Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis.

Accurate quantification based on nucleic acid amplification is necessary to avoid the spread of pathogens, making early diagnosis essential. Droplet digital PCR (ddPCR) stands out for absolute parasite quantification because it combines microfluidics with the TaqMan test. This helps deliver maximum accuracy without needing a reference curve. This study assessed the efficacy of ddPCR as a detection tool for the bovine theileriosis (BT) caused by Theileria parasites. We developed and validated a duplex ddPCR method that detects and quantifies the Theileria genus (18S rRNA) and identifies clinically significant Theileria annulata parasites (TaSP) in experimental and clinical samples. ddPCR was shown to be as effective as qPCR throughout a 10-fold sample dilution range. However, ddPCR was more sensitive than qPCR at lower parasite DNA concentrations and reliably assessed up to 8.5 copies/µL of the TaSP gene in the infected DNA (0.01 ng) samples. The ddPCR was very accurate and reproducible, and it could follow therapeutic success in clinical cases of theileriosis. In conclusion, our ddPCR assays were highly sensitive and precise, providing a valuable resource for the study of absolute parasite quantification, drug treatment monitoring, epidemiological research, large-scale screening, and the identification of asymptomatic parasite reservoirs in the pursuit of BT eradication.

Population Genetic Analysis of the Theileria annulata Parasites Identified Limited Diversity and Multiplicity of Infection in the Vaccine From India.

Background: Apicomplexan parasite Theileria annulata causes significant economic loss to the livestock industry in India and other tropical countries. In India, parasite control is mainly dependent on the live attenuated schizont vaccine and the drug buparvaquone. For effective disease control, it is essential to study the population structure and genetic diversity of the Theileria annulata field isolates and vaccine currently used in India. Methodology/Results: A total of 125 T. annulata isolates were genotyped using 10 microsatellite markers from four states belonging to different geographical locations of India. Limited genetic diversity was observed in the vaccine isolates when compared to the parasites in the field; a level of geographical substructuring was evident in India. The number of genotypes observed per infection was highest in India when compared to other endemic countries, suggesting high transmission intensity and abundance of ticks in the country. A reduced panel of four markers can be used for future studies in these for surveillance of the T. annulata parasites in India. Conclusion: High genetic variation between the parasite populations in the country suggests their successful spread in the field and could hamper the disease control programs. Our findings provide the baseline data for the diversity and population structure of T. annulata parasites from India. The low diversity in the vaccine advocates improving the current vaccine, possibly by increasing its heterozygosity. The reduced panel of the markers identified in this study will be helpful in monitoring parasite and its reintroduction after Theileria eradication.

A Real-Time PCR based assay for determining parasite to host ratio and parasitaemia in the clinical samples of Bovine Theileriosis.

Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field.

Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization.

Contagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG's and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at âˆ¼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

Hematobiochemical alterations and direct blood polymerase chain reaction detection of Theileria annulata in naturally infected crossbred cows.

AIM: The aim was to determine hemato-biochemical changes and rapid diagnosis of Theileria annulata in naturally infected crossbred cows. MATERIALS AND METHODS: Blood samples from lactating crossbred cows (n=40) between 3 and 7 years of age and showing clinical signs of tropical theileriosis were collected, with or without anticoagulant, and analyzed for tropical theileriosis by direct smear, direct blood polymerase chain reaction (PCR) detection of merozoite-piroplasm surface antigen (Tams1) gene specific amplicon, estimation of hematological and biochemical parameters. Healthy crossbred cows (n=6), examined free from hemoprotozoan infections were included as control. RESULTS: The infected crossbred cows revealed significantly (p<0.001) lower values of total erythrocytic counts (4.46±0.2 × 10(6)/µL), hemoglobin (Hb 6.025±0.39 g%), packed cell volume (17.05±1.1%), mean corpuscular volume (37.94±1.70 fL) and mean corpuscular Hb (13.5±0.48 pg; p<0.002) compared with healthy control. The serum samples of infected cows revealed profound (p<0.05) hyponatremia (Na 133.21±2.36 mEq/l) and hypocalcemia (Ca 8.39±0.34 mg%). Infected crossbred cows showed a significant increase (p<0.05) of mean serum activity of alanine aminotransferase (61.45±13.36 U/L), aspartate aminotransferase (146.1±20.97 U/L), blood urea nitrogen (28.26±3.90 mg%), creatinine (1.55±0.13 mg%), direct bilirubin (0.33±0.04 mg%; p<0.001) and lactate dehydrogenase (3001.32±167.0 U/L; p<001). Blood direct PCR revealed a 721-bp fragment amplified from the target gene encoding 30-kDa major merozoite surface antigen of T. annulata using specific primer pairs. This assay was positive for all the infected animals. CONCLUSION: The assessments of hemato-biochemical parameters in T. annulata infected crossbred cows may be useful in understanding disease pathogenesis, prognosis and corrective measures for supportive therapy. Moreover, blood direct PCR can reliably be used for rapid detection of T. annulata in conjunction with microscopic examination.

A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples.

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T(m) of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID(50)). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.