Veterinary parasitic diseases remain a major threat to livestock production, companion animal health, wildlife conservation, and public health worldwide. Conventional diagnostic methods, including microscopy, serology, and culture-based techniques, often suffer from limited sensitivity, poor specificity, prolonged turnaround time, and dependence on specialized laboratory facilities. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based diagnostics have introduced highly sensitive, rapid, and field-deployable platforms for parasite detection. This review critically summarizes the principles and recent applications of CRISPR/Cas12, Cas13, and emerging Cas14-based diagnostic approaches in veterinary parasitology, covering protozoan, helminthic, and arthropod-borne parasites. It further compares CRISPR platforms and discusses their integration with isothermal amplification, lateral flow assays, fluorescence detection, and portable biosensors to improve diagnostic performance. Moreover, current challenges, including sample preparation, assay standardization, multiplex detection, regulatory approval, and commercialization, are highlighted. Finally, future perspectives on amplification-free CRISPR assays, artificial intelligence-assisted diagnostics, and point-of-care platforms are discussed. By providing a translational perspective from laboratory innovation to field application, this review highlights the potential of CRISPR-based diagnostics to transform veterinary parasite surveillance and disease control.
Leishmania (Mundinia) species have attracted increasing attention due to their ancestral phylogenetic position, broad distribution, and ability to exploit non-sand fly vectors. However, the structure and immunomodulatory role of their surface glycoconjugates remain poorly understood. We comparatively characterized the biochemical and functional properties of glycoconjugates from representative Mundinia species during interaction with murine macrophages. Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) were extracted, purified, and preliminary characterized from Leishmania enriettii, L. orientalis, L. martiniquensis, and L. macropodum. Immunoblotting analysis of LPG using MG1 antibody detected different levels of glucose side chains branching-off the repeat units of Mundinia species, except L. enriettii. In contrast, GIPLs were conserved and galactosylated (suggestive of type II) by fluorophore-assisted carbohydrate electrophoresis (FACE). Structural polymorphisms were assessed in relation to nitric oxide (NO) production by murine macrophages, including TLR2- and TLR4-dependent responses. Both glycoconjugates induced variable levels of NO production via TLR4, with secondary involvement of TLR2. Although we found polymorphisms in the sugar moieties, a clear correlation between structure and activity could not be found.
The phylum Apicomplexa includes major protozoan pathogens of humans and animals (Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium parvum, Babesia spp., Eimeria spp., and Neospora caninum) with substantial global health and economic impact. Resistance to key chemotherapeutics, including the partner and antifolate drugs used against several of these diseases, is narrowing a limited arsenal of target-specific treatments, creating an urgent need for mechanistically distinct drug targets. Proteolytic enzymes are attractive candidates, yet antiprotease drug discovery in the Apicomplexa has concentrated on aspartyl and cysteine proteases, leaving zinc metalloproteases (ZMPs) comparatively neglected despite non-redundant roles in haemoglobin digestion, invasion, egress, organelle biogenesis, and immune evasion. This review synthesises the biology, conservation, and drug-target status of apicomplexan ZMPs (MEROPS families M1, M16C, M17, M18, M24, M41) across the six species above. Catalytic-domain conservation, genetic and pharmacological validation status, and inhibitor pharmacology (hydroxamic acid, phosphinic acid, bestatin, amino-benzosuberone, and peptide-based scaffolds) are compared family-by-family. A multi-criteria druggability framework identifies the M17 leucyl aminopeptidase and apicoplast-resident FtsH1 (M41) as the most compelling pan-apicomplexan targets, reflecting genetic essentiality, chemical tractability, and host selectivity, while the expanded M16C insulinase family of C. parvum illustrates a lineage-specific opportunity. Principal gaps include sparse genetic validation outside P. falciparum and T. gondii, near-total absence of experimental structures beyond P. falciparum, and no ZMP inhibitor yet advanced to in vivo or clinical testing. This synthesis aims to advance apicomplexan zinc metalloproteases from an underexplored genomic feature toward a validated class of antiparasitic drug targets.
Methylglyoxal (MG), a toxic metabolite of the glycolysis pathway, is converted to D-lactate in the Leishmania parasite. Our previous study shows the importance of inorganic phosphate (Pi) in MG synthesis and its intracellular accumulation. No microscopic signs of adverse effects were observed in the Leishmania parasite at high MG concentrations. We hypothesize that under hypophosphatemic (Pi-deficient) conditions, D-lactate is converted to pyruvate with the help of a functionally uncharacterized lactate dehydrogenase (LDH) like protein in the Leishmania parasite. Under the Pi-deficient state, the glycolytic pathway was skewed towards the MG production from glyceraldehyde-3-phosphate. Increased glyoxalase (GLO)-I and II, LDH-like protein, and pyruvate concentrations were found under hypophosphatemic conditions. Antimony (Sb) is toxic to the Leishmania parasite by disrupting thiol homeostasis. Sb was shown to increase MG concentrations by inhibiting the GLO pathway, and to decrease LDH-like protein and pyruvate levels. We conclude that under hypophosphatemic conditions, the glycolysis pathway is compromised, and to fulfill energy requirements, the Leishmania donovani parasite converts D-lactate into pyruvate to replenish its energy levels.
