Comparative Pathology of Experimental Avibacterium Paragallinarum Infection in Chicken and Japanese Quail

Avibacterium paragallinarum (Av.paragallinarum) is the causative agent of infectious coryza (IC) in chicken, an economically devastating disease of poultry industry. Despite the availability of effective vaccines against IC, the infection is rampant in unorganized poultry sector. Moreover, reports of wider host susceptibility in poultry farms and under eld conditions are also emerging. Av.paragallinarum affects the upper respiratory tract of birds and has a predilection for the nasal turbinates. However, no study has systematically evaluated the early pathological changes and mucosal immune responses during Av. paragallinarum infection in the nasal turbinates of chicken. Furthermore, the use of Japanese quail as an alternate experimental animal model than chicken in IC remain unexplored. Here, we examined clinical signs, gross and histopathological changes at the nasal turbinates following experimental Av. paragallinarum infection in chicken and Japanese quail. Methods We developed a comprehensive scoring system for recording gross and histopathologic lesions during infection. Our data show that chicken have a higher susceptibility to Av.paragallinarum infection than the Japanese quail. Japanese quail had signicantly lower gross as well as histopathology score in nasal turbinates as compared to infected chicken. Importantly, chicken at an early age i.e. 2.5 weeks were able to induce mucosal immune responses against Av. paragallinarum infection. Our ndings suggest that there are demonstrable differences in the disease pathology and host immune response to Av. paragallinarum infection in chicken and Japanese quail and warrant further investigation for the use of Japanese quail as an animal model for IC.


Introduction
Infectious coryza(IC),also known as Snot is a bacterial infection of poultry caused by Avibacterium paragallinarum (Av. paragallinarum). IC is a disease of economic importance in several parts of the world and predominantly affects the upper respiratory tract of chicken (Wahyuni et al. 2018;Sun et al., 2018;Balouria et al., 2019).The disease is mostly seen in intensive poultry farming operations where infectious droplets are transmitted by aerosols or through contaminated water or contact with asymptomatic carrier birds, which increases the risk of disease outbreak. Infection is known to in ict myriad of losses like drop in egg production, increased culling rates in growing chicken, poor body weight gain, especially in broiler chicken and continual problem of respiratory tract infection in birds of all ages (Droual et al., 1990;Blackall, 1999). Unorganized farming system and multispecies rearing practices are also attributed for the high risk of transmission of the disease among ocks and farms, when located within very close geographic proximity (Crispo et al., 2018).Such progressive infection is not surprising, as most of the recovered birds become carrier and persistently shed the bacteria in the environment through infected nasal secretions.
Reports of disease outbreaks of IC in poultry other than chicken have been well described (Priya et al., 2012;Wahyuni et al., 2018;Mohammad and Sridevi, 2019). Therefore, appropriate preventive measures such as vaccination is required to prevent such disease outbreaks. Despite the availability of vaccines, IC persists in several commercial farms. One of the major reasons for the failure of existing vaccines is the absence of local pathogenic serotypes of Av. paragallinarum in vaccines, which lacks cross protection between vaccine and eld strains (Bragg et al., 1997;Jacobs et al., 2003Chukiatsiri et al., 2009. The impact of Av. paragallinarum infection in chicken has been described (Anjaneya et al., 2013;Ali et al., 2013;Trujillo-Ruiz et al, 2016). However, a detailed investigation of early pathological changes at the nasal turbinates and host-induced mucosal immune responses in chicken is lacking. Earlier studies suggest that Av. paragallinarum has the potential to infect Japanese quail under eld conditions (Thenmozhi and Malmarugan, 2013;Wahyuni et al., 2018) but the gross and histopathological features of infection remain unclear. Japanese quail has a shorter generation interval and demands less housing space with meagre feed requirements than the chicken. Therefore, Japanese quail could serve as an alternative animal model to chicken for larger IC vaccination studies where a higher statistical power is desirable.
Here, we experimentally infected chicken and Japanese quail with Av. paragallinarum to investigate early pathological features of infection in the two species and to validate the potential of Japanese quail as an alternate disease model of IC. We evaluated the clinical signs post-infection and early pathological changes in nasal turbinates to establish a causal relationship between pathogen and the bird species with respect to the time of infection. Our results show that experimental Av. paragallinarum infection elicits characteristic disease pathology and induces mucosal immunity at an early age i.e. 2.5 weeks of age in chicken.

Materials And Methods
The work presented here belongs to a larger experimental study and some results obtained from the study has been published previously (Balouria et al., 2019). The information detailed here is based upon the same experimental set up, birds (chicken and Japanese quail), challenge route besides sampling programme, which was already published. However, the results presented here are novel and have not been published elsewhere. For easy understanding, some of the experimental details from the published paper are presented here to provide a complete overview of the current work.

Birds, their management and ethical approval
Healthy day-old chicken (White leghorn layer, BV-300 strain) and Japanese quail (Coturnix coturnix japonica) were procured from local unit of Venkateshwara Hatcheries Pvt. Ltd., Ludhiana, India and Central Poultry Development Organization, Northern region, Chandigarh, India, respectively. Birds were obtained on the same day to maintain age uniformity and to minimize experiment-induced biasness. Prior to their arrival, the bird cages, feeders, waterers and rooms were fumigated with mixture of potassium permagnate and formalin solution. The birds were examined for vaccination against Marek's diseases at the delivery portal. On reaching experimental site, birds were kept separately in two different rooms and provided antibiotic-free feed and water ad libitum. A strict hygienic management was performed during the period of experiment to avoid secondary bacterial infections. No birds from either species showed any respiratory signs before infection challenge. Disease-free status of the birds was con rmed after negative growth of Av. paragallinarum from infra-orbital sinus swabs of two birds from each species prior to the start of the experiment. All experimental procedures were approved by local Institutional Animal Ethics Committee (IAEC), GADVASU, Ludhiana, India and performed according to their regulations and guidelines. supplemented with 1% (v/v) lter-sterilized, heat-inactivated horse serum, and 0.0025% (w/v) reduced nicotinamide adenine dinucleotide. Ten percent sheep blood agar with Staphylococcus aureus as feeder culture was additionally used (Quinn et al., 2011) to con rm the purity of organism. The challenge bacterial suspension was prepared by inoculating dew-drop like colonies onto HTM broth at 37 • C for 24 h. The bacterial culture was centrifuged at 25 • C (4000 x g) for 10 min. The obtained bacterial pellets were washed three times in sterile PBS (pH 7.4) and subsequently ten-fold serially diluted to determine bacterial cell concentrations. The colony forming units (CFU) in bacterial suspension used for experimental infection was1.5x10 8 CFU/ml.

Experimental design
At the age of 2 weeks, chickens and Japanese quails were segregated into two groups i.e. "Mockinoculated group" containing 5 birds and "Infected group" with 23 birds. Birds were then acclimatized for 2.4. Gross and histopathological examination The pathological changes in nasal turbinates were carefully examined across all the groups including naive control at each of the mentioned time points (Table 1) and the intensity of gross lesions were subsequently recorded through semi-quantitative scoring ( Table 3).The nasal turbinates and other tissues after decalci cation with Osteosoft ® were xed in 10% neutral buffered formalin, para n embedded, and sectioned at 4-5μm thickness. The sections were stained with haematoxylin and eosin stain and evaluated for histopathological changes. The histopathological changes noted in nasal turbinates were graded(scored on a 0 to 3 scale)( Table 4).The mean severity index for each section of nasal mucosal surface based on histopathological scoring criteria from both chicken and Japanese quail was also recorded. The bright eld microscopy was performed for recording histopathologic changes with Nikon CFi (Nikon Corporation, Japan) and photographs were captured by DS Fi-2 camera (Nikon Corporation).

Statistical Analysis
The data was analyzed using Prism Software version 8.4.2 (Graph Pad Software, Inc, San Diego, USA). Prior to analysis, the data was subjected to normality test (Gaussian distribution) and found to be normally distributed. Two different statistical tests were applied. Firstly, a simple regression analysis was applied to assess the intensity (trend) of infection and the difference of intensity between species.
Secondly, the effect of independent variables(time point after inoculation and species difference) on the outcome of dependent variables(degree of gross pathology in all regions of nasal turbinates, degree of histopathologic changes within middle turbinates and infra-orbital sinus, and cellular in ltrations) was measured by multivariate linear mixed model i.e. twoway ANOVA with Sidak and Tukey as post hoc test. For the data on clinical signs, chi-square test (χ2) was applied. Statistical results were considered signi cant when p values were below 0.05.

Clinical signs development
In chickens, the clinical signs developed as early as 12 h post-infection (hpi) and were characterized by mild unilateral nasal discharges in almost half of the birds. However, only few birds exhibited moderate degree of bilateral nasal discharge. Ocular discharge was milder in form and noticed sporadically in few birds, while mild swelling of either infra or supra orbital sinus ( Figure 1A-B), mild redness of conjunctiva, and peri-orbital areas was seen in nearly 18% of the infected birds. Majority of birds had unilateral clinical signs in the beginning of infection and by 24hpi there was a slight increase in intensity of nasal discharge with bilateral involvement. A bilateral redness in conjunctiva was also common at 24 hpi ( Figure 1C-D). By 48 hpi, the intensity and regional involvement declined to mild form with unilateral appearance. The only clinical signs that prevail til 3 daypost infection (dpi) were mild nasal discharge, where both unilateral and bilateral ow of secretions was observed after pressing of nostrils. On the contrary, Japanese quail was resistant to infection and seldom showed any major clinical signs. However, conjunctival redness was noticed only in few birds 24 and 48 hpi. A statistically signi cant association of clinical signs with respect to species vulnerability was noticed in chicken (Supplementary Table 1).

Gross pathology
The chicken revealed redness of turbinates and nasal cavity along with mucus accumulation 12 hpi with varying severity index (Table 3, score level 1-3) that continued up to7 dpi. The degree of redness in nasal turbinates varied from intense (chocolate) red discoloration of mucosal folds (in turbinates) with haemorrhagic foci or haemorrhagic blotch to dark red or highly pinkish discolored turbinates. At 24 hpi, the chickens showed redness of folds (in turbinates) without haemorrhages (score level 2).The severity of lesion gradually waned down to pink coloration of mucosal folds (turbinates) (i.e. mild intensity; score level 1) at 3dpi. A copious sero-mucus accumulation within nasal passages was noted by 48 hpi (score level 3) that persisted for a day but subsequently observed as scanty secretion adhering to mucosal folds (turbinates) (score level 1),which continued upto 7 dpi. However, mucus secretions were not opaque at any of the considered time points. In chicken, mild to rare moderate degree of swelling of infraorbital sinus as well as supraorbital cavities (score level 1 and 2) was noticed at 48 hpi. On sectioning, such sinus cavities were found to be lled with sticky to clear uidly mucus exudates. Conjunctival haemorrhages appeared infrequently in chicken and were consistent on 48 hpi with diffuse to patchy redness of surface comprising haemorrhagic foci (score level 3). In contrast, the Japanese quail exhibited mild degree of redness in mucosal folds (turbinate) and nasal cavity (score level 1) that persisted upto 48hpi. No major changes were noticed thereafter in Japanese quail.
With respect to development of overall pathological changes noticed across the turbinates, signi cantly higher overall gross pathological lesion score was observed in chicken (slope, p =0.05) (goodness of t, r 2 = 0.21) as compared to Japanese quail (goodness of t,r 2 = 0.011) ( Figure 2A). Furthermore, a signi cantly higher overall pathologic lesion score was noticed at 12 (p<0.05), 24 (p<0.05) and 48 hpi (p<0.01) in chicken as compared to Japanese quail ( Figure 2B). The observed redness in the nasal turbinates and higher mucus secretion in chicken are considered to be the major factors for the visible gross pathological changes, where bacteria led to signi cant mucus secretion at 12 (p<0.05) and 48 hpi (p<0.001). The redness in nasal turbinates was however found to be host driven and was dominant at 24 hpi (p<0.05) (Supplementary Table 2).

Histopathological changes
Following infection challenge in chicken, mild haemorrhages were noticed 12 hpi within posterior turbinates and inferior nasal meatus. These haemorrhages became moderate in intensity by 24 hpi in posterior turbinates, inferior nasal meatus, and infra-orbital sinus and continued until 7 dpi. The loss of cilia from respiratory epithelium covering middle turbinates, inferior nasal meatus, and infra-orbital corresponded to an initial invasion and tissue damage by the bacteria. These changes were predominant in infra-orbital sinus and less observed in posterior turbinates. Like deciliation, a similar trend was noticed for epithelial damage as well. However at 7 dpi, a gradual improvement i.e. regeneration/strati cation of epithelium was noticed. Mucus (alveolar) gland activity in the form of glandular hyperplasia and hypertrophic changes was rst noticed at 24 hpi with maximum effect noted in the mucosal folds of middle turbinates and fairly at inferior nasal meatus, which subsequently waned after 48 hpi ( Figure 3A1-A2). Besides haemorrhages, heterophilic in ltration in mucosal folds was prominently seen in middle turbinates at 12 hpi. Later, a mild heterophilic in ltration was observed at posterior turbinates (within bowman's gland), inferior nasal meatus, and infra-orbital sinus. Among all anatomical sites, heterophilic in ltrations were predominant at middle turbinates especially at propria mucosa and around submucosal blood vessels. Lymphoid cell in ltration was noted almost in an equitable proportion to heterophils in rst 24 hpi at all sections of nasal cavity, with an exception to inferior nasal meatus, where naturally accumulated lymphoid cells as nasal associated lymphoid tissues (NALT) exists. A moderate in ltration was noted in posterior turbinates and infra-orbital sinus from 3dpi until 7 dpi ( Figure 3A3-A4). Formation of small lymphoid follicles as secondary NALT was commonly seen in propria mucosa, submucosal connective tissues of middle turbinates, nasal septum, and infra orbital sinusat48 hpi and 3 dpi ( Figure  3A4 inset).
Japanese quail also exhibited haemorrhages as foremost histopathological change from 12 hpi onwards and predominantly within the middle turbinates. No other regions of nasal cavity showed any distinct areas of haemorrhages ( Figure 3B1). The loss of cilia from epithelial surface was milder than chicken, though a moderate loss (as sloughing of super cial epithelium) was reported at 48 hpi from middle turbinates. Further, the loss of epithelium was observed in middle turbinates and fairly in infra-orbital sinus as mild degenerative changes limited to upper 1/3 rd of epithelial thickness at 12 hpi and upto 3 dpi ( Figure 3B2). Intriguingly, on 3 dpi, the Japanese quail were found to exhibit concurrent changes i.e. broplasia at submucosal connective tissue with mild epithelial cell regeneration at some places ( Figure  3B3). By 7dpi, regenerative changes was observed ( Figure 3B4). Only mild mucus gland involvement was noticed in middle turbinates, inferior nasal meatus and infra-orbital sinus post 24 hpi. Unlike chicken, a mild heterophilic in ltration was predominately noticed in inferior nasal meatus and infra-orbital sinus at 12 hpi. No in ltration of heterophils was noticed in posterior turbinates.
Though lymphoid cell in ltration was mild in nature, however, in comparison to heterophils, they were more prominent and prevalent in all the compartments of nasal cavity starting at 12 hpi until 7dpi. An insigni cant formation of secondary NALT was observed in infra-orbital sinus on 3 dpi. On comparison between birds, a strong trend in lymphoid cell activity particularly in NALT (in inferior nasal meatus) and formation of secondary NALT as protective response was noted in chicken. Similarly, lymphoid cell in ltration in other areas like propria mucosa, sub mucosal connective tissues of nasal septum, and posterior turbinates was observed. However in Japanese quail, such pathologic responses were meager during early stage of infection and remained unaffected at later stages. Heterophilic in ltration and alveolar mucus gland activity was more pronounced in chicken at an early stage of infection, which proceeded quickly to the basal level after 3dpi. In addition, the loss of epithelium was predominant in chicken than Japanese quail. Impact of infection and host response on the outcome of histopathological changes Infection with Av. paragallinarum induced distinct histopathological changes in chicken and Japanese quail especially in middle turbinates (Intercept = 9.18) ( Figure 4A) and infra-orbital sinus (Intercept = 7.99) ( Figure 4B). As compared to the middle turbinates, an increasing trend in tissue damage in infra-orbital sinus was noted in chicken as infection progressed ( Figure 4C and D).Both time as well as host factor determined the development of histopathological changes in middle turbinates and infra-orbital sinus (Supplementary Table 3), where chicken had signi cant higher histopathological scores in middle turbinates by 24 hpi (p<0.01) ( Figure 4C) and in infra-orbital sinus at 3dpi (p<0.05) ( Figure 4D). Infectiona liated changes e.g. heterophilic and lymphocytic in ltration were governed by the time of infection and species involved (Supplementary Table 4). Chicken had a signi cantly higher heterophilic in ltration than Japanese quail especially at 24hpi (p<0.001).Pathological changes like loss of epithelium, lymphoid cell in ltration, NALT hyperplasia and haemorrhages were found to be mainly host driven and were signi cantly higher at 72 hpi in birds. However, in chicken haemorhages were visible at 24 hpi that peaked on 72 hpi (data not shown).

Discussion
Av.paragallinarum infection in poultry especially in chicken has been studied extensively. However, a complete understanding of pathological changes immediately after infection is still lacking. Moreover, the disease pathology in other species of birds than chicken is not known. Here, we elucidate, for the rst time, the kinetics of pathological changes due to Av.paragallinarum infection in the nasal turbinates of chicken and Japanese quail. We developed a comprehensive scoring system for recording clinical signs, gross and histopathological lesions during Av. paragallinarum infection in chicken and Japanese quail. Following experimental infection, the prominent clinical signs in chicken included unilateral and bilateral nasal discharge while Japanese quail exhibited only conjunctival redness for a short duration. The development of clinical signs in chicken was noticed a little earlier than the previously reported studies. This could be possibly due to the use of younger birds (<2.5 weeks of age) and intra-sinus route of inoculation in our study. In chicken, ocular discharges and mild swelling of infraorbital sinus were also observed but were less severe than other reports (Anjaneya et al., 2013;Xu et al., 2019). This could be due to our use of a mild to moderate pathotype of Av. paragllinarum that did not severely affect architecture of infected organs. Therefore, the bacterial strain, pathogenicity, and host susceptibility could govern outcome of clinical signs and antagonistic effect to infection (Jacobs et al., 2003;Zhao et al., 2010). A multiple infusion of infection through direct intra-sinus inoculation of birds over a period of three days for the establishment of infection and development of clinical signs has been attempted previously (Anjaneya et al. 2013).However, we successfully reproduced reasonable level of infection in both chicken and Japanese quail without multiple inoculation of the birds.
We speculate that the lack of clinical signs in Japanese quail is predominantly associated with host factors including an increased level of lipid peroxidation by the epithelial surface and leucocytes in systemic circulation (Diptesh et al., 2020). This mechanism could promote protection of Japanese quail against infection, besides the existential anatomical complexities at nasal turbinates of Japanese quail (Cevik-Demirkan et al., 2007). Using an in silico analysis, an anti-oxidant mechanism as an early response strategy against Av. paragallinarumhas been reported in chicken (Boucher et al., 2014).In our earlier work, we found that Japanese quail is tolerant to this oxidative stress mechanism and uses an unde ned mechanism for disease resistance (Diptesh et al., 2020).
The trend in the development of gross pathological lesions in nasal turbinates was almost similar to other studies (Bragg 2002;Anjaneya et al., 2013;Ali et al., 2013) with a slight variation in severity and intensity. The severity of lesions in our study mainly ranged from mild to moderate degree with rare occurrence of severe intense lesions. In addition to the pathogenic strain type, we also speculate that some host-oriented inherent immunological factors like early serum/tissue bactericidal activity could govern the outcome of infection (Byarugaba et al., 2007). Statistical analysis during early host pathogen interaction reveals signi cant impact of Av. paragallinarum in chicken as compared to Japanese quail. In chickens, the gross pathological changes begin at 12 hpi and peaked at 48hpi, which suggest that chicken is a more susceptible host than Japanese quail. Among various host-associated pathological variables, mucus or nasal secretions were found to be markedly in uenced by the infection and a signi cantly higher mucus out ow was observed during rst 48 h. Importantly, we have previously observed lodgement of bacteria across alveolar mucoid gland, their luminal surface, and at the base of epithelial lining of glands by immunohistochemistry (IHC) (Balouria et. al. 2019;Ahmed et al, 2020). Such changes were commonly observed in middle turbinates and inferior nasal meatus. Other prominent change was redness in turbinates, an invariable host response at an early stage i.e. within 12-24 hpi following intra-sinus inoculation arising from the damage in icted upon penetration of needle. Intriguingly, redness of turbinates continued further resulting into more in ammatory disturbances at later time points i.e. 24 and 72 hpi. A similar effect was observed in Japanese quail but only for~48 h. Microscopically, the noted redness in turbinates was not merely associated with bacterial-in icted damage but also as a result of bacterial adhesion to ciliated epithelial cells resulting in epithelial cell damage and in ammation. A similar loss of epithelium was previously observed in chicken following infection of chicken with eld isolates of Av.paragallinarum and the authors suggested the epithelium loss as a possible cause of narrowing of nasal passages and airways (Anjaneya et al., 2013). However, such epithelial damage was not noticed in Japanese quail in our study, despite bacterial adhesions with ciliated surface, which implies poor bacterial attachment on the respiratory mucosal surface. Future studies are required to know if any host-derived factor is responsible for the disease resistance in Japanese quail or if the moderately pathogenic bacterial strain used in our study failed to induce adequate cellular pathological changes Av. paragallinarum is capable of producing outer membrane vesicles (OMVs) with secretory proteins that can contribute to its pathogenicity through formation of bio lms (Ramon Rocha et al., 2006).The role of OMVs in the pathogenicity of Av. paragallinarum in Japanese quail needs veri cation. On the contrary, in chicken, Av. paragallinarum can induce bio lm formation through OMVs at nasal turbinates, which augments its pathogenicity. This was well supported from our earlier histological observation (IHC) of co-aggregation of bacteria at respiratory epithelial surface, tissue edema, and partial lysis/necrosis of epithelial cells at nasal turbinates in chicken (Balouria et al., 2019;Singh, 2020).
Heterophils are the major polymorphonuclear leukocytes in birds that act against most bacterial infections and respond early to pathological insults (Krams et al., 2012). In the present study, heterophils were found as early as 12 hpi in Japanese quail than chicken, which shows differential ability of the Japanese quail in mounting a quick immunological response to combat infection. In a parallel experiment, we observed an increased tissue-a liated heterophil to lymphocyte ratio in nasal turbinates of infected Japanese quail that was associated with a rise in haemagglutinin antibody response (Diptesh et al. 2020).This is in contrast to a report where a weaker antibody response in the host (i.e. Great tits -Parus major) was reported with high heterophil to lymphocyte ratio due to infection or infectionassociated stress (Krams et al., 2012).
Unlike Japanese quail, the chickens in the present study had little higher proportion of lymphocytes as compared to heterophils. Despite this, chicken could not mount a reasonable antibody response. However, secondary lymphoid follicle formation in the form of secondary NALT was observed only in chicken. The NALT formation probably resulted from contigual presence of bacterial antigen(s) causing modi cation and re-activation of lymphoid cells for more pronounced immune protection. To the best of our knowledge, there are no reports on the formation of secondary lymphoid cell follicle as secondary NALT at sub mucosal region of nasal turbinates in IC. Therefore, while evaluating the cellular changes, we carefully excluded primary NALT from this characteristic observation. Due to the absence of local lymph nodes, avian species mostly rely on localized sub-epithelial and sub mucosal lymphoid aggregates, like primary NALT (as in case of upper respiratory tract), for antigen uptake, processing and induction of an immune response. NALT are the primary storehouse of T cells, confer cell-mediated immunity (Kuper et al., 1992;Oshima and Hiramatsu, 2000), and undergo conditional hyperplasia. However, our recent data show that NALT can extend humoral immunity to chicken through inducing B cell and IgA responses (Manuscript under review).Therefore, histologically observed NALT hyperplasia on 72 hpi and thereafter in both the species, is because of long, persistent presence of bacteria or bacterial antigens, which invariably triggered re-organization, clustering and packaging of both B and T cells for better immune protection. In addition, the presence of bacteria at later time points corroborated with our successful reisolation of bacterial colonies as late as 7 dpi. We hypothesize that Av. paragallinarum can in uence mucus secretion from the host and reduce the viscosity of mucus gel to more liquid form. A watery mucus could enable bacteria to migrate within luminal surface to reach deeper peri-glandular tissues. A similar situation has also been described for Helicobacter pylori, which hydrolyses acidic mucin into neutral mucin in gastric glands that facilitates its easy migration within mucus (Celli et al. 2009). Thus, the changes in composition of mucin might have governed direct interaction of Av. paragallinarum with tissues leading to alveolar mucus gland hyperplasia and hypertrophic changes.
Overall, our data show that chicken is the most favourable host for Av. paragallinarum infection and has a high tendency to acquire the infection from the environment. Japanese quail does not seem to act as a host species favouring the ampli cation of this pathogen. Due to the use of mild to moderate pathotype of Av. paragallinarum, we could not induce infection similar to that under eld conditions in both chicken and Japanese quail. Based on the pathological changes observed in the current study, Japanese quail could not be used as a novel animal model for IC. However, we found that younger chicken i.e. chicken