Detection of natural Trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of ITS 1 rDNA

BIRYOMUMAISHO, S., MELVILLE, S.E., KATUNGUKA­RWAKISHAYA, E. & LUBEGA, G.W. 2009. Detection of natural Trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of ITS1 rDNA. Onderstepoort Journal of Veterinary Research, 76:285–289 Different species of trypanosomes may infect their mammalian hosts both singly or in combination. This study was undertaken to determine the trypanosome species that may be afflicting pigs in Uganda. Blood was collected from pigs of all ages and sexes from two districts, Kasese in Western and Jinja in Central Uganda. Of the 133 pig blood samples from Kasese that were tested for trypano­ somes using the microhaematocrit centrifugation technique (MHCT), none was found to be infected. However, of the 253 pigs from Jinja district, nine were infected with trypanosomes of which three had T. vivax as determined by MHCT. However, application of the ITS1 rDNA PCR test revealed that eight pigs had T. vivax in mixed infections and one pig had T. vivax monolithic infection. These observa­ tions show that under certain circumstances, pigs may be important reservoirs for, as well as hosts to, T. vivax, contrary to earlier reports.


INTRODUCTION
Trypanosomes remain a constant threat to the lives of humans, cattle and other domesticated animals, throughout large regions of Africa and South Amer ica.In Africa, the disease in domestic animals is mainly caused by T. congolense and T. vivax, with T. congolense being the most pathogenic species in East Africa and T. vivax in West Africa (Kaaya, Win qvist & Johnson 1977).Methods of screening ani mals for trypanosomosis for epidemiological studies include direct microscopic observation of parasites in the buffy coats following concentration of blood using the microhaematocrit centrifugation technique (MHCT), or serological tests that include the com plement fixation test and the card agglutination test (Claes, Ilgekbayeya, Verloo, Saidouldin, Geerts, Buscher & Goddeeris 2005).However, recently a variety of polymerase chain reaction (PCR)based techniques have been proposed.Unlike the sero logical tests, PCR methods have largely overcome difficulties of sensitivity and specificity associated with direct parasite examination.
A number of PCR primers have been developed for detection of species and subspecies of T. congo lense (Savannah) (Masiga, Smyth, Hayes, Bromidge & Gibson 1992); T. congolense (Kilifi) (Masiga et al. 1992) and T. vivax (Masake, Majiwa, Moloo, Makau, Njuguna, Maina, Kabata, JoleMoiYoi, & Nantulya 1994).Since these are speciesspecific tests, sev eral primer sets would be required to test each sam ple for all species of trypanosomes.Attempts have been made to develop a single molecular marker to accurately distinguish between species in a single PCR test using the internal transcribed spacers (ITS) located within the ribosomal RNA genes, both for trypanosomes and a number of other organisms (Schlotter, Hauser, von Haeseler & Tautz 1994) and Samuel (1998).
It has been proposed previously that the principal hosts of T. vivax are cattle, sheep, goats, horses and camels, but not dogs and pigs (Losos 1986).Stephen (1986) suggested that pigs are refractory to T. vivax infection since when he challenged pigs with wildcaught tsetse flies infected with T. vivax and T. congolense in a ratio 2:1, no T. vivax para sites were subsequently detected in the blood of these animals.However, a recent study by Ng'ayo, Ngiru, Eucharia, Muluvi, Osir & Masiga (2005) in western Kenya showed that pigs can, indeed, be infected with T. vivax.In their study intended to de termine the animal reservoirs of human sleeping sickness, of the ten pigs infected with trypano somes, three carried T. brucei and five had T. vivax.Similarly, in a study of trypanosome molecular dif ferential diagnosis in livestock in South Western Uganda, Balyeidhusa, Enyaru, Matovu, Nerima, Akol & Sebikali (2006) detected three T. vivax infec tions in 22 pigs.
The present study, therefore, was designed to con duct further investigations of the trypanosome spe cies that affect pigs reared in two distinct agroeco logical zones in Uganda.

Collection and handling of pig blood samples
Blood was collected from pigs of all ages and both sexes on smallholder farms in the districts of West ern (Kasese) and Central (Jinja) Uganda.Fewer pigs than cattle were kept by farmers in the two districts; therefore, all pigs that were reared in villages where the cattle and goats were bled, were also bled.A total of 133 and 253 pigs were bled from Kasese and Jinja districts, respectively.In larger pigs (weigh ing more than 50 kg) blood was obtained from the marginal ear vein and in smaller pigs, from the ante rior vena cava or the cutaneous abdominal vein.Two to five millilitres of blood were dispensed into EDTAcoated collection tubes.Of this blood, 20 µℓ was spotted onto Whatman FTA cards.The remain ing blood was used for preparation of microhaema tocrit tubes for subsequent centrifugation and exam ination for trypanosomes as described by Mur ray, Mur ray & McIntyre (1977).

Speciation of trypanosomes with microhaematocrit centrifugation/dark ground microscopy technique (MHCT)
Capillary tubes were centrifuged at 1 020 g for 5 min and the presence of trypanosomes detected by ob servation of parasite motion just above the buffy coat.In a sample where trypanosomes were detect ed, the microhaematocrit tube was cut just below the buffy coat to include 1 mm of the erythrocyte layer, and the contents including about 1 cm of plas ma were transferred to a glass slide and covered with a cover slip.The wet smear was observed for trypanosomes under the microscope with reduced illumination and classified into species basing on their movements whereby T. congolense vibrates without progressing, T. brucei swims and T. vivax moves fast across the field (Radostatis, Gay, Blood & Hinchcliff 2000).

Extraction of DNA from blood spotted on FTA cards
In this study, DNA was extracted from trypanosome positive pigs because we intended to identify sam ples for further molecular studies (results not pre sented in this paper).DNA was extracted from dry blood spotted on FTA cards using the QIAamp® mini kit (2003) protocol and subsequently used for speciation of infecting trypanosomes by PCR ampli fication of ITS1 rDNA.Briefly, three or four 2 mm diameter circles of whole dried blood spot were punched out of the FTA cards and placed in a 1.5 mℓ microfuge tube, covered with cell lysis buffer and incubated at 85 °C for 10 min.Genomic DNA was extracted by adding proteinase K and incubating the extract at 56 °C for 1 h.The DNA was precipi tated with ethanol and subsequently eluted in 110 µℓ of buffer AE, aliquoted and stored at -20 °C until re quired for use as template DNA for testing individual DNA extracts for infecting trypanosome species by ITS1 rDNA PCR.

Amplification of the rDNA intergenic sequence by the polymerase chain reaction (ITS1-PCR)
The 25 µℓ total volume PCR mix contained 2.5 µℓ of 10x PCR buffer (15 mM MgCl 2, 0.25 µℓ of each10 mM dNTP solution), 0.5 µℓ containing 10 mM (10x mix ture) of the forward and reverse ITS1 rDNA primers, 0.125 µℓ of HotStar Taq DNA polymerase and 5 µℓ of the DNA extracted from FTA blood spots.The ITS1 rDNA primers used were: • Forward (CF) 5'CCGGAAGTTCACCGATATTG • Reverse (BR), 5'TTGCTGCGTTCTTCAACGAA Reactions were performed in a PTC100 Peltier Thermal Cycler which was programmed as follows: lid preheating at 96 °C for 15 min, followed by 38 cycles each consisting of denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min and extension at 72 °C for 90 s.The mixture was incubated at 72 °C for 10 min for the final extension step.The products were left at 4 °C in the thermocycler pending electro phoresis in a 1.5 % agarose gel containing 0.5 µg/mℓ ethidium bromide and flooded with 1x TBE buffer.Electrophoresis was performed at 55 V for 30 min.

The positive control trypanosome DNA
The T. brucei positive control DNA was extracted from the procyclic form of strain 927 (T.brucei TREU 927/4 PAL/KE/70/EATRO 1534).Trypano soma con golense and T. vivax DNA were extracted from strains IL 3000 and IL 2569, respectively, which were kindly provided by Dr Phelix Majiwa.

Identification of infecting trypanosomes using MHCT and ITS1 rDNA PCR
The results of MHCT trypanosome speciation from pig blood samples (Table 1) based on movement characteristics of trypanosomes showed three spe cies, namely, T. brucei, T. congolense and T. vivax.The movement characteristics of trypanosomes from pig no.128 were not characteristic of any of the three species; hence the infecting trypanosomes species were not assigned.All infections were confirmed by retesting with ITS1 rDNA PCR.
Species identification using the ITS1 rDNA PCR anal ysis detected more species of trypanosomes in individual samples compared to the MHCT except for pig number 164.Trypanosoma vivax was detect ed by both techniques; T. brucei was the most com mon trypanosome and was detected in eight of the nine samples by ITS1 rDNA PCR, mainly in mixed infections.The MHCT detected only three mixed in fections compared to eight with the molecular tech nique.
Determination of the trypanosome species by PCR analysis was achieved by comparing individual PCR amplicons with the corresponding band size of the positive control (Tb, Tc and Tv) bands (Fig. 1).Pres ence of more than one band denoted presence of mixed trypanosome infections while single bands denoted monolithic infections.Of the nine pig sam ples, three showed banding patterns corresponding Although agarose gels as used here cannot unequi vocally resolve band sizes that vary by less than 100 bp (to distinguish between the T. congolense subgroups), Fig. 1 clearly shows that each band ob tained from the Ugandan isolates corresponded in size with one of the positive control samples.

DISCUSSION
The lack of sensitive and suitable diagnostic tests hinders the collection of accurate epidemiological data and institution of control programmes for live stock diseases.This study focused on accurate de tection of parasites and how various species of trypanosomes could simultaneously be detected from blood samples of porcine hosts.Notable was the detection of T. vivax in pigs that have hitherto been reported to be refractory to the infection.
There have been previous reports of T. vivax not being a parasite of pigs (Losos 1986;Stephen 1986;Desquesnes & Davilla 2002), but rather of cattle as the principal hosts.To date, pigs have always been excluded from trypanosomosis control exercises (personal obser vations), but these data suggest it may be neces sary to treat pigs in areas where prevalence of cat tle trypanosomosis is relatively high (exceeding 10 % prevalence).At present we do not know to what extent infection with T. vivax causes symptomatic disease in pigs.It is possible that pathology is less severe but that it plays a more important role as a reservoir for infec tion of tsetse and onward transmission to more sus ceptible species, thus aiding the maintenance of this parasite in the ecosystem.Future clinical pa thology studies could be helpful in defining the im portance of infection of pigs.

FIG. 1
FIG. 1 Agarose gel (1.5 % w/v) banding pattern after amplification of ITS1 rDNA of trypano somes from pigs bled in Jinja district, central Uganda.M = Lambda ladder size marker (100 base pairs); Tb = T. brucei; Tc = T. congolense; Tv = T. vivax; ve = negative con trol (i.e. containing no DNA).The numbers assigned to each amplified DNA sample on the gel reflect the order in which pigs were bled

TABLE 1
Speciation of trypanosomes isolated from pigs in Jinja district to mixed trypanosome infections comprising of T. vivax, T. congolense and T. vivax; four had T. vivax and T. brucei; one T.brucei and T. congolense while one is T. vivax monolithic infection.