ORCID Profile
0000-0002-8787-6755
Current Organisation
National University of Ireland
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Host-parasite interactions | Invertebrate biology | Animal protection (incl. pests and pathogens) | Zoology
Publisher: Elsevier BV
Date: 02-2023
Publisher: Cambridge University Press (CUP)
Date: 12-03-2019
DOI: 10.1017/S0031182019000258
Abstract: Bovine trichomoniasis is a notifiable, reproductive disease of cattle caused by the parasite Tritrichomonas foetus. Culturing with modified Diamond's medium (MDM) is required to increase the low number of organisms received from a preputial s le, but is limited in application to remote areas as it requires continuous cold chain storage. This study utilized lyophilization to sustain the viability of MDM during transport in lieu of a continuous cold chain. All lyophilized MDM was able to sustain T. foetus after storage for 42 days at 24 °C, and the results demonstrated that lyophilized MDM was equally as viable as refrigerated liquid MDM. Storage of lyophilized MDM at room temperature for 1 and 7 days did not impact T. foetus yield, both with and without exposure to light. A limitation of the lyophilized MDM was demonstrated with a significant decrease in T. foetus yield when the media was stored at 37 and 58 °C. The lyophilization of MDM provides a robust method of transporting and storing medium prior to reconstitution and inoculation, for use in T. foetus diagnosis and surveillance in remote areas.
Publisher: Cambridge University Press (CUP)
Date: 2022
DOI: 10.1017/S0950268822001078
Abstract: During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated in iduals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.
Publisher: Microbiology Society
Date: 23-07-2021
DOI: 10.1099/JMM.0.001400
Abstract: Introduction. Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus Bartonella . Mixed Bartonella infections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification of Bartonella species in single and mixed infections is valuable, as different Bartonella species have varying impacts on infected hosts. Gap Statement. Current diagnostic methods are inadequate at identifying the Bartonella species present in mixed infections. Aim. The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identify Bartonella species and demonstrate that this approach can resolve mixed Bartonella infections. Methodology. We used Illumina PCR licon NGS to target the ssrA and gltA genes of Bartonella in fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixed Bartonella s les to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing. Results. In total, we identified 74 Ctenocephalides felis , two Ctenocephalides canis, two Pulex irritans and three Archaeopsylla e. erinacei fleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for Bartonella , all of which were cat fleas. Sanger sequencing of the ssrA and gltA genes confirmed the presence of Bartonella henselae , Bartonella clarridgeiae and Bartonella koehlerae . Illumina NGS of ssrA and gltA licons further confirmed the Bartonella species identity in all 12 flea s les and unambiguously resolved the artificially mixed Bartonella s les. Conclusion. The adaptation and multiplexing of existing PCR assays for ersity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing for Bartonella speciation and resolving mixed Bartonella infections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.
Publisher: Public Library of Science (PLoS)
Date: 15-09-2017
Publisher: ZappyLab, Inc.
Date: 18-09-2017
Publisher: Elsevier BV
Date: 07-2020
Publisher: ZappyLab, Inc.
Date: 18-09-2017
Publisher: ZappyLab, Inc.
Date: 18-08-2017
Publisher: Springer Science and Business Media LLC
Date: 22-05-2018
DOI: 10.1007/S00436-018-5926-3
Abstract: Commonly employed diagnostic methods for Fasciola spp., such as a traditional sedimentation and faecal egg count, or a commercially available coprological ELISA, have limitations in their sensitivity or ability to differentiate species. A reliable DNA isolation method coupled with real-time PCR addresses these issues by providing highly sensitive and quantitative molecular diagnosis from faecal s les. The current study evaluated a standard benchtop vortex for F. hepatica egg disruption in sheep and cattle faecal s les and determined the minimum faecal egg load required for a positive result from un-concentrated (raw) faecal s les. The minimum faecal egg load for a positive real-time PCR result from 150 mg raw faecal s le was 10 and 20 eggs per gram for sheep and cattle, respectively. No significant difference (P = 0.4467) between disruptions on a benchtop vortex for 5 or 10 min was observed when compared to 40 s of disruption at 6.0 m/s in a benchtop homogeniser.
Publisher: Unpublished
Date: 2016
Publisher: Elsevier BV
Date: 02-2020
Publisher: Elsevier BV
Date: 2021
Publisher: Springer Science and Business Media LLC
Date: 09-08-2021
DOI: 10.1186/S13071-021-04896-Y
Abstract: Canine heartworm ( Dirofilaria immitis ) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D . immitis and associated risk factors for canine heartworm antigen positivity—and thus disease—in Australia is scarce or outdated. The current reference method for D . immitis diagnosis in dogs is via the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of D . immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed. Blood s les ( n = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D . immitis DNA using a modified Knott’s test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models. A total of 16 dogs (9.6% 95% confidence interval [CI]: 5.9–15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D . immitis antigen within s les (Cohen’s kappa = 0.98), but the optical density was significantly increased in paired plasma s les for D . immitis antigen-positive s les (Wilcoxon matched-pairs signed rank test, two-tailed P 0.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be D . immitis antigen positive (odds ratio: 4.39 95% CI: 1.26–13.51). All s les positive for the modified Knott’s test were also positive for D . immitis DNA by PCR. This study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of D . immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity.
Publisher: MDPI AG
Date: 31-12-2020
DOI: 10.3390/PATHOGENS10010025
Abstract: Dogs and cats play an important role as reservoirs of vector-borne pathogens, yet reports of canine and feline vector-borne diseases in Saudi Arabia are scarce. Blood s les were collected from 188 free-roaming dogs and cats in Asir (70 dogs and 44 cats) and Riyadh (74 dogs), Saudi Arabia. The presence of Anaplasma spp., Bartonella spp., hemotropic Mycoplasma spp., Babesia spp., and Hepatozoon spp. was detected using a multiplex tandem real-time PCR. PCR-positive s les were further examined with specific conventional and real-time PCR followed by sequencing. Dogs from Riyadh tested negative for all pathogens, while 46 out of 70 dogs (65.7%) and 17 out of 44 cats (38.6%) from Asir were positive for at least one pathogen. Positive dogs were infected with Anaplasma platys (57.1%), Babesia vogeli (30%), Mycoplasma haemocanis (15.7%), and Bartonella henselae (1.4%), and cats were infected with Mycoplasma haemofelis (13.6%), Candidatus Mycoplasma haemominutum (13.6%), B. henselae (9.2%), and A. platys (2.27%), all of which are reported for the first time in Saudi Arabia. Co-infection with A. platys and B. vogeli was detected in 17 dogs (24.28%), while coinfections were not detected in cats. These results suggest that effective control and public awareness strategies for minimizing infection in animals are necessary.
Publisher: Springer Science and Business Media LLC
Date: 12-2017
Publisher: Elsevier BV
Date: 02-2022
DOI: 10.1016/J.VETPAR.2022.109663
Abstract: 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.
Publisher: Elsevier BV
Date: 02-2020
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.VETPAR.2018.01.004
Abstract: 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 s le 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 s les provides increased s le 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 lification (LAMP), have been shown to detect F. hepatica DNA as early as 1 week post infection (WPI). In this study, faecal s les were collected weekly from 10 experimentally-infected Merino lambs and subjected to diagnosis via traditional sedimentation, coproELISA and real-time PCR. S les were first considered positive at 6-8 WPI by coproELISA, real-time PCR and sedimentation, respectively. At 9 WPI 100% of s les 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 s le preparation were compared at 2 WPI: (i) 150 mg raw faecal s les and (ii) 3 g faecal starting volume prior to sedimentation and pelleting. Neither method of s le preparation yielded positive results at 2 WPI suggesting that DNA lification by real-time PCR is associated with faecal egg load.
Publisher: MDPI AG
Date: 30-09-2022
Abstract: The antioxidant superoxide dismutase (SOD) catalyses the dismutation of superoxide, a dangerous oxygen free radical, into hydrogen peroxide and molecular oxygen. Superoxide generation during the oxidative burst of the innate immune system is considered a key component of the host defence against invading pathogens. We demonstrate the presence and differential expression of two SODs in Fasciola hepatica, a leaderless cytosolic (FhSOD1) and an extracellular (FhSOD3) form containing a secretory signal peptide, suggesting that the parasites exploit these enzymes in distinct ways to counteract reactive oxygen species (ROS) produced by cellular metabolism and immune defences. Both enzymes are highly expressed by the infective newly excysted juvenile (NEJ) stages and are found in abundance in their excretory–secretory products (ES), but only FhSOD1 is present in adult ES, suggesting that the antioxidants have different functions and pathways of secretion, and are under separate temporal expression control during the migration, growth, and development of the parasite. Functionally, the recombinant FhSOD1 and FhSOD3 exhibit similar activity against superoxide to their mammalian counterparts. Confocal immuno-localisation studies demonstrated the presence of FhSOD1 and FhSOD3 on the NEJ tegument and parenchyma, supporting our suggestion that these enzymes are secreted during host invasion to protect the parasites from the harmful oxidative bursts produced by the activated innate immune response. By producing superoxide enzymatically in vitro, we were able to demonstrate robust killing of F. hepatica NEJ within 24 h post-excystment, and that the lethal effect of ROS was nullified with the addition of SOD and catalase (the antioxidant enzyme responsible for the dismutation of hydrogen peroxide, a by-product of the SOD reaction). This study further elucidates the mechanism by which F. hepatica protects against ROS derived from cellular metabolism and how the parasite could mitigate damage caused by the host’s immune response to benefit its survival.
Start Date: 2024
End Date: 12-2026
Amount: $455,563.00
Funder: Australian Research Council
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