Search

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.

Staphylococcus aureus is an opportunistic bacterium known to cause severe infections in humans and animals. It is one of the major bacteria causing subclinical and clinical mastitis, leading to significant economic losses in livestock industry. In this study, we have isolated and characterized 80 S. aureus clinical isolates from mastitis-infected animals. The analysis of antimicrobial susceptibility, molecular typing, biofilm production and genetic determinants was performed to understand molecular and phenotypic features of the prevalent pathogen. Our antibiotic susceptibility assays showed the majority (57.5%) of isolates to be multidrug-resistant (MDR), 38.75% resistant and 3.75% sensitive. We found 25% isolates to be methicillin-resistant S. aureus (MRSA) based on oxacillin susceptibility assays. In the MRSA group, maximum isolates (95%) were MDR compared to 45% in MSSA. Multilocus sequence typing (MLST) revealed 15 different STs; ST-97 was the most common ST, followed by ST-2459, ST-1, ST-9 and ST-72. The agr typing showed agr-I as the most common type, followed by type II and III. Most isolates developed biofilms, which ranged in intensity from strong to weak. The presence or absence of lukS, a virulence-related gene, was found to have a substantial relationship with the biofilm phenotype. However, no significant association was found between biofilm formation and antimicrobial resistance or other virulence genes. We also found four MRSA isolates that were mecA negative based on molecular assays. Our findings reveal the prevalence of multidrug-resistant S. aureus clinical isolates in India that are biofilm positive and have critical genetic factors for disease pathogenesis causing bovine mastitis. This study emphasizes the need for the comprehensive surveillance of S. aureus and other mastitis-causing pathogens to control the disease effectively.

Tropical theileriosis caused by Theileria annulata infection is a significant livestock disease affecting cattle health and productivity resulting in substantial monetary losses in several countries. Despite the use of an effective vaccine for disease control still, a high incidence of infection is reported from India. One of the many reasons behind the ineffective disease control can be the existence of genetically diverse T. annulata parasite population in India. Therefore, studies focusing on understanding the genotypes are warranted. In this study, we have performed a genetic analysis of the Indian T. annulata field cell lines and the vaccine line using microsatellite markers, Genotyping based sequencing (GBS) and tams1 gene polymorphism. The degree of allelic diversity and multiplicity of the infection was determined to be high in the Indian population. No geographical sub-structuring and linkage disequilibrium were observed in the population. High population diversity was found which were similar with countries like Oman, Tunisia, and Turkey in contrast to Portugal and China. The presence of multiple genotypes as determined by microsatellite marker genotyping, GBS analysis and tams1 gene polymorphism point toward a panmictic parasite population in India. These findings are the first report from India which would help in understanding the evolution and diversity of the T. annulata population in the country and can help in designing more effective strategies for controlling the disease.

Plasmodium, Toxoplasma, Cryptosporidium, Babesia, and Theileria are the major apicomplexan parasites affecting humans or animals worldwide. These pathogens represent an excellent example of host manipulators who can overturn host signaling pathways for their survival. They infect different types of host cells and take charge of the host machinery to gain nutrients and prevent itself from host attack. The mechanisms by which these pathogens modulate the host signaling pathways are well studied for Plasmodium, Toxoplasma, Cryptosporidium, and Theileria, except for limited studies on Babesia. Theileria is a unique pathogen taking into account the way it modulates host cell transformation, resulting in its clonal expansion. These parasites majorly modulate similar host signaling pathways, however, the disease outcome and effect is different among them. In this review, we discuss the approaches of these apicomplexan to manipulate the host–parasite clearance pathways during infection, invasion, survival, and egress.