This study was conducted to assess the humoral immune response to Peste des petits ruminants (PPR) vaccination in West African Dwarf (WAD) sheep experimentally infected with Trypanosoma congolense (Tc). Twenty adult WAD sheep, assigned to five groups of four sheep each were used. Group A sheep were neither vaccinated nor infected, while all sheep in groups B-E were vaccinated using PPR vaccine. Sheep in groups D and E were then infected with 5 x 105 trypanosomes per ml of Tc one (1) week post vaccination (PV) while sheep in groups C and D were treated at 3 weeks post infection with diminazene aceturate. A prepatent period of 12-14 days was observed and antibody titres detectable at one (1) week PV in all vaccinated sheep. The antibody levels progressively increased to a peak Geometric mean titre (GMT) values of 181.0, 222.9, 78.8 and 4.0 for groups B, C, D and E respectively at 8 week PV. Parasitaemia was followed by a depressed sero-conversion rate of antibody. Treatment however enabled the vaccinated Tc infected sheep to mount antibody responses that was superior to that of the infected untreated group. The effectiveness of PPR control programme in trypanosomosis endemic areas could be undermined by trypanosome-induced immunosuppression. Even so, this could be ameliorated by effective trypanocide chemotherapy.
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
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.
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.
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).
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.
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.
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).
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
|Week||Group A||Group B||Group C||Group D||Group E|
* Vaccination day; ** Infection day; *** 1st treatment day; ****2nd 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).
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.
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.
We are grateful to Professor M.C.O. Ezeibe for the generous gift of the Aluminium magnesium silicate (AMS) used in this study.