Introduction

The immune system is the surveillance and defense system of the body (Nash, 2007). Certain disease-causing organisms such as trypanosomosis can impair the immune system and immune response (Magez et al., 2002). Trypanosomes are held responsible for producing a state of severe immunosuppression, which renders the infected host more susceptible to secondary infection, produce poor immune response to vaccinations (Tabel et al., 2008; Bisalla et al., 2009) and also facilitate the establishment and dissemination of the parasite in the infected host (Goni et al., 2002). Whether clinically or experimentally induced, African trypanosomosis produces numerous aberrations in the immune system of the infected host (Bagasra, et al., 1981). It has been reported that the underlying cellular mechanism(s) responsible for immunosuppression could be due to trypanosome-derived B lymphocyte mitogen responsible for the polyclonal B lymphocyte responses occurring during the disease and possibly, for the ultimate suppression of B lymphocyte responses (Gomez-Rodriguez et al., 2009). However, earlier workers have reported rapid restoration of immune competence after trypanocidal drug treatment in infected mice, dog and cattle (Murray et al., 1974; Rurangirwa et al., 1978; Whitelaw et al., 1979; Anene et al., 1989). This study thus investigated the impact of trypanosome infection and diminazene aceturate treatment on PPR vaccination in the WAD sheep.

Materials and Methods

Experimental Animals

Twenty adult male West African Dwarf (WAD) sheep weighing between 9-13kg were used in this experiment. They were acclimatised for two weeks during which routine treatment developed at National Animal Production Research Institute, Zaria (NAPRI, 1984) and modified by Aye(1998) was applied. The sheep were kept in a fly-proof well ventilated house. They were fed on cut and carry grasses consisting of guinea grass (Panicum maximum), elephant grass (Pennisetum purpureum) as is the usual practice in this eco-zone. Water was available ad libitum. Each sheep was identified using neck tag. The experimental sheep were handled in compliance with the guidelines for the humane treatments of animals during experimentation in the University of Nigeria.

Trypanosomes                                                                                         

CT70 strain of Trypanosoma congolense was obtained from the Nigeria Institute for Trypanosomosis Research (NITR) Vom, Plateau State, Nigeria. The parasites were first isolated from a cow in Zaria and were maintained in rats. They were passaged in donor rats from where the experimental sheep were infected.

Vaccine/Antigen- PPR Homologous Vaccine (PPRV)

Peste des petits ruminants homologous vaccine (PPRV 75/1) was obtained from the Nigeria Veterinary Research Institute (NVRI) Vom, Plateau State, Nigeria. A 50-dose vial of the vaccine was reconstituted with 50ml of distilled water and each animal received 1ml subcutaneously in accordance to the manufactures recommendation.

Drug Treatment

Diminazene aceturate (Trypanzen®, Veterinary Pharmaceutical, Pantex Holland) was reconstituted according to the manufacturer recommendation by dissolving a sachet containing 2.36g of diminazene aceturate in 12.5ml of distilled water. The volume administered was calculated from their body weight at the dose of 7mg/kg via the intramuscular route.

Infection of Experimental Sheep

Infected blood from the donor rats were obtained from the retrobulbar plexus via the median canthus of the eyes into a sample bottle containing ethylene diamine tetra acetate (EDTA). Infected blood was then diluted in phosphate buffer saline (PBS). Estimated 1.0 × 10⁶ T. congolense suspended in 0.5ml of PBS was used to infect each sheep via intravenous route. The quantity of parasite inoculated was estimated using the rapid matching method of Herbert and Lumsden (1976).

Experimental Design

The twenty WAD sheep were assigned to five groups (A-E) of four sheep each. Group A sheep were neither vaccinated nor infected while all sheep in groups B-E were vaccinated against PPR. Sheep in groups D and E were inoculated with T. congolense one week post vaccination. Sheep in groups C and D were treated with 7mg/kg diminazene aceturate intramuscularly 3 weeks post infection and repeated 2 weeks later.

Blood Sample Collection

Whole blood (3ml) was collected from all experimental sheep prior to the commencement of the study and thereafter weekly via jugular venipuncture for serology. The samples were allowed to clot, centrifuged at 3000rpm for 15minutes and sera harvested. Sera were stored at -20ºC until analysed.

Parameters

Parasitaemia

The parasites were detected by wet blood film (Woo, 1970) and buffy coat dark phase contrast microscopy method (Murray et al., 1983), while counts were estimated using the rapid matching technique of Herbert and Lumsden (1976).

Determination of PPR Antibody

Preparation of Chicken Erythrocytes

Washed chicken red blood cells were prepared and 0.6% concentration was calculated as described by Wosu (1984).

Standardization of the Peste des Petits Ruminants (PPR) Haemagglutinin Antigen

Peste des petits ruminants (tissue cultured mono-specific) vaccine was reconstituted as recommended for use in sheep and goat. This was standardized as described by Ezeibe et al. (2010). Briefly, one millilitre (1ml) of each reconstituted vaccine was mixed with one gram (1g) Aluminium Magnesium Silicate (AMS). The vaccine-chemical mixtures were incubated at room temperature for one hour (1hr) before they were centrifuged at 3000rpm for 10 minutes. The supernatants were subjected to same procedure twice. The resultant supernatant was then used as the antigen for haemagglutination (HA) test.

Determination of PPR Viral Titre using Haemagglutination (HA)

30µl of PBS was added into each well in the rows of V bottom microtitre plate. Next, serial dilution of 30µl of the standardised PPR antigen was made in the first well and the last aliquot discarded. The third row was the RBC control of 30µl of PBS + 30µl of washed chicken RBCs. The set up was left on the bench for one hour. The result was read only when the RBC control had fully settled at the bottom of the wells. Reciprocal of the highest dilution factor is taken as the viral titre. Subsequently the 4 HU was determined using the equation: e.g. If the HA result was 32, the 4 Haemagglutination units (HU) would be 32/4=8. Therefore, the viral titre would be diluted in a ratio of 1 part of the antigen to 7 parts of PBS.

Determination of PPR Antibody Titre using Haemagglutination Inhibition (HI) Test

30µl of PBS was added into each well in the rows of V bottom microtitre plate. Next, serial dilution of 30µl of the test serum was made + 30µl of the 4 HU PPR antigen in each well of the first row. Next, 30µl of known PPR antiserum was added + 30µl of the 4 HU PPR antigen in each of the second row. Then next 30µl of washed chicken RBC was added + 30µl of the 4 HU PPR antigen in each well of the third row. The whole set up was thoroughly mixed and left for 45minutes for adequate antigen-antibody reaction. Finally, 30µl of chicken RBC was added to each well in all the rows. The whole set up was incubated overnight at 4°C. The result was read the following day only when there is complete sedimentation of the RBCs in the RBC control and clear inhibition in the row containing the specific antiserum. Reciprocal of the highest dilution factor was considered as the HI result.

Statistical Analysis

Parasitaemia value was subjected to One Way Analysis Of Variance (AVOVA). Probability of less than 0.05 (p ≤ 0.05) were considered significant and variant means were separated using Duncan multiple range test (Duncan, 1996). Haemagglutination inhibition titre was calculated with the Geometric mean titre (GMT) using the Tube dilution method and Table provided by Villegas and Purchase (1989).

Results

Parasitaemia

All the infected sheep became parasitaemic by day 14 post infection (PI) and the parasitaemia was sustained until day 21 PI when treatment was administered (Fig. 1).

Fig. 1: Parasiteamia of WAD sheep immunized against PPR and infected with Trypanosoma congolense and treated with diminazene aceturate

The parasites cleared in the infected and treated group (D) within 24 hours of treatment and remained aparasitaemic throughout the experiment. The sheep in group E (infected untreated) remained parasitaemic till the end of the experiment.

Geometrical Mean Titre of Ppr Antibody

The geometrical mean titre (GMT) 1.2 which was recorded on week 0 when the experiment commenced was maintained in group A (unvaccinated and uninfected) up to the end of the experiment by week 10 post vaccination (Table 1).

Table 1: Geometric Mean Titre (GMT) of WAD sheep immunized against PPR and infected with Trypanosoma congolense and treated with diminazene aceturate

*   Vaccination day; ** Infection day; *** 1^st treatment day; ****2^nd treatment day

Group A- Unvaccinated and uninfected; Group B- Vaccinated and uninfected; Group C- Vaccinated, uninfected and treated; Group D- Vaccinated, infected and treated; Group E- Vaccinated, infected and untreated

The vaccinated groups (B, C, D and E) recorded progressive increase in their GMT levels and peaked to GMT values 181.0, 222.9,  78.8 and 4.0 respectively by week 8 post vaccination. The increase in GMT was more pronounced in the vaccinated uninfected groups (B and C) (GMT 181.0 and 222.9 respectively) compared with the vaccinated infected groups (D and E) (GMT 78.8 and 4.0 respectively). By week 4 (i.e. 3 weeks post-infection), the GMT values of the vaccinated uninfected groups (B and C) were 27.9 and 22.6 respectively while the vaccinated infected groups (D and E) were 2.8 and 2.5 respectively. From week 5 (i.e. 1 week post re-treatment) to the end of the experiment, the infected and treated group (D) recorded a progressively higher antibody GMT level  (from GMT value 11.3 to 78.8) than the infected untreated group (E) (from GMT value 2.8 to 4.0).

Discussion

In this study, the standardization of the PPR haemagglutinin antigen by incubating the cultured PPR viral sample with AMS was to activate the haemagglutinin. It has been reported that all the PPR vaccine (cultured virus) were haemagglutination negative and that incubation with AMS can be adopted to convert cultured PPR virus to standard PPR haemagglutinin antigen for use in haemagglutination and haemagglutination inhibition test to confirm diagnosis of PPR (Ezeibe et al., 2010).

Increased antibody titre detected as early as one week post-vaccination is in agreement with the report of Sinnathamby et al. (2001) in goats and Banik et al. (2008) in sheep and goat but contrasts with the finding of Khan et al. (2009) and Intizar et al. (2009) who detected antibodies two weeks post immunization in sheep and goat. This difference may be attributed to the strain of virus used in producing the vaccine, the breed of animal involved and the sensitivity of technique used in detecting the antibody. The peak antibody titre reached by 8 weeks post-vaccination also agrees with the findings of Sinnathamby et al. (2001), Khan et al. (2009) and Intizar et al. (2009). The peak antibody titres were maintained in the infected vaccinated sheep up to 10 weeks post immunization when the study was terminated. However persistence of antibodies in the serum of vaccinated animals for longer periods has been demonstrated in previous studies. The presence of antibody in the serum for up to 63 days corresponding to the end of his experiment (Intizar et al., 2009), 5 months (Sinnathammby et al., 2001), 6 months (Olugasa and Anderson, 2012), 12 months (Awa et al., 2002; Rashid et al., 2010) and 3 years (Zahur et al., 2014) post immunization have been reported. This may be a reflection of the efficacy and potency of the vaccine, and the duration of immunity it confers. It has been reported that a single immunization with PPR vaccine (Nigeria strain 75/1) conferred solid immunity and has protective efficacy for at least 3 years duration (OIE, 2013; Zahur, 2014).

Following the manifestation of Trypanosoma congolense in the blood stream of vaccinated infected sheep by 12-14 day post infection, there was a depression in the sero-conversion rate as seen in the antibody GMT. This is in agreement with Mwangi et al. (1990) who reported depression in antibody response to anthrax spore vaccine in T. congolense infected goats; to haemorrhagic septicaemia vaccination in T. evansi infected buffalo-calves (Singla et al., 2010), to Foot and Mouth disease and clostridal vaccination in Trypanosoma infected cattle (Scott et al., 1977), and Ilemobade et al. (1982) who also reported a slight depression in antibody response to contagious bovine pleuropneumonia vaccination in Trypanosoma infected cattle. This may be attributed to the immunosuppressive effect of the parasite on the host immune defence possibly mediated through a B-lymphocyte defect (Murray et al., 1974). Rurangirwa et al. (1983) reported that B-cells appeared to be one of the targets of immunosuppression as evidenced by greatly reduced IgG1 and IgG2 responses in cattle following vaccination against Brucella abortus. Also, Gómez-Rodríguez et al. (2009) reported that the underlying cellular mechanism(s) responsible for immunosuppression could be due to trypanosome-derived B lymphocyte mitogen responsible for the polyclonal B lymphocyte responses occurring during the disease and possibly, for the ultimate suppression of B lymphocyte responses.

Trypanocidal administration enabled the Trypanosoma infected sheep to mount PPR antibody responses superior to that of the infected untreated group but inferior to vaccinated uninfected groups. This is in agreement with Singla et al. (2010) and Mwangi et al. (1990). The partial recovery of immune responsiveness recorded in this study post trypanocidal therapy suggests that the immune depression was in some ways associated with the presence of living trypanosomes (Eze et al., 2011) in the host. Also, the vaccinated uninfected but treated group experienced a boost in their antibody GMT level. This could be attributed to other therapeutic activity of Diminazene aceturate.

Conclusion

In conclusion, vaccination of WAD sheep against PPR using the homologous PPR vaccine (Nigerian 75/1 strain) caused a progressive increase of the PPR antibody titre up to a peak level at 8 weeks post vaccination. The presence of trypanosomes in the blood stream of the WAD sheep depressed the humoral antibody response to PPR immunization. Diminazene aceturate treatment cleared the parasites from the blood of the infected sheep leading to some improvement in antibody titre levels.

Acknowledgement

We are grateful to Professor M.C.O. Ezeibe for the generous gift of the Aluminium magnesium silicate (AMS) used in this study.

Reference

1. Anene BM, Chukwu CC and Anika, SM. 1989. Immunosuppression of humoral immune response in canine trypanosomosis. Microbios Lett., 40: 37-46.
2. Awa DN, Ngagnou A, Tefiang E, Yaya D and Njoya A. 2002. Post vaccination and colostral Peste des petits ruminantsI antibody dynamics in research flocks of Kirdi goats and Foulbe sheep of North Cameroon. Preventive Veterinary Medicine, 55: 265-271.
3. Aye PA. 1998. The effect of two management systems on some physiological parameters and growth rate of the West African Dwarf goats. M. Tech. Thesis. Federal University of Technology Akure, Nigeria.
4. Bagasra O, Schell RF and Le Frock JL. 1981. Evidence for Depletion of Ia+ Macrophages and Associated Immunosuppression in African Trypanosomiasis. Infection and Immunity, 31: 188-193.
5. Banik SC, Podder SC, Samad MA and Islam MT. 2008. Sero-surveillance and immunization in sheep and goats against Peste des petits ruminantsI in Bangladesh. Bangladesh J. Vet. Med., 6 (2): 185-190.
6. Bisalla M, Adamu S, Ibrahim ND, Lawal IA and Esievo KAN. 2009. Effect of immunomodulation with Levamisole on the course and pathogenesis of acute experimental Trypanosoma congolense infection in sheep. Afr. J. Biotech., 8(5): 827-834.
7. Duncan OD. 1996. Path analysis: sociological examples. J. Soc., 72: 1-16.
8. Eze JI, Ngene AA, Anosa GN and Ezeh IO. 2011. Immunomodulatory effect of dietary selenium supplementation on serum antibody response and leukocytic profile of Trypanosoma brucei brucei. Afr. J. Biotech., 10(56): 12098-12106.
9. Ezeibe MCO, Eze JI, Ijabo O, Ngene AA, Okoroafor ON, Chukwu OS, Ukomadu NM, Sanda EM, Eze IC and Ugonabo JAC. 2010. Standardization of the Peste des petits ruminantsI (PPR) haemagglutinin antigen for haemagglutination- inhibition test. Appl. Ani. Res., 38: 113-115.
10. Gómez-Rodríguez J, Stijlemans B, De Muylder G, Korf H, Brys L Berberof, M, Darji A, Pays E, De Baetselier P and Beschin A. 2009. Identification of a Parasitic Immunomodulatory Protein Triggering the Development of Suppressive M1 Macrophages during African Trypanosomiasis. J. Inf. Dis, 200 (12): 1849-1860. doi: 10.1086/648374
11. Goni O, Alcaide P and Fresno M. 2002. Immunosuppresion during acute Trypanosoma cruzi infection: involvement of Ly6G(Gr1⁺) CD11b⁺ immature myeloid suppression cell. Int. Immunol., 14(10) 1125-1134. doi: 10.1093/intimm/dxf076.
12. Herbert WJ and Lumsden WHR. 1976. Trypanosoma brucei: a rapid matching for estimating the host’s parasitaemia. Exp. Parasitol., 40: 427-432.
13. Ilemoboda AA, Adegboye DS, Onoviran O and Chima JC. 1982. Immune depressive effects of trypanosomal infection in cattle immunized against contagious bovine pleura-pneumonia. Parasite Immunol., 4 (4): 273-282.
14. Intizar M, Ahmad MD, Anjum AA and Henif A. 2009. Comparative efficacy of Peste des petites ruminants (PPR) vaccines available in Pakistan in Sheep and Goats. Pakistan Vet. J., 29 (4):202-205.
15. Khan HA, Siddique M, Arshad M, Abubakar M, Akhtar M, Arshad MJ and Ashraf M. 2009. Post-vaccination antibodies profile against Peste des petits ruminantsI (PPR) virus in sheep and goats of Punjab, Pakistan. Trop. Ani. Hlth Prod., 41 (4): 427-430.
16. Magez S, Stijlemans B, Brad T and de Baetselier P. 2002. DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi and T brucei is mitogenic for B lymphocyte and stimulates macrophage expression of interleukin-12, tumour necrosis factor alpha and nitric oxide. Microbes Infect., 4: 999-1006.
17. Murray M, Trail JCM, Turner DA and Wissocq N. 1983. Livestock productivity and trypanotolerance. Network training manual. ILCA, Addis Ababa, Ethiopia.
18. Murray PK, Jennings FW, Murray M and Urquhart GM. 1974. The nature of immunosuppression in Trypanosoma brucei infections in mice. 11. The role of T and B lymphocytes. , 27 (5): 825-840.
19. Mwangi MD, Munyua WK, Nyaga PN. 1990. Immunosuppression in caprine trypanosomiasis: Effects of acute T congolense infection on antibody response to anthrax spore vaccine. Trop. Ani. Hlth Prod., 22 (2): 95-100.
20. NAPRI 1984. Highlights of Research Achievements on Animal Production. Science and Technology Briefing, Lagos December 1984; pp3-17.
21. Nash H. 2007. The immune system. Pet Education.com, 1997 #45; 1 jan.2007. http://www.peteducation.com/article.cfm?cls=2&cat=1614&articleid=957
22. OIE 2013. Peste des petits ruminantsI. Chapter 2.7.11. version adapted by the World Assembly of Delegates of the OIE in May 2013.
23. Olugasa BO and Anderson JRN. 2012. Assessment of seroconversion against peste des petits ruminantsI vaccine among sheep and goats in Buchanan, Liberia. Sokoto J. Vet. Sci., 10 (2): 56-60.
24. Rashid AH, Hussain A and Asim MA. 2010. Evaluation of Peste des petits ruminantsI cell culture vaccine in sheep and goats in Pakistan. Vet. Italiana, 46 (3): 315-318.
25. Rurangirwa FR, Musoke AJ, Nantulya VM and Tabel H. 1983. Immune depression in bovine trypanosomiasis: Effects of acute and chronic Trypanosoma congolense and chronic Trypanosoma vivax infections on antibody response to Brucella abortus Parasite Immunol., 5: 267-276.
26. Rurangirwa FR, Tabel H, Loses G, Masiga WN and Mwambu P. 1978. Immunosuppressive effect of Trypanosoma congolense and vivax on the secondary immune response of cattle to Mycoplasma mycoides sub specie mycoides. Res. Vet. Sci., 25: 395-397.
27. Scott JM, Pegram RG, Holmes PH, PayTWF, Knight PA, Jennings FW and Urquhart 1977. Immunosuppression in bovine trypanosomiasis: Field studies using foot-and-mouth disease vaccine and clostridial vaccine. Trop. Ani. Hlth Prod., 9 (3):159-165.
28. Singla LD, Juyal PD and Sharma NS. Immune responses to haemorrhagic septicaemia (HS) vaccination in Trypanosoma evansi infected buffalo-calves. Trop. Ani. Hlth Prod.,42 (4):589-595.
29. Sinnathamby G, Renukaradhya GJ, Rajasekhar M, Nayak R and Shaila MS. 2001. Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of Peste des petits ruminantsI virus: identification of a T cell determinant Vaccine, 19: 4816–4823.
30. Tabel H, Wei G and Shi M. 2008. T cell and immunopathogenesis of experimental African trypanosomosis. Immunol. Rev., 225: 128-139.
31. Villegas P and Purchase HG. 1989. Titration of biological suspension. In Purchase, HG; Arp, LH; Domermuth, CH; Pearson, JE. (Eds). A laboratory manual for isolation and identification of avain pathogens. AAAP, Kenneth square, P.A. USA.
32. Whitelaw DD, Scott JM, Reid HW, Holmes PH, Jennings FW and Urquhart GM. 1979. Immunosuppression in bovine trypanosomiasis: Studies with louping-ill vaccine. Research in Veterinary Science, 26: 102-107.
33. Woo PTK. 1970. The haematocrit configuration technique for the diagnosis of Africa trypanosomiasis. Acta Tropica, 27: 384-386.
34. Wosu LO. 1984. Standardization of red blood cells for haemagglutination test and removal of natural agglutinins. Nigerian Veterinary Journal, 13 (1): 39-42.
35. Zahur AB, Irshad H, Ullah A, Afzal M, Latif A, Ullah RW, Farooq U, Samo MH, Jahangir M, Ferrari G, Hussain M and Ahmad MM. 2014. Peste des petits ruminantsI Vaccine (Nigerian Strain 75/1) confers protection for at least 3 years in sheep and goats. Journal of Bioscience and Medicine, 27-33. DOI: 10.4236/jbm.2014.26005.