Anti-parasitic drugs and drug development 2

Theileriosis, or Bovine Anaemia due to Theileria orientalis Genotypes (BATOG), is an emerging intra-erythrocytic piroplasmic vector borne disease affecting Australian cattle. Annual industry losses have been estimated at over $20m, with the endemic 3-host tick, Haemaphysalis longicornis identified as the disease vector. Producers seek reliable control measures, however despite research efforts, no registered treatments are currently available. Two small scale pilot trials were conducted to determine the efficacy of several existing veterinary medicines with known anti-protozoal activity, against T. orientalis Ikeda infection. In the first study, all calves infected by inoculation with infected blood became PCR positive by SD46 post infection. Four calves per group were treated with either buparvaquone (BPQ, 2.5 mg/kg), imidocarb dipropionate (5 mg/kg), oxytetracycline (20 mg/kg), tulathromycin (2.5 mg/kg), or were untreated. The only treatment that significantly reduced the gene copy numbers of the parasite by PCR was BPQ, but animals remained carriers. Efficacy of buparvaquone for the treatment of T. orientalis is proven, however due to long drug residues this treatment is non-viable for use commercially. In the second small trial, thirty calves were infected with T. orientalis ikeda through application of infected unfed female adult H. longicornis ticks. All calves became PCR positive by SD13 post tick application. Group 1 were treated with toltrazuril (15 mg/kg) on SD 3 and Group 2 untreated. Calves were weighed, and blood taken and analysed for PCV, qPCR (T. orientalis Ikeda), Theilerial ELISA and piroplasm counts at various intervals up to SD64 post infection. No statistically significant differences in group mean bodyweight, PCV, qPCR ct value, Theilerial ELISA or piroplasms per HPF were observed any time-point, except PCV at SD 20. At this timepoint, mean PCV was significantly higher in the toltrazuril treated group. Further dose determination work is ongoing.

Hi, I'm Susan, veterinarian, parasitologist, wife, mum, cat whisperer. I graduated from Sydney University in 2003, and since have worked in small animal practice and in industry registering medicines for pets and farm animals. Inspired by the persistence of the  flea and the life history of intestinal worms, I became a member of the ANZCVS in Veterinary Parasitology in 2018. Currently I'm researching Theileria orientalis Ikeda strain in Australian cattle, also at Sydney University.

Introduction: Glucokinase (GLK) and Hexokinase (HK) are key enzymes involved in glycolysis and pentose phosphate pathway (PPP), and have been characterized as essential targets in Trypanosoma cruzi (Tc)-mediated infection. A recent study reported the propensity of the concomitant inhibition of TcGLK and TcHK by compounds GLK2-003 and GLK2-004, thereby presenting an efficient approach in Chagas disease treatment. 

Computational Methods: In this study, we set out to investigate this therapeutic possibility using advanced bioinformatics techniques coupled with atomic and molecular scaling methods. 

Results: Sequence alignment of TcGLK and TcHK revealed that both proteins shared approximately 33.3% homology in their glucose/inhibitor binding sites. Conserved residues in TcGLK/TcHK include PRO92/PRO163, ASN130/ASN213, ASP131/ASP214, GLY188/GLY238, SER189/239, GLN236/GLN300, ASP29/ASP90, GLY31/GLY92, THR33/THR94, ARG36/ARG97, GLY184/GLY236 and THR185/THR237. The total binding free energies of GLK2-003 and GLK2-004 were favorable in both proteins (TcGLK-GLK2-003= -28.71 kcal/mol, TcGLK-GLK2-004=-31.54 kcal/mol, TcHK-GLK2-003= -36.15 kcal/mol, and TcHK-GLK2-004=-33.51 kcal/mol). 

Discussion: We discussed the importance of amino acid residues PRO92 and THR185 which were pivotal to the binding and stabilization of compounds GLK2-003 and GLK2-004 in TcGLK, likewise their conserved counterparts; PRO163 and THR237 in TcHK. The induction of similar pattern of perturbations in both TcGLK and TcHK secondary structure by GLK2-003 and GLK2-004 was also elaborated. Pharmacokinetic (ADMET) profiling of both compounds further revealed their compliance to the Lipinski’s rule of 5, indicative of their drug-likeness.

Findings from this study therefore provide insights into the underlying mechanisms of dual inhibition exhibited by the compounds. These results can pave way for the discovery and optimization of novel dual Tc inhibitors with improved pharmacokinetics eventuating in the mitigation of Chagas disease.

Folu holds a bachelor's and master's degree in Biochemistry from the University of Ibadan. She proceeded to University of KwaZulu Natal, South Africa for another master's degree in Pharmaceutical Chemistry with South African National Research Foundation scholarship. Presently, her PhD thesis in the same department and institution is under examination. Folu does research in computer-aided drug design for the treatment of neglected tropical parasitic diseases and has published in peer-reviewed journals of international repute. She is happily married to a consummate parasitologist. 

Folu is open to post-doctoral positions in the field of parasitology especially in computer-aided drug design.

Control of gastrointestinal nematodes (GINs) infections in humans and animals is seriously threatened by global development of anthelmintic resistance. In Sudan, resistance to benzimidazoles has been reported recently in cattle and goats from South Darfur. The efficacy of ivermectin was evaluated in goats in three study areas in South Darfur. The faecal egg count reduction test (FECRT) was used to evaluate the efficacy of ivermectin in goats naturally infected with GINs in Bulbul (n=121), Kass (n=33) and Nyala (Domaia: n=72, university farm: n=52). Ivermectin was injected subcutaneously at the dose recommended for goats (0.4 mg/kg body weight) or 0.8 mg/kg bw, and the efficacy was evaluated 12 days after treatments. Nematodes surviving ivermectin treatment in goats in Bulbul and Nyala were harvested and larvae used to infect worm free male sheep (n=6, ≤6 months old). From day 24 sheep were dosed subcutaneously with ivermectin every eight days with increasing doses from 0.2 mg/kg to 1.6 mg/kg bw. Reduced ivermectin efficacy was identified in goats in Bulbul and Nyala. The paired and unpaired FECRs for 0.4 mg/kg revealed reductions of 72.6/76.4%, 75.7/86.9% and 85.3/83.1% in Bulbul, Domaia and university farm, respectively, while a double dose in Bulbul showed an efficacy of 86.4% and 90.4%. In Kass, ivermectin was 95.6% effective. Eggs of strongyles, Strongyloides papillosus and Skrjabinema ovis were identified before treatment, while only strongyle eggs were found after treatments. After treatment, all larvae were Haemonchus spp. Ivermectin efficacy in sheep using different doses, including 1.6 mg/kg was from 33.9 – 85.6%. In necropsies after treatment with 1.6 mg/kg dose, only adult Haemonchus contortus were found. For the first time ivermectin resistant H. contortus populations have been identified in goats in Sudan, and resistance was experimentally confirmed in sheep.

My name is Khalid M. Mohammedsalih from Sudan. I am Assistant Professor of Veterinary Pharmacology at University of Nyala, Sudan. My position now is a director of Ruminant Research Center, University of Nyala. I received my PhD from University of Khartoum (Sudan) in 2018. My experience during the last years is in the control of parasitic infections in ruminants. I received advanced training, during 2017 - 2020 (short visits), on detection of molecular mechanisms of anthelmintic resistance at Institute for Parasitology and Tropical Veterinary Medicine, Freien Universität Berlin, Germany, under Supervision of Prof. Dr. Georg von Samson-Himmelstjerna. 

Introduction: Most grazing ruminants are infected by helminth parasites that have a massive impact on their welfare and production. In that context, diseases caused by gastrointestinal parasitic nematodes are an important animal health issue. The main mean to control such parasites relies today on the use of anthelmintic drugs. However, intensive use of chemicals has selected drug-resistant parasite populations in many animal species. This includes resistance to the broad-spectrum macrocyclic lactones (MLs) such as ivermectin (IVM). There is now considerable evidence that ML resistance is a polygenic trait in nematodes, but the molecular mechanism(s) of ML resistance remains to be resolved. The problem is worsened by the lack of efficient tools to track resistance. Materials and Methods: In that context, we have developed a stable fluorescent probe of IVM, IVM-FLUO, and by using confocal imaging, we were able for the first time to visualize, map and quantify IVM in the whole worm Caenorhabditis elegans. We have compared the distribution and signal intensity of IVM-FLUO in susceptible and ML-resistant C. elegans strains. Results: Some specific fluorescent spots detected all along worm body, revealed IVM location in strategic organs. Signal quantification revealed a higher global IVM amount in wild-type animals compared to the IVM-resistant worms, in line with a link between IVM concentration in the worm and IVM resistance status. Discussion: Our datas shed light on IVM distribution in C. elegans and they contribute to the understanding of IVM resistance mechanisms. IVM-FLUO is an innovative tool that meets the expectations of clinicians and breeders concerned about the loss of drug efficacy. Its development will allow evaluating the efficacy of anthelmintic drugs and the resistance status of pathogenic parasites in livestock. It represents a valuable asset for the research of drug resistance mechanisms in general.

Mélanie Alberich is a research engineer at the French National Institute for Research on Agriculture, Food and the Environment (INRAE, Toulouse, France) in the Intheres unit (Therapeutic innovation and resistance). Resistance to anthelmintic macrocyclic lactones (MLs) is a major global health problem. The mechanisms of resistance to MLs in nematodes remain to be proven. It is in this context that Melanie works on (i) producing fluorescent anthelmintic probes in order to (ii) understand the behaviour of the molecules in nematodes, and (iii) decipher the molecular mechanisms of resistance to MLs in resistant nematodes.