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Our laboratory's vaccine development strategy against the livestock parasite Fasciola hepatica centres around disrupting key biological processes by combining groups of antigens with similar/complementary functional actions into a single vaccine cocktail. In this study the focus was on antioxidant protein vaccines and a protease inhibitor vaccine aimed at disrupting the parasite's ability to defend against oxidative stress and protease-inhibitor balance, respectively. Two combinations of recombinantly expressed antioxidants were assessed, namely peroxiredoxin (rFhPrx), thioredoxin (rFhTrx) and thioredoxin-glutathione reductase (rFhTGR) (Group 1) and rFhPrx, rFhTrx, and two superoxide dismutases (rFhSOD1 and rFhSOD3) (Group 2). The protease inhibitor vaccine cocktail included representatives of each of the key secreted protease inhibitor families, namely a Kunitz-type inhibitor (rFhKT1), a serpin (rFhSrp1) and a stefin, (rFhStf1) (Group 3). The vaccine combinations were formulated in adjuvant Montanide 61VG administered at five timepoints; two before experimental challenge with 60 F. hepatica metacercariae and three after infection. The vaccine combinations did not reduce the liver fluke burden, and only Group 2 displayed a marginal reduction in egg viability (8.2%). Despite previous results showing an effect of liver fluke vaccines on overall weight gain in infected animals, no significant (P value >0.05) impact on weight gain was observed in this study. Antibodies were elicited against all the vaccine antigens within the cocktails and were maintained at high levels to the end of the trial, due to our strategy of continuing vaccine administration after infection. However, these responses were not boosted by the challenge F. hepatica infection. A comparative analysis with previous vaccine data using a protease inhibitor vaccine found no repeat of the promising outcomes associated with this vaccine, indicating that the addition of rFhSrp1 to the vaccine cocktail did not improve vaccine efficacy. Assessment of liver pathology across the two trials using a modified liver enzyme score (glutamate dehydrogenase to platelet ratio) at eight weeks post infection suggests an association with liver fluke burden above 45 flukes, which could be used to predict liver pathology in future trials. The results reported in this study highlight the ambiguousness in liver fluke vaccine development and the difficulty in obtaining consistent and repeatable protection. This work stresses the need for repetition of trials and the use of sufficiently sized groups to assess vaccine efficacy with adequate statistical power., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Canine roundworm, Toxocara canis, is considered ubiquitous but patent infections are rare in adult owned urban dogs. Hepato-pulmonary migration of T. canis is common in young dogs, but in adult dogs, the migration of T. canis is arrested in tissues and larvae are inhibited. During this somatic migration, T. canis release excretory-secretory (E/S) larval antigens against which the host mounts an immune response. Detection of anti-T. canis E/S immunoglobulins is considered a proxy for the presence of arrested somatic T. canis larvae. By screening several cohorts of dogs in New South Wales (NSW), Australia, we determined the seroprevalence of anti-T. canis E/S in urban owned dogs visiting a veterinary teaching hospital in Sydney to be 3.8 % (n = 53), which was significantly lower (two-proportion z-test, P < 0.05) than the seroprevalence in pet dogs in regional western NSW (22.2 %, n = 63), and rehomed greyhounds (53.6 %, n = 28). Using a logistic regression model, the risk of testing positive in regional pet dogs (odds ratio [OR] = 37.0) and rehomed greyhounds (OR = 81.0) was significantly higher than in urban dogs (P < 0.05). Although routine deworming of dogs eliminates patent infection, our data show a low number of urban dogs with anti-T. canis E/S antibodies, which implies that the majority of these dogs were not exposed to T. canis previously, do not possess inhibited T. canis larvae, and in the case of intact females, will not transmit it to their puppies., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In Australia, Cooperia spp. are often overshadowed by parasites believed to be more pathogenic production-limiting nematodes. A rise in anthelmintic resistance and reports of reduced growth rates attributed to infection with Cooperia spp. in Europe increases the need to be able to monitor the presence of C. pectinata, C. punctata and C. oncophora in Australian cattle. Here, we present the first molecular confirmation of C. pectinata and C. punctata in Australian cattle using ITS2 rDNA and COXII mtDNA. Cultured larvae were morphologically differentiated to the genus level with the aid of iodine solution and their DNA was screened using a cattle nematode MT-PCR panel. By isolating individual iodine stained and morphologically identified nematode larvae, we demonstrated the presence of C. pectinata and C. punctata using a generic ITS2 rDNA qPCR assay following DNA amplicon sequencing. A novel suite of COXII mtDNA species/genus-specific PCR assays for Cooperia speciation from complex nematode samples enabled us to detect all three species (C. oncophora, C. pectinata, C. punctata) in Australia cattle samples. Our approach, utilising traditional techniques coupled with the manipulation of individual nematode larvae, provides a foundation for the inclusion of Cooperia spp. into existing high throughput molecular diagnostic panels for cattle nematode surveillance., Competing Interests: Declaration of Competing Interest No conflict of interest declared., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fasciolosis due to infection with Fasciola hepatica, Fasciola gigantica or their hybrids is a significant global cause of livestock production loss. Infection is commonly diagnosed by a labour-intensive sedimentation and faecal egg count (FEC), which has limited throughput and is only applicable after completion of the 8-12 week pre-patent period (PPP). A commercially-available ELISA for the detection of coprological antigen (coproELISA) enables detection prior to the completion of the PPP and is suitable for diagnosis of larger sample sizes, although the sensitivity reported under experimental infection settings can be difficult to replicate in the field, particularly in cattle. A recently-published real-time PCR workflow for the sensitive detection of Fasciola spp. DNA in faecal samples provides increased sample throughput, although the point at which this technique is first able to diagnose infection remains unknown. Other tools for the molecular diagnosis of fasciolosis, such as conventional PCR and loop-mediated isothermal amplification (LAMP), have been shown to detect F. hepatica DNA as early as 1 week post infection (WPI). In this study, faecal samples were collected weekly from 10 experimentally-infected Merino lambs and subjected to diagnosis via traditional sedimentation, coproELISA and real-time PCR. Samples were first considered positive at 6-8 WPI by coproELISA, real-time PCR and sedimentation, respectively. At 9 WPI 100% of samples were positive by all three methods. To evaluate the capacity of the real-time PCR approach to detect infection prior to completion of the PPP, two methods of sample preparation were compared at 2 WPI: (i) 150 mg raw faecal samples and (ii) 3 g faecal starting volume prior to sedimentation and pelleting. Neither method of sample preparation yielded positive results at 2 WPI suggesting that DNA amplification by real-time PCR is associated with faecal egg load., (Copyright © 2018 Elsevier B.V. All rights reserved.)