Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples

We developed a TaqMan real-time polymerase chain reaction (PCR) assay for the quantitative detection of Theileria equi from the in vitro-cultured parasite and field blood samples collected from horses living in Ghana and Brazil. The detection limit for the assay was determined to be 1.5 parasites/microl per sample, and the quantitative capacity was demonstrated using the in vitro-cultured parasite. For field applications, the real-time PCR assay was compared to a previously established nested PCR assay used as the gold standard for the real-time PCR assay. Of 65 field blood samples, 46 samples were T. equi-positive in the nested PCR assay, while the real-time PCR assay also detected the parasite in all 46 of the nested PCR-positive samples but did not detect T. equi in the remaining 19 negative blood samples. This quantitative real-time PCR assay provides a valuable tool for fast laboratory diagnostic assessment of T. equi infection in horses.     

Development of two immunochromatographic tests for the serodiagnosis of bovine babesiosis

In this study, we developed two immunochromatographic tests (ICTs), which are nitrocellulose membrane-based immunoassays for the convenient and rapid serodiagnosis of bovine babesiosis caused by Babesia bovis (BoICT) and Babesia bigemina (BiICT). The efficacy of two ICTs was evaluated using 13 positive sera from experimentally infected cattle with B. bovis or B. bigemina. Clear results showed that the BoICT and ELISA detected antibodies in sera collected from 14 to 93 days post-infection, while BiICT and ELISA detected from 13 to 274 days post-infection. In additon, non-infected cattle, Neospora caninum, and Cryptosporidium parvum were negative in two ICTs. To evaluate the field utility of the ICTs, we tested 186 field bovine sera collected from cattle living in Yanbian (China) and Mato Grosso do Sul (Brazil). The results of ICTs were compared to those of classical serodiagnostic methods, enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence assay (IFAT). The overall concordances of BoICT were determined as 92.5 and 90.3% when the results of ELISA and IFAT were set as the reference standards, respectively. In contrast, those of BiICT showed 96.8 and 92.5% relative to the results of standard ELISA and IFAT, respectively. Conventional and rapid diagnosic devices for bovine babesiosis may provide a valuable tool in clinical and field applications.     

Comparison of polymerase chain reaction methods for the detection of <Emphasis Type="Italic">Theileria equi</Emphasis> infection using whole blood compared with pre-extracted DNA samples as PCR templates

Rapid, efficient, and reproducible procedures for isolating DNA before PCR gene amplification are essential for the diagnosis of piroplasms. In this study, we evaluated the ease and reliability of detecting Theileria equi by PCR using pre-extracted DNA samples (by QIAamp DNA Mini Kit and phenol-chloroform methods) compared with blood spotted on FTA cards as PCR templates. Although minimal variations in limit of detection were observed among the methods compared, overall, the use of pre-extracted DNA samples and blood spotted on FTA cards had comparable detection limits. These results indicate that T. equi infection can be efficiently detected directly from FTA cards by PCR without the need for pre-extraction of DNA from blood samples.     

The Effect of Intercropping Lablab purpureus L. with Sorghum on Yield and Chemical Composition of Fodder

In two years the growth and composition of mixtures of sorghum with Lablab purpureus as strips, paired rows and alternate rows were compared with those of sorghum and lablab monocrops. In the first year, when sowing did not occur until 7 August, the sorghum yields were low in the monocrop and even less in the mixed crops. Lablab yield was also reduced in mixtures compared with the monocrop, but total forage yield was greater for the mixed crops compared with sorghum alone. Of the mixed cropping systems, a reduction in the yield of lablab plants and in the phosphorus and potassium content of shed lablab leaves in paired rows suggested that there was more competition for nutrients between lablab plants grown in this treatment. In the second year, earlier sowing increased sorghum growth at the expense of lablab yield in the mixed cropping systems, with the result that total forage yield was not increased when sorghum was intercropped with lablab. However, the crude protein content of sorghum stems and leaf yield were increased in mixed crops, particularly in paired and alternate rows rather than strips, demonstrating that close configuration of the legume and cereal are necessary for the cereal to obtain most benefit from nitrogen fixed by the legume. It is concluded that, when conditions are favourable for rapid sorghum and lablab growth, the sorghum will benefit more when it is grown in paired rows with lablab rather than in strips. However, the close spacing normally adopted for paired rows may encourage competition between lablab plants and increase the requirements for phosphorus and potassium fertilizer.     

Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5%), 20/21 (95.2%) and 42/42 (100%) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2%), 13/21 (61.9%) and 30/42 (71.4%) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.     

The Effects of Timing of an Interim Harvest on the Yield and Composition of Lablab purpureus

Lablab purpureus was grown in two field experiments in consecutive years to evaluate the effects of the timing of an interim harvest on the yield and nutritive value of the harvested material and the subsequent regrowth, which was determined from the second and final harvest. Delaying the first harvest to 50–70 days post-sowing increased the total (first plus second harvests) dry matter (DM) yield, with a greater first harvest and reduced second harvest DM yield. The delay also reduced the crude protein concentration of the first harvest and tended to increase its modified acid-detergent (MAD) fibre concentrations. The delay increased the crude protein concentration and decreased the MAD fibre concentration of the second harvest. The total crude protein yield of both harvests increased with late interim harvesting. The first harvest plant calcium concentration increased and phosphorus concentration decreased with a delay in the interim harvest. It is concluded that, in the difficult growing conditions of the Sahelian zone of sub-Saharan Africa, delaying the interim harvest of Lablab purpureus until 50–70 days post-sowing will have beneficial effects on total dry matter and crude protein yields.     

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood

With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.