The virulence pattern of Avibacterium paragallinarum isolates from Indian field conditions demonstrates the existences of all three serotypes i.e. serotype A, B and C was studied. Bragg’s ‘in-contact’ challenge model was applied to study the virulence pattern of these fifteen field isolates. In this model, one bird from each group (10 birds / group) was challenged with pathogen suspension and left for natural infection in the group. The substantial disease causing ability was observed in all field isolates except, one isolate belonging to serotype A. It was also further observed that, isolates belonging to serotype C are more virulent than isolates from serotype A and B. First time serotype B of Avibacterium paragallinarum was found from Indian field isolates. Similar to natural infection pattern, most of the isolates showed two disease peaks in their disease profile. Elevated virulence and variations in virulence pattern among field isolates of Avibacterium paragallinarum indicate the change in prevalence of this pathogen and emergence of potent variants of Avibacterium paragallinarum in India.
Infectious coryza (IC) is a severe respiratory disease of chickens caused by the bacterium Avibacterium paragallinarum. The causative agent is non-motile gram negative bacilli and it shows worldwide distribution, mainly in countries having intensive poultry industry (Vargas and Terzolo, 2004). The economic loss associated with infectious coryza results from poor growth performance in growing birds and marked reduction (10–40%) in egg production in layers (Blackall and Soriano, 2008). The typical symptoms associated with infectious coryza are nasal discharge, conjunctivitis, swelling of face and sinus and swelling of wattles. Some of the other common symptoms include decrease in feed intake and water consumption which consequently retard growth of the birds (Blackall, 1989).
Apparently, Infectious coryza gets complicated by the presence of other pathogens such as Mycoplasma gallisepticum, Mycoplasma synoviae, Escherichia coli, Pasteurella multocida, and viral pathogens like infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILT) and fowl pox virus (FPV) (Anjaneya et al., 2013). The duration of such complicated infectious coryza cases prolong up to 7 weeks (Vargas and Terzolo, 2004). The first report of experimental infection to chicken with Avibacterium paragallinarum was reported by Nelson in 1933. Since then many researchers worked on pathogenicity of Avibacterium paragallinarum in different parts of world. Yamaguchi et al. (1990) reported that there is variation in virulence within a serovars of Avibacterium paragallinarum. The difference in virulence was also seen among NAD dependant and NAD independent strains of Avibacterium paragallinarum (Bragg, 2002b, c). In recent years, Anjaneya et al., 2013, Durairajan et al., 2013 and Kaur et al., 2005 have contributed in establishing the virulence pattern of Indian field isolates of Avibacterium paragallinarum and observed alterations in the pathogenicity of those field isolates. The probable reason for this change in virulence is due to altered prevalence of strains over a time. Hence, it is essential to keep an observation on virulence pattern of field isolates of pathogens to reduce the chances of vaccine failure and re-emergence of severe form of the infectious coryza disease.
The present study was undertaken in this context to review the pathogenicity of Indian field isolates collected between the years 2013 to 2015. The present evaluation permits the construction of disease profile which can be further used to draw a comparison between different field isolates.
Materials and Methods
Fifteen field isolates of Avibacterium paragallinarum used in this experiment were collected, isolated, identified and serotyped as explained by Patil et al., 2016. The isolates were referred as Isolate 1 to Isolate 15. Out of which Isolate 1 to Isolate 5 were serotype A, Isolate 6 & 7 were serotype B whereas Isolate 8 to Isolate 15 were serotype C.
All isolates were retrieved from storage and maintained on TM/SN medium before further use. The pure colonies of isolates were inoculated in Brain- heart infusion supplemented with 1% Sodium Chloride, 0.0025% (W/V) reduced NAD and 1% (V/V) filter sterilized chicken serum (Soriano et al., 2004) and incubated under reduced O2 tension for 48 hours. ‘In-contact’ challenge model described by Bragg and Greyling (1999) was adopted for evaluation of virulence of isolates.
Before experiment the poultry sheds were disinfected with suitable disinfectants and kept unused for two months. The setup of experimental design was prepared as per challenge model described by Bragg, 2002. 160 unvaccinated seven week old layer pullets were procured from commercial poultry having no pre-history of infectious coryza. The birds were randomly allocated into sixteen groups (10 birds/ group) and each group was housed in a separate experimental cage. Each cage was provided with common feed and water facility. One bird was randomly picked up from each group and challenged with 0.2 ml Avibacterium paragallinarum suspension (108cfu) of field isolate via intersinus route. The procedure was repeated for all isolates. The remaining birds in the group were then infected via an in-contact natural challenge route. The last group was used as unchallenged control.
Scoring of Clinical Signs
The post challenge study was carried out for 20 days and clinical signs were recorded and evaluated in a scored table for infectious coryza on a daily basis. The clinical signs were evaluated in following manner-
0 Score: no clinical signs,
1 Score: mild (slight nasal discharge),
2 Score: moderate (slight facial swelling and nasal discharge) and
3 Score: severe (severe facial swelling with lacrimation and anorexia).
Lastly the total disease score for each group for each day was calculated and the mean daily disease score was calculated for each day by dividing the daily disease score by the total number of chickens (Bragg, 2002).
The unvaccinated chickens with no pre-history of infectious coryza were challenged with all fifteen Indian field isolates of Avibacterium paragallinarum simultaneously. The disease causing ability was observed in all the field isolates. The average disease score of each isolate was calculated and presented in Table 1. Graphical comparisons of the virulence pattern of Indian field isolates belonging to each serotype are illustrated in Figure 1( a-c). In the groups infected with Avibacterium paragallinarum serotype A (Isolate 1- 5) the initial disease peak was observed in between 7 to 9 DPI in majority of groups and subsequent disease peak on 15 DPI. The groups inoculated with Avibacterium paragallinarum serotype B (Isolate 6 & 7) showed delayed initiation of clinical manifestation. The signs began from 5 DPI. Isolate 6 showed two disease peaks, first on 9 DPI and next peak on 15 DPI, whereas in Isolate 7 only one peak was observed. In the groups inoculated with Avibacterium paragallinarum serotype C (Isolate 8 – 15) prominent and high disease scores were observed. The disease peaks were identified in between 7 to 9 DPI and 15 DPI respectively.
No challenged bird was dead in this experiment and no clinical signs were recorded in the unchallenged control group of chickens.
Table 1: Average disease scores obtained when chicken groups (n=10) are inoculated with Indian field isolates of Avibacterium paragallinarum by using Bragg’s ‘in-contact’ challenge model
|Average Disease Score
Fig 1a-c: Disease profiles obtained when seven week old unvaccinated chickens challenged with field isolates of Avibacterium paragallinarum by using Bragg’s “in-contact” challenge model.
1a: Mean disease scores of Avibacterium paragallinarum belong to serotype A.
1b: Mean disease scores of Avibacterium paragallinarum belong to serotype B.
1c: Mean disease scores of Avibacterium paragallinarum belong to serotype C.
Infectious coryza is a cosmopolitan disease caused by Avibacterium paragallinarum wherever poultry farming is carried out. Understanding of pathogenicity of field isolates is becoming an important aspect to know the change in prevalence of pathogen and for avoidance of vaccine failure. Classically, two animal experimental infection models are in function to study virulence of Avibacterium paragallinarum viz., artificial intrasinus-injection-route model and ‘in-contact’ challenge model (Shi-jin-Jiang et al., 2009). Artificial intrasinus-injection-route model is one of the common and age old method primarily used to evaluate infectious coryza vaccine (Matsumoto and Yamamoto, 1971) and with slight modifications for virulence characterization (Rimler et al.,1977). But, this particular model does not give much idea about pathogen’s ability to spread the disease as well as no direct comparison of virulence of different isolates can be drawn. On the contrary, ‘in-contact’ challenge model is better in which one bird from a group is inoculated with pathogen culture and left for natural transmission of disease in the group. The clinical signs are scored numerically and due to that a graphical representation of disease profile of isolate becomes possible (Bragg, 2002).
The attempts to study pathogenicity of Avibacterium paragallinarum isolated from Indian field conditions are very limited and in all these reports, evaluation of virulence was predominantly based on clinical signs and pathological findings (Anjaneya et al., 2013). In the said work, we have attempted to use Bragg’s ‘in-contact’ challenge model to study and evaluate virulence of Avibacterium paragallinarum isolated from Indian field conditions.
It was observed that all Indian field isolates except Isolate 5 are significantly virulent and able to cause disease in chickens. Similar findings were demonstrated by Anjaneya et al. (2013), Durairajan et al. (2013) and Kaur et al. (2005). Isolates belonging to serotype C were more virulent than isolates of serotype A and B. This is in accordance with the study of Byarugaba et al. (2007), Soriano et al. (2004) and Bragg (2002).
Most of the isolates demonstrated formation of unique two disease peaks in their disease profiles. After first peak the disease scores were sharply declined in between the DPI 11 to DPI 13 and raised for short time before final lapse. Such disease peaks were also observed by Shi-jin-Jiang et al. (2009) and Bragg (2002) in their virulence studies. This phenomenon was also been observed in natural outbreaks of infectious coryza. Whereas sharp decline in between two peaks was not observed in Isolate 7 and Isolate 12. But no other significant differences in disease pattern were observed in them.
Present study throws light on virulence pattern of Avibacterium paragallinarum field isolates. It has been found that there is considerable difference in virulence of Avibacterium paragallinarum field isolates belonging to all serotypes. Expect one isolate that belongs to serotype A, all remaining isolates are having good potential to cause disease in chickens. Bragg’s ‘in-contact’ challenge model is easy and suitable experimental infection model to represent disease profiles of field isolates. High level virulence of field isolates indicates drastic change in prevalence of pathogen over years in India. This scenario pinpoints towards emergence of potent disease causing variants of Avibacterium paragallinarum in India which further needs more characterization.
The authors deeply acknowledge the support and research facilities provided to them by concerned authorities.