Protective Efficacy of Chitosan Coupled Johne’s Disease Vaccine

Johne’s disease (JD), also called as Paratuberculosis is a chronic disease affecting domestic and wild ruminants. JD, caused by Mycobacterium avium subsp paratuberculosis (MAP) is characterized by weight loss, reduced milk yield, diarrhea and granulomatous enteritis. JD is worldwide in distribution and indeed endemic in India. Prevalence of JD has been reported in several states of India and the rate of prevalence has a wide variation. In small ruminants, the prevalence percentage ranged between 3.5 and 42.3. In large ruminants the prevalence was 1.8 to 34.1 per cent (Singh et al., 2009). JD is an economically important disease and the loss could be attributed to meat and milk production loss, early culling of reactors and loss due to mortality.
       
With no systematic vaccination programme in place, high prevalence and a long, silent sub-clinical phase have placed JD control strategies in a difficult situation. Though live vaccines are available and being used for the control of JD world over, they do not give 100% protection necessitating the need for more vaccines which can prevent infection and shedding of the bacteria with longer duration of immunity. Moreover, MAP is an intracellular pathogen and effective immunization strategies aim at vaccine candidates that stimulate a robust Th1 response (Seder and Hill, 2000). Use of local isolates of pathogens in vaccine preparations has always been a preferred approach (Uzonna et al., 2003). Moreover, vaccine delivery also plays a major role in inducing a good protective immune response. Recent attempts involving the use of nanoparticles as vaccine carriers have yielded good results prompting the use of nanoparticles as a vaccine delivery system (Santos et al., 2019). A heat killed, chitosan nanoparticle coupled JD vaccine developed with an Indian MAP isolate has proved to be effective in goats under in house conditions. To further validate the potency of the vaccine and duration of immunity, the present study has been done with limited challenge studies. 

Johne’s disease is highly endemic in India. Developed countries follow the strategy of testing, culling and vaccination for the control of JD. In India, culling of positive animals will be very challenging due to socioeconomic issues. With regular vaccination being the only option, availability of a potent and cheap vaccine inducing a good Th1 response is of paramount importance. In this context, we tested the potency and duration of immunity of a heat killed, chitosan coupled vaccine.
 
Pre challenge immune responses
 
MAP is a slow-growing intracellular pathogen and a good Th1 response than the Th2 response is required to overcome the invasion of intracellular pathogen. Th1 response for MAP vaccine was assessed by studying IFN-g, IL-2 and IL-12 cytokine gene expressions and Th2 response by IL-10 expression. Cytokine gene expression following immunization indicated better Th1 response 3 weeks (wk) post immunization (PI). IFN-g and IL-2 expressions were found to be the highest at 3wk PI (Fig 1) in immunized goats. IL-12 has been found to promote cell mediated immune response by stimulating IFN-g production (Wojno et al., 2019). Innate ability of dendritic cells to recognize pathogens lead to IL-12 production which is responsible for activating CD4-positive T cells to differentiate into Th1 cells that produce IFN-g. Though significant increase in IL-12 response was noticed at 3 wk PI, the response peaked at 14 wk PI, probably since it is also involved in maintenance of IFN-g producing Th1 cells (Uzonna et al., 2003). As expected IL-10, the Th2 response indicator increased at 3 wk PI and peaked at 14 wk PI in immunized animals (Fig 1). Moreover, in accordance with the fact that IL-10 might have a role in immunity during later stage of exposure (Park and Scott, 2001), the response was found to be stable even at 23 wk PI (Fig 1).
 

 
Post challenge immune responses
 
Post challenge (PC) studies have indicated an increase in IFN-g, IL-2 and IL-12 responses one wk after challenge (Fig 2, 3 and 4). A slight decrease in IFN-g and IL-12 responses were noticed at 2 wk PC and 2,8 wk PC respectively. Several studies have indicated that IL-12 is the key cytokine that initiates and maintains Th1 responses (Park and Scott, 2001). All the three Th1 cytokines (IFN-g, IL-2 and IL-12) were seen up regulated up to the observation period of 34 weeks PC indicating a strong Th1 response in the immunized animals. With increased levels in immunized goats, IFN-g might have played a vital role in the protective immunity of the animals following challenge with MAP by IFN-g mediated signalling of macrophages (Kumanan et al., 2009).
       

 

 

 
Earlier studies with MAP have suggested a dominant Th1 response initially and a predominant Th2 response at the later stage of the disease. This could be due to the ability of extracellular MAP to persist outside of macrophages and results in a changeover from cellular immunity to humoral immunity (Magombedza et al., 2014). In our study too, though a dip in Th2 response was noticed until 2 wk PC as evidenced by a decreased IL-10 response (Fig 5), the expression increased in immunized animals from 8 wk PC onwards. Nevertheless, both Th1 and Th2 responses were significantly higher in immunized animals at 23 and 34 wk PC indicating a protective immune response.
 

       
Initially, the protective immune response to primary vaccination was assessed for a period of up to six months. Following challenge, the immune response was tested up to a period of 9 months to assess the duration of immunity. It is clear from our studies that our vaccine induced a good Th1 response as required in case of intracellular pathogens like MAP as evidenced by a decent IFN-g, IL-2 and IL-12 responses up to 14 wk PI. This initial Th1 response was followed by a good Th2 response with a better IL-10 response than the IFN-g and IL-2 responses in the vaccinated animals at 23 wk PI. In addition to our immunological findings, we also observed that, six months post challenge, un immunized control animals developed occasional mild diarrhoea, whereas immunized animals showed no such symptoms. Post challenge, there was weight loss in un immunized control animals compared to the immunized animals.

Cytokine gene expression studies have indicated a good Th1 response until 14 wk PI. This initial Th1 response was followed by a good Th2 response with a better IL-10 response than the IFN-g and IL-2 responses in the vaccinated animals at 23 wk PI. Following challenge, both Th1 and Th2 responses were significantly higher in immunized animals at 23 and 34 weeks post challenge indicating a protective immune response.

The authors thank the Department of Biotechnology, GoI, New Delhi for funding this work. The authors also thank the Dean, FBS, TANUVAS; The Professor and Head, PGRIAS, Kattuppakkam, The Professor and Head, URF, Madhavaram and the Professor and Head, Department of Animal Biotechnology, Madras Veterinary College for providing facilities.