Detailed post mortem analyses of 68 free-ranging, slaughter-age pigs from two sites in the Banke District of Nepal identified 36% as being infected with Echinococcus granulosus. The cysts ranged from infertile, immature cysts a few millimetres in diameter to fertile cysts >10 cm in diameter. PCR RFLP and DNA sequencing identified the cysts as being E. granulosus sensu stricto. The Banke district has recently been identified as having a high prevalence of porcine cysticercosis. These data suggest that cestode zoonoses in this, and possibly other parts of Nepal may be a serious concern for human health. An assessment of the level of human cystic echinococcosis and neurocysticercosis, in the region is warranted and the introduction of control measures are required to limit the parasites' transmission.

Parasitic roundworms (nematodes) cause substantial morbidity and mortality in animals worldwide. Anthelmintic treatment is central to controlling these worms, but widespread resistance to most of the commercially available anthelmintics for veterinary and agricultural use is compromising control, such that there is an urgency to discover new and effective drugs. The purpose of this article is to review information on parasitic nematodes, the treatment and control of parasitic nematode infections and aspects of discovering new anthelmintics in the context of anthelmintic resistance problems, and then to discuss some progress that our group has made in identifying selected compounds with activity against nematodes. The focus of our recent work has been on discovering new chemical entities and known drugs with anthelmintic activities against Haemonchus contortus as well as other socioeconomically important parasitic nematodes for subsequent development. Using whole worm-based phenotypic assays, we have been screening compound collections obtained via product-development-partnerships and/or collaborators, and active compounds have been assessed for their potential as anthelmintic candidates. Following the screening of 15,333 chemicals from five distinct compound collections against H. contortus, we have discovered one new chemical entity (designated SN00797439), two human kinase inhibitors (SNS-032 and AG-1295), 14 tetrahydroquinoxaline analogues, one insecticide (tolfenpyrad) and two tolfenpyrad (pyrazole-5-carboxamide) derivatives (a-15 and a-17) with anthelmintic activity in vitro. Some of these 20 'hit' compounds have selectivity against H. contortus in vitro when compared to particular human cell lines. In our opinion, some of these compounds could represent starting points for 'lead' development. Accordingly, the next research steps to be pursued include: (i) chemical optimisation of representative chemicals via structure-activity relationship (SAR) evaluations; (ii) assessment of the breadth of spectrum of anthelmintic activity on a range of other parasitic nematodes, such as strongyloids, ascaridoids, enoplids and filarioids; (iii) detailed investigations of the absorption, distribution, metabolism, excretion and toxicity (ADMET) of optimised chemicals with broad nematocidal or nematostatic activity; and (iv) establishment of the modes of action of lead candidates.

Parasitic roundworms (nematodes) are significant pathogens of humans and animals and cause substantive socioeconomic losses due to the diseases that they cause. The control of nematodes in livestock animals relies heavily on the use of anthelmintic drugs. However, their extensive use has led to a widespread problem of drug resistance in these worms. Thus, the discovery and development of novel chemical entities for the treatment of parasitic worms of humans and animals is needed. Herein, we describe our medicinal chemistry optimization efforts of a phenotypic hit against Haemonchus contortus based on a pyrrolidine-oxadiazole scaffold. This led to the identification of compounds with potent inhibitory activities (IC50 = 0.78-22.4 μM) on the motility and development of parasitic stages of H. contortus, and which were found to be highly selective in a mammalian cell counter-screen. These compounds could be used as suitable chemical tools for drug target identification or as lead compounds for further optimization.

Newborn mammals have an immature immune system that cannot sufficiently protect them against infectious diseases. However, variation in the effectiveness of maternal immunity against different parasites may couple with temporal trends in parasite exposure to influence disparities in the timing of infection risk. Determining the relationship between age and infection risk is critical in identifying the portion of a host population that contributes to parasite dynamics, as well as the parasites that regulate host recruitment. However, there are no data directly identifying timing of first infection among parasites in wildlife. Here, we took advantage of a longitudinal dataset, tracking infection status by viruses, bacteria, protists and gastro-intestinal worms in a herd of African buffalo (Syncerus caffer) to ask: how does age of first infection differ among parasite taxa? We found distinct differences in the age of first infection among parasites that aligned with the mode of transmission and parasite taxonomy. Specifically, we found that tick-borne and environmentally transmitted protists were acquired earlier than directly transmitted bacteria and viruses. These results emphasize the importance of understanding infection risk in juveniles, especially in host species where juveniles are purported to sustain parasite persistence and/or where mortality rates of juveniles influence population dynamics.

Specimens of four genetically distinct groups of Macropostrongyloides baylisi Wood, 1930 were analysed morphologically. Each genotype was found to represent a morphologically distinct species: Ma. baylisi from Osphranter robustus woodwardi (Thomas) and Osphranter robustus erubescens (Sclater); Ma. spearei n. sp. from Osphranter robustus robustus (Gould) and O. r. erubescens; Ma. mawsonae n. sp. from Macropus giganteus Shaw and Ma. woodi n. sp. from Osphranter rufus (Desmarest). The new species described here are differentiated primarily by several male-specific features that have been overlooked in previous taxonomic revisions. These features include striations on the terminal part of the spicule ala, the papillae surrounding the genital cone and the bursal striations. Furthermore, scanning electron photomicrographs have revealed greater details of previously undefined structures within the buccal cavity that warrant further investigations.

Inhibition of α-glucosidase enzyme is of prime importance for the treatment of diabetes mellitus (DM). Apart of many organic scaffolds, pyridine based compounds have previously been reported for wide range of bioactivities. The current study reports a series of pyridine based synthetic analogues for their α-glucosidase inhibitory potential assessed by in vitro, kinetics and in silico studies. For this purpose, 2-amino-4-aryl-6-(phenylthio)pyridine-3,5-dicarbonitriles 1-28 were synthesized and subjected to in vitro screening. Several analogs, including 1-3, 7, 9, 11-14, and 16 showed many folds increased inhibitory potential in comparison to the standard acarbose (IC50 = 750 ± 10 µM). Interestingly, compound 7 (IC50 = 55.6 ± 0.3 µM) exhibited thirteen-folds greater inhibition strength than the standard acarbose. Kinetic studies on most potent molecule 7 revealed a competitive type inhibitory mechanism. In silico studies have been performed to examine the binding mode of ligand (compound 7) with the active site residues of α-glucosidase enzyme.

Tick infestation is a leading cause of tick-worry and tick-borne diseases in livestock and associated economic losses in tropical and subtropical regions of the world. The cattle and buffalo populations in Pakistan are exposed to tick infestation throughout the year, but very little is known about the biology, diversity and distribution of tick species across different agro-ecological zones (AEZ) of the country. The present study aimed to investigate the prevalence (number of bovines infested with ticks out of the investigated population) and diversity of hard ticks infesting bovines in 30 villages located in five distinct AEZs (i.e. Arid, Indus delta, Northern irrigated plain, Sandy desert and Southern irrigated plain). We collected a total of 774 ticks (adult and nymphs) from cattle (n = 116) and water buffaloes (n = 88) on small-holder dairy farms (with <10 bovids per establishment) from September to November 2017. The overall tick prevalence was 46.1% (cattle: 47.9%; buffaloes: 44%), which varied significantly from 22.2% in the Indus delta to 70.5% in the Sandy desert. Tick prevalence was slightly higher in female (46.5%) than male animals (45%), and higher in calves (i.e. ≤ 1 year of age) (55%) than in young animals (i.e. up to 3 years of age) (39%) and adults (48%). Five tick species - Hyalomma anatolicum, Hyalomma hussaini, Hyalomma scupense, Rhipicephalus microplus and Rhipicephalus annulatus - were identified morphologically and then genetically. Genetic identification, achieved using the sequences of two mitochondrial (cytochrome c oxidase subunit 1 and 16S) and one nuclear ribosomal (second internal transcribed spacer) regions, was consistent with the morphological findings. Phylogenetic analyses of the DNA sequence data sets showed that the five species of tick identified here were closely related to the same species or closely related species from within and outside of Pakistan. Of five presently recognised taxa within the R. microplus complex, two were identified herein, including the R. microplus clade C and R. annulatus. This investigation provides the first genetic evidence of the occurrence of R. annulatus in Pakistan as well as Hy. hussaini and Hy. scupense in bovines specifically in the provinces of Sindh and Punjab, respectively. The present findings emphasise the importance of combining morphological and molecular approaches to study the diversity of ticks. Further longitudinal studies are required to establish seasonal variations in the prevalence and distribution of bovine ticks in different AEZs of Pakistan.

A series of 1-methyl-1H-pyrazole-5-carboxamides were synthesized as potent inhibitors of the parasitic nematode of sheep, Haemonchus contortus. These compounds did not show overt cytotoxicity to a range of mammalian cell lines under standard in vitro culture conditions, had high selectivity indices, and were progressed to an acute toxicity study in a rodent model. Strikingly, acute toxicity was observed in mice. Experiments measuring cellular respiration showed a dose-dependent inhibition of mitochondrial respiration. Under these conditions, potent cytotoxicity was observed for these compounds in rat hepatocytes suggesting that the potent acute mammalian toxicity of this chemotype is most likely associated with respiratory inhibition. In contrast, parasite toxicity was not correlated to acute toxicity or cytotoxicity in respiring cells. This paper highlights the importance of identifying an appropriate in vitro predictor of in vivo toxicity early on in the drug discovery pipeline, in particular assessment for in vitro mitochondrial toxicity.

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

The strongyloid nematode subfamily Phascolostrongylinae comprises parasites of the large intestine and stomach of Australian macropods and wombats. In this study, we tested the phylogenetic relationships among the genera of the Phascolostrongylinae using the first and second internal transcribed spacers of the nuclear ribosomal DNA. Monophyly was encountered in the tribe Phascolostrongylinea comprising two genera, Phascolostrongylus and Oesophagostomoides, found exclusively in the large intestine of wombats. The tribe Hypodontinea, represented by the genera Hypodontus and Macropicola from the ileum and large intestine of macropods, was also found to be monophyletic. The tribe Macropostrongyloidinea, comprising the genera Macropostrongyloides and Paramacropostrongylus, was paraphyletic with the species occurring in the stomach grouping separately from those found in the large intestines of their hosts. However, Macropostrongyloidesdissimilis from the stomach of the swamp wallaby and Paramacropostrongylus toraliformis from the large intestine of the eastern grey kangaroo were distinct from their respective congeners. This study provided strong support for the generic composition of the tribe Phascolostrongylinea. The unexpected finding of M. dissimilis and P. toraliformis being distantly related to their respective congeners suggests a requirement for future taxonomic revision that may warrant separation of these species at the generic level.

Nematodes of the genus Macropostrongyloides inhabit the large intestines or stomachs of macropodid (kangaroos and wallabies) and vombatid (wombats) marsupials. This study established the relationships of seven species of Macropostrongyloides using mitochondrial (mt) protein amino acid sequence data sets. Phylogenetic analyses revealed that species of Macropostrongyloides (M. lasiorhini, M. baylisi, M. yamagutii, M. spearei, M. mawsonae and M. woodi) from the large intestines of their hosts formed a monophyletic assemblage with strong nodal support to the exclusion of M. dissimilis from the stomach of the swamp wallaby. Furthermore, the mitochondrial protein-coding genes provided greater insights into the diversity and phylogeny of the genus Macropostrongyloides; such data sets could potentially be used to elucidate the relationships among other parasitic nematodes of Australian marsupials.

Context: Calves left with their dam to suckle will consume ~7–12 L/day; however, the amount of milk provided to dairy young calves removed from their dams may often be as low as 4 L/day, or 10% of their bodyweight. Aims: This study compared once and twice daily feeding, as well as feeding levels of 10 and 20% of bodyweight and studied the effect on behaviour and metabolic indicators indicative of hunger. Methods: Forty-six male dairy calves were allocated to one of three treatments from 3 to 8 days of age: (i) 10% of bodyweight offered daily as one meal (1 × 10%, n = 16); (ii) 10% of bodyweight offered daily over two meals (2 × 5%, n = 15); or (iii) 20% of bodyweight offered over two meals (2 × 10%, n = 15). Behaviour during and after feeding was observed by video, and blood samples taken on selected days were analysed for glucose, nonesterified fatty acids (NEFA), cortisol and packed-cell volume. Key results: Milk intake on Days 3 and 4 was lower in those calves fed once daily compared with calves fed twice daily. Calves fed at 20% bodyweight had higher milk intake compared with calves fed at 10% bodyweight on all days other than Day 3 and growth was higher in those calves. Non-nutritive sucking was mainly associated with feeding times and it was highest in calves fed 10% bodyweight over two meals, with a suckling pattern that suggested that feeding at 10% bodyweight satisfied feeding motivation less than feeding at 20% bodyweight. Play behaviour was reduced in calves fed once daily, suggesting hunger and reduced welfare. There were significant effects on physiological indicators of metabolic state. NEFA concentrations were significantly higher in calves fed once daily and calves fed 10% of bodyweight at certain time points, indicating a lower energy balance. Conclusions: Feeding twice daily offers benefits to calves up to Day 4 of life whereas feeding 20% of bodyweight was beneficial after Day 4 to increase satisfaction of feeding motivation and nutrition for growth. Although metabolic variables were within normal physiological range for all treatments, the effects on feed intake, growth and non-nutrient sucking suggest marked effects on hunger when calves are fed the lower milk allowance. Implications: The fairly common practice of feeding dairy calves 10% of bodyweight, in one or two daily meals may leave calves hungry, and an increase in milk allowance should be considered.