Peste des Petits Ruminants serological survey in Karamoja sub region of Uganda by competitive ELISA

Following the historical reports of mysterious illnesses and deaths in goats in the Karamoja sub-region in April, 2007 and subsequent confirmation of Pest des Petitis Ruminants in July, 2007; we carried out a serological survey to determine the indicative caprine PPRV exposure rate by 2009. We sampled 280 goats from Moroto, Nakapiripirit, Abim and Kotido Districts of North-eastern Uganda to detect antibodies against PPRV using competitive enzyme linked immunosorbent assay (cELISA). The prevalence of PPRV antibodies in the districts of Moroto, Nakapiripirit, Kotido and Abim was 63.2% (CI = 95%, 58.0 – 68.0%), 72.0% (CI = 95%, 65.6 78.4%), 85% (CI = 95%, 81.0 – 88.9%) and 1.6% (CI = 95%, -0.01 – 3.22%) respectively. The overall prevalence of antibodies against PPRV in Karamoja sub-region was found to be 57.6 % (CI = 95%, 48.8 – 66.4%). The high prevalence of antibodies against PPRV suggests that active infection may still be present and therefore the need to institute disease control measures. More studies should be undertaken to characterize the viruses involved and the epidemiology of PPR in Uganda.


Introduction
ently recognized as being endemic in west and central Africa (Scott 1981).It has also been reported in Sudan Peste des petits ruminants (PPR) is a contagious (Taylor 1984) and in East Africa in Kenya (Wamwayi viral disease of small ruminants (Furley et al., 1987(Furley et al., ). et al., 1995)).

The causative virus (PPRV) belongs to the genus
In small ruminants, symptoms of sudden Morbillivirus of the family Paramyxoviridae, closely depression, fever, nasal and ocular discharges, related to the Rinderpest virus of bovines, distemper diarrhea and occasional death are common (Gibbs et virus of domestic and wild carnivores (Murphy et al., al., 1979).Peste des petits ruminants virus needs close 1999).It's also closely related to the human measles contact between infected and susceptible animals to virus and Morbilliviruses of marine mammals spread (Lefevre et al., 1994).In Uganda, goats were (Yayehrad 1997).Sero -conversion to PPRV by goats having severe diarrhea which was not responding to and sheep protects in contact bovines from natural anti-helmentic and antibiotic treatment (P.Panvuga, infection which may also interfere in tissue culture personal communication).In an attempt to know the rinderpest virus (TCRPV) vaccine response exact cause, serum samples from symptomatic goats (Sudharshan et al., 1995).Peste des petits ruminants, were subjected to cELISA test which confirmed PPR also known as goat plague is of increasing importance in Karamoja sub-region.This follow up study was in Africa and Asia where small ruminants form an conducted to investigate the seroprevalence of PPR important component of agricultural food production virus many months after the initial evidence of (OIE, 2002).The virus has been circulating in parts of sub Sahara Africa for several decades and in the antibodies against PPRV in goats within Karamoja Middle East and southern Asia since 1993, although sub-region (MAAIF, 2009).the first description of the virus in India dates way
This virus was first reported in West Africa in the Sampling and sample size determination : There early 1940s (Gargadennec et al.,1942) and was later are approximately one million four hundred and found in Senegal (Gilbert et al,1962) and subsequninety nine thousand nine hundred and six estimated goats in the Districts of Moroto, Nakapiripirit, Abim the case was for the Karamoja region goats.For this and Kotido Districts (MAAIF, 2008).
reason we used end-point titration of PPR to detect PPRV antibodies in the 280 goats sampled.The seroprevalence of PPR in the districts near Briefly, rinderpest virus (RPV) antigen stock those in the current study was recently reported to be was reconstituted by addition of RPV antigen stock in about 36% (Ruhweza et al, 2010).Taking the reference coating buffer (1/100).Therefore, 60ul of RPV stock goat population at 1,499,906 (MAAIF, 2008) and the were put in 6ml of coating buffer.To all ELISA plate background PPRV seroprevalence to be 36% (Ruhweza wells, 50µl of diluted antigen were added.The plates et al., 2010 ) we determined the sample size (n) using Win were then taped to spread the antigen.They were episcope 2.0 software (http://www.clive.ed.ac.uk/ immediately sealed and incubated for one hour at 37 cliveCatalogueItem.asp?id=B6BC9009-C10F-4393-ºC on an orbital shaker.Thereafter, the plates were A22D-48F436516AC4) by setting the confidence at washed three times by filling and emptying the wells 95% and error term at 5% on the sample size with distilled water and blotted to dry on an absorbent determination by percentage tab.The sample size was paper.Enough blocking buffer was prepared the tests calculated as thus; sampling fraction= 0.024, sample to be done; that is, for one plate; 25ml of coating buffer size n = 354, adjusted sample size n (a) = 353, used was mixed with 25µl Tween 20 and 75µl bovine serum value of n = 353 goats.
(Negative serum).40µl of blocking buffer was added There is high level insecurity in the Karamoja sub region due to animal rustling that has for long to each of the wells with an extra 10µl in the defined the lifestyle of the Karamajong and the monoclonal antibody (Mab) control wells (F1, F2, G1 neighboring Kenyan Pastoral tribes.This is one of the and G2).10ul of test serum samples were added to each well in duplicate and 10µl of each control sera, reasons why diseases like PPR still persist in this part that's to say; positive wells B1, B2 and C1, C2, weak of the country.We therefore set out to sample 354 positive D1, D2 and E1, E2, Negative control H1and goats in these districts but we could only access 280 H2.A working dilution of monoclonal antibody was goats during the study period.All goats that we came immediately prepared in the blocking buffer (1:100 across in the protected kraals (all animals are communally dilution i.e. 60µl Mab in 6ml blocking buffer; for one grazed and held in protected kraals in this region) plate) and added to 50µl of Mab in all wells of the plate established to minimize animal rustling were sampled.
except the conjugate controls.Incubation and washing We therefore recognize possible under or over were repeated as previously described.Before the end estimation of PPRV antibodies due to taking slightly of Serum/monoclonal antibody incubation; the less goats than were required but to our knowledge this working dilution of the conjugate in the blocking study sets a stage for more exhaustive studies done buffer sufficient for all micro plates was prepared under more elaborate sampling designs.
(approximately 5 minutes before).For one plate 6µl Blood samples from the jugular vein were taken of the conjugate was added to 6ml blocking buffer.from a total of 280 goats.2mls of serum were obtained Then, to all wells, 50µl of anti-mouse conjugate in from each goat blood sample and subjected to cELISA blocking buffer re added.test to determine the seroprevalence of PPRV in the The resultant preparation was incubated for one four districts (table 1) found in Karamoja sub-region hour at 37 ºC.After the incubation, the plates were (table 1), North Eastern Uganda.By the time sampling washed three times and blotted to dry.Immediately was done, all the animals in the sub-region had not before the end of the conjugate incubation, a working been vaccinated against PPR virus, implying that dilution of the substrate (H O )/chromogen (OPD) was The plates were put on an orbital shaker at room c-ELISA for PPR sero-surveillance is even higher temperature to ensure uniform mixing.All plates were (95.4%) if the target population is non-vaccinated as covered to protect them from excessive light and then This increased the levels of the disease not until the allowed to stand at room temperature for 10 minutes true diagnosis was established.Lack of quarantine of for color development.After color development, the the infected animals (FAO, 1999) as the practice of reactions are stopped by adding 50µl of stopping nomadic pastoralism where the nomads from north solution (1 M sulphuric Acid) to all wells of the test western Kenya, the Turkanas, used to graze from these plate and to the first column of the blanking plate.The districts with their infected animals as pastoralists did plates were now ready for reading using an electro not know anything about clinical signs and spread of photometer at the wavelength of 492nm.
the disease, hence mixing the infected with the non The intensity of the developed color is directly infected ones.Transmission of PPR virus involves proportional to the optical density as read by the close contact between susceptible and infected electro photometer which provided results in form of animals in the febrile stage (Braide, 1981) and percentage inhibitions (PI).
therefore rampant practice of communal grazing and PI = 100 -(Absorbance of the test wells)/(Absorbance watering by pastoralists in Karamoja sub-region, is of the Mab control wells) × 100 one of the reasons for the shooting PPR virus antibody A sample was considered positive if it had a PI levels revealed in this study.The discharges from eyes, value of greater than 50% and it was considered nose, mouth and the loose feaces contain large negative if it had a PI value of less than 50%.amounts of virus.Fine droplets released in air from secretors and excretion, particularly when affected

Results and Discussion
animals cough or sneeze (Taylor., 1984;Badza et al., 1988) and when animals in close contact inhale the Of the 280 serum samples examined for the droplets and are likely to become infected.Abim presence of PPR virus antibodies from Karamoja subdistrict had the lowest seroprevalence of PPR virus region, 160 (57.6%) and 120 (42.4%) serum samples (1.6% -0.01 -3.22) as compared to other districts in were positive and negative for PPRV antibodies Karamoja sub-region this can be explained the fact that respectively.
it is located in the interior of Karamoja Sub-region.The seroprevalences of PPR virus antibodies varied By the time this study was carried out, the between districts of Karamoja sub-region with the disease had not spread much in the interior of Uganda highest seroprevalence recorded in Kotido district and therefore, there is need for a nationwide surveillance 85.2% (CI = 95%, 81.0 -88.9) and the lowest in Abim program to ascertain the extent of spread of the PPR district 1.6% (CI = 95%, -0.01 -3.22) (Table 1 Tanzania also had a prevalence of already spread in the surrounding districts of karamoja 45.8 which also doesn't vary much from the observed et al., sub-region.There is a need to isolate and characterize overall prevalence from this study ( the circulating PPR viruses in Uganda.2009).Explanation for the shooting seroprevalence of PPR in these districts can be due to misdiagnosis of the