Characterisation of the gene encoding a 104-kilodalton micronemerhoptry protein of Theileria parva

Iams, Keith P.; Young, J. R.; Nene, Vishvanath; Desai, Jagruti; Webster, Paul; ole-MoiYoi, Onesmo K.; Musoke, A.J.

doi:10.1016/0166-6851(90)90007-9

Cited by 48 publications

(25 citation statements)

References 33 publications

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“…The nested PCR (nPCR) amplifications were performed in a total volume of 25 µl containing 0.25 units of Taq DNA polymerase, (Promega), 1X PCR buffer (500 mM KCl/ 100 mM Tris-HCl [pH 8.3]/0.01% (w/v) gelatin), 200 mM of each dNTP and 25 ng of primers and 1.5 mM of MgCl 2 . The sequences of the nested forward and reverse primers were 5′-GGC CAA GGT CTC CTT CAG ATT ACG-3′ and 5′-TGG GTG TGT TTC CTC GTC ATC TGC-3′, respectively, and were used to amplify a 277-bp internal fragment located between bases 2784 and 3061 of the p104 gene (Iams et al 1990). Cycling profile conditions for the secondary PCR were as described for the primary amplification (Skilton et al 2002) except that the reaction was run for 30 cycles, and the annealing temperature was 55°C (Odongo et al 2009).…”

Section: Single Primer Pair P104 Pcr Combined With Hybridizationmentioning

confidence: 99%

“…Recently, speciesspecific and PCR-based RLB hybridization methods have been developed for the detection and identification of Theileria and Babesia species (Gubbels et al 1999), and the technique has been applied to simultaneously detect and differentiate tick-borne protozoan parasites in the blood of carrier cattle (Oura et al 2004;Altay et al 2008). The p104 PCR assay (Skilton et al 2002) is a T. parva speciesspecific assay based on primers derived from conserved region of a 104-kDa antigen gene (p104) of T. parva (Iams et al 1990;Skilton et al 2002). This assay was previously extensively validated and shown to be sensitive with a detection threshold of 2 parasites/µl of infected blood (Skilton et al 2002).…”

Section: Introductionmentioning

confidence: 99%

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A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals

et al. 2009

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Theileria parva causes East Coast fever, an economically important disease of cattle in sub-Saharan Africa. We describe a nested polymerase chain reaction (nPCR) assay for the detection of T. parva DNA in cattle blood spotted onto filter paper using primers derived from the T. parva-specific 104-kDa antigen (p104) gene. The sensitivity of this assay was compared to a previously described p104-based PCR and also the reverse line blot (RLB) technique, using serial dilutions of blood from a calf with known T. parva piroplasm parasitaemia. The relative sensitivities of the three assays were 0.4, 1.4 and 4 parasites/microl corresponding to blood parasitaemias of 9.2 x 10(-6)%, 2.8 x 10(-5)% and 8.3 x 10(-5)%, respectively. The three assays were applied to samples from two calves infected with the T. parva Muguga stock. Parasite DNA was consistently detectable by the two p104 PCR assays until 48 and 82 days post-infection, respectively, and thereafter sporadically. RLB detected parasite DNA in the two infected calves until days 43 and 45. Field samples from 151 Kenyan cattle exhibited 37.7% positivity for T. parva by regular p104 PCR and 42.3% positivity using p104 nPCR. Among 169 cattle blood samples from Southern Sudan, 36% were positive for T. parva using nPCR. The nPCR assay represents a highly sensitive tool for detection and monitoring of asymptomatic carrier state infections of T. parva in the blood of cattle.

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Section: Single Primer Pair P104 Pcr Combined With Hybridizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals

et al. 2009

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“…Determination of T. parva Infection Levels in Infected Bovine Blood, Engorged Nymphs, and Adult Salivary Glands. Before quantifying the level of T. parva in the blood, engorged nymphs, and the salivary gland of adult ticks, we determined the presence of the parasite genomic DNA by nested PCR (nPCR) targeting the single copy gene encoding the p104 antigen (GenBank accession M29954) (Iams et al 1990). PCR conditions and the primers in the primary reaction mixture were as described previously (Skilton et al 2002).…”

Section: Methodsmentioning

confidence: 99%

“…The data seem to show both differences in susceptibility (proportion of ticks that acquire infection) and in efÞciency (the number of infected acini within an infected tick) between these two tick strains (Young et al 1995). To make a more detailed comparison of the pathogenesis of infection in these two strains we have developed a quantitative real-time PCR (qPCR) assay based on the gene encoding T. parva p104-kDa antigen (Iams et al 1990) for detection and quantiÞcation of T. parva in guts of engorged nymphs and dissected salivary glands of adult ticks. The data conÞrm that the Kiambu tick strain is highly susceptible to salivary gland infection, whereas the Muguga tick strain is signiÞcantly less susceptible, and provide the Þrst quantitative evidence that this may be related to events in the gut.…”

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confidence: 99%

Quantification of <I>Theileria parva</I> in <I>Rhipicephalus appendiculatus</I> (Acari: Ixodidae) Confirms Differences in Infection Between Selected Tick Strains

Odongo

Ueti

Mwaura

et al. 2009

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Theileria parva is the etiologic agent of East Coast fever, an economically important disease of cattle in sub-Saharan Africa. This protozoan parasite is biologically transmitted by Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae). An understanding of the vector-parasite interaction may aid the development of improved methods for controlling transmission. We developed quantitative polymerase chain reaction (qPCR) and nested PCR (nPCR) assays targeting the T. parva-specific p104 gene to study T. parva pathogenesis in two strains of R. appendiculatus that had previously been selected to be relatively more (Kiambu) or less (Muguga) susceptible to infection. Nymphs from both strains were fed simultaneously to repletion on acutely infected calves. Nymphs from the Kiambu strain showed significantly higher engorgement weights compared with Muguga strain nymphs. Immediately after engorgement qPCR confirmed that nymphal Kiambu ticks had significantly higher parasite loads at repletion than Muguga nymphs. By 12 d postengorgement, parasites were below quantifiable levels but could be detected by nPCR in 83-87% (Muguga and Kiambu, respectively) of nymphs. After the molt, adult feeding on naïve cattle stimulated parasite replication in the salivary glands. PCR detected significantly more infected ticks than microscopy, and there was a significant difference between the two tick strains both in the proportion of ticks that develop salivary gland infections, and in the number of parasites within infected salivary glands. These data confirm that although both tick strains were competent vectors, Kiambu is both a significantly more susceptible and a more efficient host for T. parva than Muguga. The mechanisms that contribute to the levels of susceptibility and efficiency are unknown; however, this study lays the groundwork for a comparison of the transcriptome of these tick strains, the next step toward discovering the genes involved in the tick-parasite interaction.

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“…The nested p104 polymerase chain reaction (nPCR) was used to screen all field samples for the presence of T. parva. Primers derived from the T. parva-specific 104-KDa antigen (p104) gene were used in the PCR amplification as previously described by Odongo et al (2010) and Iams et al (1990). The sequences of the forward and reverse primers were 5'ATT TAA GGA ACC TGA CGT GAC TGC 3' and 5' TAA GAT GCC GAC TAT TAA TGA CAC C 3', respectively, for first the round and 5' GGC CAA GGT CTC CTT CAG AAT ACG3' and 5'TGG GTG TGT TTC CTC GTC ATC TGC 3', respectively, for second the round.…”

Section: Genomic Dna Extraction and Detection Of Theileria Parvamentioning

confidence: 99%

Tick burden and acquisition of immunity to Theileria parva by Tarime cattle in comparison to Sukuma cattle under different tick control regimes in the Lake Zone of Tanzania

Laisser¹,

Chenyambuga²,

Karimuribo³

et al. 2016

J. Vet. Med. Anim. Health

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This study was conducted to determine tick burden and immunological parameters of resistance to East Coast fever (ECF) in Tarime and Sukuma cattle. Tick load, packed cell volume (PCV), Theileria parva (T. parva) specific antibody percent positivity (PP), and prevalence of T. parva parasites were studied in relation to dipping regime, strains, and season. A total of 50 experimental cattle were included in this study. Tick load was determined by whole body counts, antibody percent positivity was determined by the polymorphic immunodominant molecule (PIM)-based T. parva enzyme-linked immunosorbent assay (ELISA), and prevalence of T. parva parasites was detected by a nested polymerase chain reaction (PCR) based on the p104 gene. Dipping frequency on tick burden showed no statistically significant differences when cattle of either strain were dipped either once every 2 or 3 weeks in the dry and wet seasons. However, Tarime cattle had higher (p<0.05) tick count than Sukuma cattle and non dipped groups maintained high tick infestation throughout the experimental period. The PCV values were within the physiological range, although this parameter was lower in Tarime cattle (p<0.05). All cattle regardless of strain were seropositive, although Tarime cattle maintained higher PP compared to Sukuma by 15%. Conversely, the prevalence of T. parva parasites was lower in Tarime (38%) compared to Sukuma cattle (38.5%), but the difference was not significant (p>0.05). During the study period, 20% (5/25) of Sukuma cattle contracted ECF, but none of the Tarime cattle showed clinical signs for the disease. The differences between the two strains shown in terms of PP and T. parva parasite prevalence may indicate the ability of individual cattle to resist tick infestation and ECF infection under natural challenge. Higher antibody levels but lower parasite prevalence attained by Tarime cattle, suggests inherent ability of Tarime cattle to resist clinical development of ECF infection, but to remain as T. parva carriers.

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Characterisation of the gene encoding a 104-kilodalton micronemerhoptry protein of Theileria parva

Cited by 48 publications

References 33 publications

A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals

A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals

Quantification of <I>Theileria parva</I> in <I>Rhipicephalus appendiculatus</I> (Acari: Ixodidae) Confirms Differences in Infection Between Selected Tick Strains

Tick burden and acquisition of immunity to Theileria parva by Tarime cattle in comparison to Sukuma cattle under different tick control regimes in the Lake Zone of Tanzania

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