected at 0, 2nd, 4thand 6th dpi. With the fact of high level of clinical presentation between 4-6 dpi, maximum evidences were observed at the tissue collected at 6th dpi. The infiltration of lymphocytes, prominent blood vessels, haemorrhages of blood vessels with oedematous fluid in surrounding area were observed in the proventriculus, kidneys and caecal tonsils tissues collected from group A. While, similar type of lesions was also observed in the trachea and lung tissues collected from group B. These findings were also recorded in trachea, kidneys, proventriculus and caecal tonsils of group C and D.

Comparably, these all histopathological findings with sloughing of ciliary cells of trachea were observed in all tissue collected from group E. As compared to all groups, no histopathological changes were observed in any tissue collected from group F (Fig. 3).

Immunohistochemical evidences of the viral antigens in different collected tissues: Tissues collected at 6th dpi were used for the evidences of viral antigen distribution through immunohistochemistry. Overall, strong immune-labelling was evident in the tissue of co-infected birds of group C, D and E as compared to individual virus challenged group A and B. The viral antigen was present in several organs of birds inoculated with NDV and IBV. Mostly, the presence of IBV antigens was highly observed in hyperplastic epithelial cells and submucosa of the trachea in the group E and B, moderate to low existence in group C and D. Likewise, the presence of IBV antigens was highly observed in epithelium of air capillaries and necrotic debris in lungs in the group E and B, moderate to low existence in group C and D.

Whereas, NDV antigen positive cells were highly detected in the proventriculus, kidney and caecal tonsils of group E and moderate in group C, D and A but not in group B and F. In the kidney, antigen was detected in tubular epithelial cells in most of the groups with varying degree. Antigen positive tubular epithelial cells or glandular caecal tonsil were more intensely labelled in group E and A compared with the other groups (C and D). The expression of viral antigens was absent in group F (Fig. 4).


The respiratory diseases caused by viruses have a marked influence on the poultry production and, in a disease endemic setting such as Pakistan, may further enhance economic losses (Yashpal et al., 2004). A co-infection in poultry not only may cause difficulty in disease diagnosis by an exhibition of varying clinical manifestation but, limited information regarding interaction between co-infecting viruses such NDV and IBV, may make situation further worse than before (Costa-Hurtado et al., 2014). Over the last few decades, NDV and IBV infections has accounted for a significant losses to the poultry sector in Pakistan (Ahmed et al., 2007; Shabbir et al., 2012). Although, natural or experimental infectious potential of NDV and IBV in commercial poultry are well established when studied alone (Qamar-un-Nisa et al., 2018; Kanwal et al., 2018; Aziz-ul-Rahman et al., 2018, 2019) however, information related to pathobiology of these viruses as a result of co-infection is scarce.

This is important because co-infection may cause huge economic losses to the farmers in the form of disease progression and condemnation of the morbid bird meat. Therefore, it is imperative to elucidate aspects of disease pathogenesis among birds upon exposure to either alone or in combination of both NDV and IBV (co-infection) under the field circumstances. Certain control measures are adopted to control individual pathogen in commercial poultry farms; however, such strategies may not be equally applicable in case of infection caused by two or more than two closely related pathogens. With this background, the current study was designed to investigate individual as well as co-infection of NDV and IBV in broiler chickens. In the present study, clinical exhibition was not limited to severe respiratory signs but mild to moderate nervous signs were also observed in case of birds challenged with both NDV and IBV which is in agreement to observation reported elsewhere (Kouakou et al., 2015).

Indeed, similar to finding reported in the previous studies, the clinical signs and lesions were severe in co-infected birds than those infected with each of the individual virus (Kouakou et al., 2015; Shirvani et al., 2018) possibly due to exaggeration of inflammatory responses (Dwars et al., 2009). This is important because the infection caused by one virus in a host may have similar tissue tropism and, therefore, is very likely to provoke or enhance an ongoing infection upon exposure to another virus (Kimura et al., 1976).Contrary to this, there are multiple mechanisms that could be involved in resultant interference among the viruses. Hence, the replication of new virus might be interfered by an earlier replication of a previous virus at the same location in the body due to activation of the immune system and subsequent immune responses to earlier viral infection at the same site (Pantin-Jackwood et al., 2015).

Considering the ongoing situation in the country, an exposure to IBV is almost unavoidable for commercial poultry setting where there is every possibility of occurrence of co-infections with NDV simply due to the fact that chances of viral infection sharing similar tissue tropism (e.g., NDV, IBV and AIV) get further enhanced in a disease endemic setting (Naguib et al., 2017). The observed microscopic changes or histopathological lesions were suggestive of classical lesions due to infection of NDV and IBV in commercial poultry (Qamar-un-Nisa et al., 2018; Kanwal et al., 2018; Aziz-ul-Rahman et al., 2018, 2019). The observed lesions were not only closely related to those reported previously during field outbreak (Miller et al., 2013; Kanwal et al., 2018) but also those where they were observed while performing a challenge-protection related experiments (Shabbir et al., 2016; Amarasinghe et al., 2018).

For instance, the findings of haemorrhages in trachea and lungs in infected birds of present study are in agreement to evidence observed in experimental poultry (Purcell et al., 1976). Subsequent to co-infection of NDV and IBV in birds of group C, D and E, severe haemorrhages in submucosa of trachea that progressed to congestion and tracheitis were observed constantly. Such findings further support the hypothesis that NDV and/or IBV play a vital role in resultant severity of clinical sings in respiratory system, possibly via impairment of other viral infection in the air way tract of bird facilitating the entry of other pathogen (Haghighat-Jahromi et al., 2008). Additionally, owing to high degree of infection, the congestion in the different tissues collected from co-infected birds indicates the deciliation and leukocytic infiltration which is in agreement to previously reported observations (Nili and Asasi, 2002).

The lungs, kidneys, proventriculus and caecal tonsil tissues had remarkable congestion followed by atrophy. Such findings indicate that, while dissemination throughout the body via blood or lymph vessels, the virus had infected immune cells (Kwon et al., 2008). Here it is important to indicate that we excluded brain tissue from histopathological processing simply because we studied co-infection of both viruses where IB does not have potential to affect brain tissues but respiratory, GIT and urinary tract. On the basis of the antigen-antibody reactions, NDV and IBV antigens were successfully detected in different tissues using IHC technique (Igwe et al., 2018). Not only that it indicate a wide spectrum of tissue tropism of each of the virus individually but also the detection of high level of NDV and IBV antigens in trachea, lungs and proventriculus collected from birds of group C, D and E indicate the analogous tissue tropism of both viruses in a pattern reported previously by others (Naguib et al., 2017).

Conclusions: The current study concluded that co-infection of NDV and IBV cause severe infection in the commercial poultry. The clinical, histopathological and immunohistochemical evidences revealed a high degree of infection in those birds challenged with both NDV and IBV simultaneously and sequentially as compared to those birds challenged with individual virus.

Conflict of interest: All authors declared no competing interest regarding this article


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Author:M. S. Imran, A. Aslam, M. Y. Tipu and T. Yaqub
Publication:Journal of Animal and Plant Sciences
Geographic Code:9PAKI
Date:Oct 31, 2019

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