Concentration Dependent Antigenic Response to Formalin Inactivated Streptococcus equi Isolates in Rabbits

The present study was conducted to evaluate concentration dependent immune response of Streptococcus equi in laboratory rabbits to gauge the immunogenic potential of Streptococcus equi for the development of future vaccine against strangles in equines. Streptococcus equi the causative agent of Strangles in young foals was isolated from the pus of submandibular abscess of strangles affected foals. Streptococcus equi was characterized on the basis of morphological, cultural and biochemical on 5% horse serum Sodium azide blood agar base and by Prolax streptococcal latex system and Analytical Profile Index (API) System. Three concentrations of formalized Streptococcus equi @ 4 × 107, 4 × 109 and 4 × 1011 were inoculated in four groups, one group as control negative, each group containing four adult male laboratory rabbits and sera of the inoculated rabbits were subjected to Indirect Heamagglutination Assay by using M-Protein of Streptococcus equi as antigen. It was found that the rabbits which were given concentration of Streptococcus equi @ 4 × 109 and 4 × 1011 cells per ml showed the highest Indirect Heamagglutination (IHA) antibody titres with GMT as 16.0.


Introduction
Strangles (also equine distemper) is a contagious upper respiratory tract infection of young horses and other equines caused by a bacterium, Streptococcus equi (not to be confused with Streptococcus equinus). Strangles is one of the first diseases in veterinary scientific literature. It has low mortality 1-2% but morbidity goes as high as 100%. It has a great threat to healthy horses kept in stables and horses used for breeding and race purposes [1,2]. Streptococcus equi is sensitive to the majority of antibiotics but in vivo treatment gets fail in both early (Before the development of lymph node abscesses) as well as in later stages (After the development of lymph node abscesses) as declared by Harington et al. and Sweeney et al. [3,4]. In early stages, though treatment is capable to clear all the Streptococcus equi from animal's body but as the treatment is withdrawn, again animal is caught by the disease because of absence of adequate immunity against Streptococcus equi. ln later stages when abscessation has been developed then antibiotic provision fails to combat with the infection because of non-availability of sufficient vascularity to the abscess site and ultimately antibiotic remains unable to reach at the site of action and therapeutic levels of the drug cannot be achieved the site of action. Considering the importance of quines and coming to know about the nature of severity of strangles and facts about the antibiotic treatment the only solution of strangles was considered to adopt prophylactic measures in the form of a vaccine development against this disease. In this respect, Bazley in 1940, 1942 and 1943 prepared an effective vaccine by using young encapsulated cells from rapidly growing cultures of Streptococcus equi, which had been heat killed. This vaccine was used extensively in army horses with considerable success. An alum hydroxide adjuvanted vaccine was also prepared by inactivating a Streptococcus equi culture with formalin, and adsorbing it with sulphate ions free aluminium hydroxide gel [5,6]. From 1940 to 1985, lot of vaccines came in picture including Strepvax®, Strep guard® and Equibac® etc., but failed to provide adequate protective immunity and certain other local reactions were also seen with these vaccines [7]. Though the progress in the development of an effective vaccine against strangles remained slow but efforts could not stop and these efforts included a recombinant S. equi hyaluronate associated protein (HAP) [8], a live attenuated intranasal vaccine [9], Equilis Strep E but all the vaccines failed to provide protective immunity. In Pakistan, this disease is a regular visitor among young equine population in spring season and after finding a fact that this disease comes with a combination of two pathogens S. equi and S. equisimillis [10] a need for bivalent vaccine containing both streptococcal species (S. equi and S. equisimillis) has been felt but after finding no data regarding immunogenic property of indigenous isolate of S. equi and S. equisimillis the need of present work has been felt. This paper describes the comparative immune response to various concentrations of formalin-inactivated S. equi antigen in laboratory rabbits.

Isolation and bio characterization of field isolates
Isolation and bio characterization of bacterial isolates from 70 foals showing sub-mandibular lymph node abscessation suffering from strangles was conducted as mentioned in Buxton, Fraser et al. and Cruickshank et al. [11,12] in the Institute of Microbiology, University of Agriculture, Faisalabad (Pakistan). The confirmation of Lancefield group C and streptococcal species was made by Prolax ® streptococcal grouping latex kit and API ® system [10] and the purified S. equi isolate was preserved in Trypticase Soy broth (Difco; Michigan, USA) containing 20% glycerol and kept at -20 o C.

Preparation of formalin-inactivated Streptococcus equi antigen
Selected Streptococcus equi (S. equi) isolate was inoculated in 500 ml flask having Modified Todd-Hewitt broth supplemented with 5% horse serum. It was kept on an orbital shaker at 60 rpm for 48 hours.
After that formalin (0.2%) was added to kill the S. equi isolate. The bacterial isolate was kept stable for 24 hours for proper action of Formalin. The killed organisms were harvested by centrifugation at 6000 xg for 1 hour at 4 o C. Two washings with sterile PBS (p H 7.2) were done. The pellet thus obtained was re-suspended in PBS. Three concentrations of S. equi were adjusted as 4 × 10 7 per ml, 4 × 10 9 per ml and 4 × 10 11 per ml by spectrophotometer. These preparations were stored at 4 o C until utilized. Sterility was checked by streaking a loopful of the killed S. equi onto blood agar, MacConkey agar plates and Thioglycolate broth and incubating for 24-48 hour at 37°C [10].

Antigenic Rresponse to formalin-inactivated Streptococcus equi in rabbits
A total of 16 adult healthy rabbits, divided randomly into 4 groups containing 4 rabbits each, were utilized in this study. The rabbits of groups A, B, C and D were used for evaluating the concentration dependent immune response to S. equi antigen. The antibody titre was determined by indirect Heamagglutination (IHA) method [13]. Formalised inocula of S. equi containing 4 × 10 7 cells/ml, 4 × 10 9 per ml and 4 × 10 11 per ml were injected subcutaneously in group A, B and C respectively. The rabbits of group D were kept as un-inoculated control. Serum samples were collected at weekly intervals for four consecutive weeks for indirect Heamagglutination (IHA) antibody titres to find out the optimum antigenic concentration/dose for S. equi antigen.

Indirect heamagglutination test (IHA)
Indirect heamagglutination test was performed as depicted by Michael et al. [14] but antigen (M-Protein) to be coated on sheep RBC's was extracted from S. equi as follows.

Preparation of formalin-inactivated Streptococcus equi antigen for IH assay
Antigen to be coated on Sheep RBC's has to be extracted from S. equi and the most immunogenic portion of S. equi is M-Protein [15] which has been extracted as follows.

Dilute-Nitrous acid extraction of M-protein from S. equi
Streptococcal cells from 250 ml Todd-Hewitt broth overnight cultures were collected, washed two times in saline, and then suspended in 0.5 ml of saline. The cell suspension was cooled to 0°C, and 200 µl of 4 M sodium nitrite was also precooled to 0°C, added, and mixed thoroughly. Then 100 µl of glacial acetic acid (room temperature) was added and mixed immediately. The HNO 2 reaction was allowed to occur for 5 min in an ice bath at 0°C. Nitrous acid reaction was stopped by raising the p H to 7 to 7.4 with 5 M sodium hydroxide. The reaction time was calculated from the time the last drop of acetic acid was added and promptly mixed. Supernatants represented the extracts and were collected at 3,000 rpm for 30 min and tested for precipitation. The extracts to be tested for anti-opsonic properties were dialyzed against 0.01 M phosphate buffer (p H 7 to 7.4) overnight at 4°C by using Spectrapor membranes (6,000 to 8,000 molecular weight cut-off; Spectrum Medical Inc., Los Angeles, Calif.) [16].

Results
The study was undertaken to evaluate the immune response of formalin inactivated S. equi in rabbits. The S. equi was isolated on the basis of cultural, morphological and bio chemical characteristics. The isolates of S. equi were G+Ve, Cocci, non-motile and arranged in chains. On blood agar it produced translucent round beta haemolytic colonies.

Concentration dependent antigenic response to S. equi antigen
Sera samples of rabbits of group A (Inoculated with 1 ml Dose with concentration @ 4 × 10 7 cells/ml per rabbit) indicated progressive increase in GMT with maximum value of 11.3 at 21st day as shown in Table 1. GMT (Geometric Mean Titre) was calculated as depicted by Brugh [17].    Group C (Concentration @4 × 10 11 /ml per rabbit) was given a higher dose than Group B and it showed increase in GMT with maximum value of 16.0 at 21 st day again as shown in Table 3, which indicated a positive concentration dependent antigenic response of S. equi whereas rabbits of group D showed no increase in titres as shown Table 4.

Discussion
The ultimate objective of such studies is to evaluate the immunity of causative agent of Strangles in equines which are the actual host of this disease. However preliminary trials are pre-requisites before its final commencement in Horses. That is why the present study was conducted in laboratory rabbits. S. equi is a causative agent of Strangles in young foals as already the similar kind of study has been elaborated by A. Shakoor et al. and Sohail Manzoor [18,19]. The establishment of infection not only depends upon the toxigenic capacity of the infecting bacteria but the environmental temperature is also involved e.g this disease is most prevalent in spring season as compared to any season. The usual media for Streptococcus specie is blood agar but Sodium Azide @0.2% was used as cross contaminant to avoid the growth of Staphylococcus and to enhance the growth of Streptococcus equi 5% horse serum was used. The positive pus samples along with scrapings of submandibular lymph node abscesses were streaked on Sodium Azide Blood agar base supplemented with 5% horse serum. This medium is highly selective for S. equi as Sodium Azide is cross contaminant and allows only G+Ve Catalase-Ve [10]. S. equi isolate was selected after studying the morphological and bio-chemical characteristics of the isolates. The selected isolates of S. equi when subjected to morphological and cultural examination, all showed as gram-positive cocci arranged in chains. The size of organism ranged between 0.5-1.5 µm. These variants were non-motile and non-spore bearing. These isolated variants produced transparent, moist and dew drop like colonies on blood agar with beta haemolysis around colonies. The colony size ranged between 1-2 mm after 48 hour incubation.
Indirect Heamagglutination (IHA) assay was used for the determination of concentration dependent immunity of S. equi. A total of 16 healthy rabbits divided randomly into 4 groups, containing 4 rabbits, each were utilized in this study. The rabbits of groups A, B, C and D were used for evaluating the concentration dependent immune response.
Groups A, B and C resulted in progressive increase in titres with maximum value at day 21 with GMT 11.3, 16.0 and 16.0 respectively. While sera of group D (negative control) showed no increase in titres. This indicated a positive progressive concentration dependent antigenic response of S. equi isolate [20]. This is pertinent to mention here that concentration of S. equi at 4 × 10 9 /ml and 4 × 10 11 /ml produced equal immune response depicting that in future vaccine concentration of S. equi at 4 × 10 9 /ml should be preferred over 4 × 10 11 /ml [21]. This indicated that the selected field isolate of S. equi is antigenic in nature and it produced concentration dependent immune response in laboratory animals, therefore indigenous isolate of S. equi at 4 × 10 9 /ml is recommended for the preparation of successful strangles vaccine [22].

Conclusion
The preparation of S. equi antigen showed antigenic response in the experimental animals (rabbits) and was found safe and no untoward reaction was observed in any rabbit. The antigenic response to the preparation of S. equi was found concentration/dose dependent. S. equi concentration at 4 × 10 9 /ml should be preferred over 4 × 10 11 /ml in future strangles vaccine as both concentrations produced similar immune response.