Pyrantel resistance in canine hookworms in Queensland, Australia.

Hookworms are the most common intestinal nematode parasites of dogs in Australia. The control of these parasites relies mostly on regular deworming with anthelmintics, with pyrantel-based dewormers being a relatively low cost and readily-available option for dog owners. Pyrantel resistance in canine hookworms in Australia was first reported in 2007, however pyrantel-based dewormers are still used against hookworm infection in dogs across Australia. The present study was conducted to evaluate the efficacy of pyrantel against hookworms infecting dogs housed in a shelter facility in Southeast Queensland which receives rescued or surrendered animals from greyhound rescue centres and dog shelters across this region. A total of 10 dogs were examined using the faecal egg count reduction test (FECRT). There was no reduction in FEC in any of the dogs following pyrantel treatment, with drug efficacies ranging from -0.9% to -283.3%. Given that these dogs originated from various sites across Southeast Queensland, the present study suggests that pyrantel resistance is widespread in this region, and hence this anthelmintic may not be a useful option for treatment of hookworm infections in dogs.

Investigation of cattle plasma proteome in response to pain and inflammation using next generation proteomics technique, SWATH-MS.

Pain assessment in farm animals has primarily relied on a combination of behavioral and physiological responses, although these are relatively subjective and difficult to quantify. It is essential to develop more effective biomarkers of pain in production animals since they are frequently exposed to routine surgical husbandry procedures. More effective biomarkers of pain would improve welfare, limit the loss of productivity associated with pain and permit better assessment of analgesics. This study aimed to investigate the use of a modern mass spectrometry data independent acquisition strategy, termed Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS), to detect candidate protein biomarkers that are known to associate with nociceptive and inflammatory processes in cattle, which could then be used to assess the efficacy of potential analgesics. Calves were randomly divided into two groups that were either surgically dehorned or subjected to restraint stress, without provision of anaesthesia or analgesia in accordance with current industry standards. Samples were analysed before and after dehorning at multiple timepoints. Significant changes in protein concentrations were detected predominantly at 24 and 96 h following dehorning, including kininogens, proteins associated with the coagulation and complement cascades and serine protease inhibitors. Gene ontology analysis revealed that the identified candidate biomarkers were associated with stress, wound healing, immune response, blood coagulation and the inflammatory and acute phase responses, which could be expected following surgical damage to tissues, but can now be more objectively assessed. These results offer more definitive and quantitative monitoring of response to tissue injury induced pain and inflammation.

Leveraging homologies for cross-species plasma proteomics in ungulates using data-independent acquisition.

The collection of blood plasma is minimally invasive, and the fluid is a rich source of proteins for biomarker studies in both humans and animals. Plasma protein analysis by mass spectrometry (MS) can be challenging, though modern data acquisition strategies, such as sequential window acquisition of all theoretical fragment ion spectra (SWATH), enable reproducible quantitation of hundreds of proteins in non-depleted plasma from humans and laboratory model animals. Although there is strong potential to enhance veterinary and translational research, SWATH-based plasma proteomics in non-laboratory animals is virtually non-existent. One limitation to date is the lack of comprehensively annotated genomes to aid protein identification. The current study established plasma peptide spectral repositories for sheep and cattle that enabled quantification of over 200 proteins in non-depleted plasma using SWATH approach. Moreover, bioinformatics pipeline was developed to leverage inter-species homologies to enhance the depth of baseline libraries and plasma protein quantification in bovids. Finally, the practical utility of using bovid libraries for SWATH data extraction in taxonomically related non-domestic ungulate species (giraffe) has been demonstrated. SIGNIFICANCE: Ability to quickly generate comprehensive spectral libraries is limiting the applicability of data-independent acquisition, such as SWATH, to study proteomes of non-laboratory animals. We describe an approach to obtain relatively shallow foundational plasma repositories from domestic ruminants and employ homology searches to increase the depth of data, which we subsequently extend to unsequenced ungulates using SWATH method. When applied to cross-species proteomics, the number of proteins quantified by our approach far exceeds what is traditionally used in plasma protein tests.

Identification and Prevalence of a Gammaherpesvirus in Free-Ranging Northern Brown Bandicoots (Isoodon macrourus).

Herpesviruses have been reported in several Australian marsupial species, with an overt, sometimes fatal disease described in macropods. Our study identifies a gammaherpesvirus in northern brown bandicoots (Isoodon macrourus) and provides virus prevalence data for bandicoots in southeast Queensland, Australia. Herpesvirus DNA was detected using pan-Herpesviridae family primers in a nested PCR format. Samples from 35 northern brown bandicoots were screened, including whole blood (n=29), oropharyngeal swabs (n=34), urine (n=22), and feces (n=23). Combining all sample types, herpesvirus DNA was detected at a total prevalence of 51% (18/35). Whole blood and oropharyngeal swabs proved to be the optimal samples for detection of this virus, with prevalences of 34% and 38%, respectively. Herpesvirus DNA was detected in 4.5% (1/22) of urine samples and not at all in fecal samples. Detection of herpesvirus was more likely in males than females. Animals were trapped at eight different locations, and at all but one location at least one herpesvirus positive animal was detected. This study indicates a high prevalence of the virus within northern brown bandicoot populations in southeast Queensland. Further research is required to understand the clinical manifestations, if any, of herpesvirus infection in this species and how this may affect populations in the face of stressors such as land clearing and habitat fragmentation.

CLINICAL PATHOLOGY OF CAPTIVE ENDANGERED MAHOGANY GLIDERS (PETAURUS GRACILIS) IN RESPONSE TO ENVIRONMENTAL CHANGE.

The impetus of this study was the imperative to establish blood biomarker values for clinically healthy mahogany gliders (Petaurus gracilis) in order to monitor the health status of eight captive individuals during their movement to a new facility. The study established ranges for 18 hematologic and 21 biochemical blood biomarkers for healthy individuals in a captive environment. The reported values are consistent with those published for other Australian glider and possum species. No statistically significant differences were found between the sexes, but significant age effects were observed. Specifically, subadult animals reported significantly higher total white cell counts, lymphocyte counts, alkaline phosphatase, creatine kinase, and glucose and chloride levels, compared to adult animals. Although there were no clinically significant changes in blood biomarkers associated with the relocation, many of the hematologic and biochemical biomarkers demonstrated the expected changes associated with the physiological stress of relocation. Specifically, triglycerides, glucose, globulins, creatinine kinase, aspartate transferase (AST), total protein, urea, phosphorous, potassium, sodium, chloride, neutrophils, and hematocrit showed changes with the large environmental change. The majority of the blood biomarkers returned to baseline levels 5 wk postrelocation, with all but one aged animal showing no signs of chronic health derangements following the relocation. The abnormal blood biomarker profiles of two geriatric individuals, one male diagnosed with pericloacal and adrenal gland tumors at the beginning of the study, and one female diagnosed with chronic urinary tract infections and suspected bone marrow disease following the relocation, are presented. The findings of this study inform the health monitoring of native gliders in captivity, rehabilitation, and clinical research scenarios. These findings also provide useful baseline data to aid in the health assessment of captive-bred individuals during their reintroduction into free-living populations.

Sharing Ideas and Practice: Institutional Partnership Influences Change in Approaches to Teaching to Enhance Veterinary Education in Vietnam in Conjunction with an OIE Veterinary Education Twinning Project.

A World Organisation for Animal Health (OIE) Veterinary Education Twinning Project was established between the veterinary schools at Nong Lam University (NLU) in Ho Chi Minh City, Vietnam, and the University of Queensland, Gatton, Australia, as part of the scheme established to promote high-quality veterinary services through improved veterinary education. Included in the partnership's primary aims were building the capacity of veterinary teaching staff with respect to general teaching practice and also in response to identified deficiency areas, and to develop outcome assessment processes. One challenge facing the project was the different approaches and experiences of teaching and learning for the faculty and students between the two widely different historical and cultural contexts of Australia and Vietnam. The project enhanced the pedagogy capability in NLU faculty and introduced student-focused approaches to teaching. The NLU staff involved in the project strongly embraced a student-centered approach to learning and case-based teaching in particular, adopting these strategies in their own teaching. An analysis of students' approach to learning demonstrates that the majority preferred a deep approach to learning and that these students valued case studies, problem-solving exercises, and working in small groups during teaching sessions more than students who took a surface approach to learning. An improved recognition of the ways the Vietnamese students approach their learning in their home country will guide future teaching design, as well as give insight into the approaches to teaching for Southeast Asian students within the Australian veterinary science programs.

A Novel Transdermal Ketoprofen Formulation Provides Effective Analgesia to Calves Undergoing Amputation Dehorning.

There is a critical need to ensure that all cattle undergoing surgical husbandry procedures are provided effective pain relief. Non-steroidal anti-inflammatory drugs (NSAIDs) are most commonly used, and typically are administered by intramuscular (IM) injection. However, administration of NSAIDs via this route to large numbers of cattle which are handled only once or twice a year, typical of many rangeland beef production systems, presents significant occupational health and safety and mis-administration risks. To address this, a novel transdermal (TD) formulation of ketoprofen was developed, and its efficacy assessed in a study of 36 Holstein-Friesian calves which were assigned to a placebo (n = 10), a TD ketoprofen (n = 10), an IM ketoprofen (n = 10) and sham dehorned group (n = 6). TD ketoprofen significantly reduced plasma cortisol concentrations between 1 to 4 h after dehorning compared to placebo treated calves, with concentrations at 2 and 4 h being very similar to those for sham dehorned calves. The expected log count of positively associated pain variables (ear flick, tail wag, ruminating, head shake, lying down, grooming and neck extending) in the TD group was reduced by 42%, compared to placebo calves, with an overall significant (p < 0.05) treatment effect. The IM group exhibited similar responses and both TD and IM cattle had a higher BW gain at 2 and 5 (p < 0.05) weeks post-dehorning, compared to placebo. This study has shown that TD administered ketoprofen was at least as effective as IM to control pain associated with dehorning and facilitates the administration of analgesic drugs prior to the surgical husbandry procedures being performed.

Hematology and Plasma Biochemistry of Wild Spectacled Flying Foxes ( Pteropus conspicillatus) in Australia.

The spectacled flying fox ( Pteropus conspicillatus) is listed as vulnerable to extinction in Australia. The species' restricted population is in decline, putatively attributed to decreasing habitat, climatic extremes, anthropogenic activities, and more recently, mass mortality events associated with tick paralysis and neonatal cleft palate syndrome. Knowledge of fundamental physiologic parameters of the species is limited. To address this knowledge gap, we sampled 50 wild-caught adult spectacled flying foxes in June (winter) in Far North Queensland, Australia. Hematologic and plasma biochemistry reference ranges were established, and a suite of urine biochemistry analytes were measured. Analyte values were compared within spectacled flying fox sex cohorts and between the spectacled flying fox and the paraphyletic black flying fox ( Pteropus alecto). Significant differences in multiple analytes (including erythrocyte, leucocyte, plasma, and urine biochemistry) were found between spectacled flying fox sex cohorts. The majority of spectacled flying fox analyte values did not differ significantly from black flying fox values. Of those analytes that differed between species (erythrocyte, platelet, eosinophil, liver enzyme, and triglyceride levels), the majority were plausibly explained by intraerythrocyte parasite burden and food resource type. Our findings provide baseline data essential to measure and meaningfully interpret flying fox population health in ecologic, conservation, and epidemiologic contexts.

Gastrointestinal Parasites in Shelter Dogs: Occurrence, Pathology, Treatment and Risk to Shelter Workers.

Dogs entering shelters can carry gastrointestinal parasites that may pose serious risks to other animals, shelter staff and visitors. Shelters provide an environment that could facilitate the spread of parasitic infections between animals. Nematodes and protozoa that transmit through ingestion or skin penetration are major enteric parasites of concern in shelter settings. Ancylostoma spp., Uncinaria stenocephala, Toxocara canis, Toxascaris leonina, Trichuris vulpis and Dipylidium caninum are the major helminths while Giardia, Cryptosporidium, Isospora spp. and Sarcocystis spp. are the most prevalent protozoan parasites in shelter dogs. The prevalence of gastrointestinal parasites in shelter dogs is typically higher than in owned dogs. A range of cost-effective drugs is available for prevention and control of helminths in shelters, notably fenbendazole, pyrantel, oxantel, and praziquantel. Parasiticide options for protozoan parasites are often cost-prohibitive or limited by a lack of veterinary registration for use in dogs. Environmental control measures reliant upon hygiene and facility management are therefore a mainstay for control and prevention of protozoan parasites in shelters. This philosophy should also extend to helminth control, as integrated parasite control strategies can allow anthelmintics to be used more sparingly and judiciously. The purpose of this article is to comprehensively review the current knowledge on the prevalence of gastrointestinal parasites most commonly found in dogs in shelters, canvass recommended treatment programs in shelter dogs, and to explore the likelihood that parasiticide resistance might emerge in a shelter environment.

Physiological stress and Hendra virus in flying-foxes (Pteropus spp.), Australia.

Pteropid bats (flying-foxes) are the natural reservoir of Hendra virus, an emergent paramyxovirus responsible for fatal infection in horses and humans in Australia. Pteropus alecto (the Black flying-fox) and the paraphyletic P. conspicillatus (the Spectacled flying-fox) appear to be the primary reservoir hosts. Previous studies have suggested that physiological and ecological factors may underpin infection dynamics in flying-foxes, and subsequent spillover to horses and in turn humans. We sought to examine temporal trends in urinary cortisol concentration in wild Australian flying-fox populations, to elucidate the putative relationship between Hendra virus infection and physiological stress. Pooled and individual urine samples were non-invasively collected from under roosting flying-foxes at two latitudinally disparate regions in the eastern Australian state of Queensland. Hendra virus detection, and (in individual urine samples) sex and species determination were PCR-based. Urinary cortisol measurement used a validated enzyme immunoassay. We found no direct correlation between increased urinary cortisol and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in P. alecto in the lower latitude Southeast Queensland roosts. We hypothesize an indirect association between low winter temperatures and increased Hendra virus infection and excretion, mediated by the physiological cost of thermoregulation. Our findings and our approach are directly relevant to elaboration of the disease ecology of Nipah virus and other emerging henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics across the wildlife/livestock/human interface.

Tracking food intake as bites: Effects on cognitive resources, eating enjoyment, and self-control.

While monitoring food intake is critical for controlling eating, traditional tools designed for this purpose can be impractical when one desires real-time feedback. Further, the act of monitoring can deplete valuable cognitive resources. In response to these concerns, technologies have been developed to aid those wanting to control their food intake. Of note, devices can now track eating in number of bites taken as opposed to more traditional units such as pieces or volume. Through two studies, the current research investigates the effects of tracking food portions at the bite level on cognitive resources, enjoyment of the eating experience, and objective and subjective self-control. Results indicate that using wearable technology to track bite portions, as compared to doing so mentally, (1) reduces cognitive resource depletion, (2) is equally as effective for allowing users to successfully achieve eating goals, and (3) does not reduce enjoyment of the eating experience. These results support the viability of tracking food intake at the bite level, which holds a number of potential implications for eating and weight management.

PHYSIOLOGIC BIOMARKERS AND HENDRA VIRUS INFECTION IN AUSTRALIAN BLACK FLYING FOXES (PTEROPUS ALECTO).

Bats of the genus Pteropus (Pteropodidae), colloquially known as flying foxes, are recognized as the natural reservoir of Hendra virus, a zoonotic paramyxovirus responsible for mortality in horses and humans. Some previous studies have suggested that physiologic and ecologic factors promote Hendra virus infection in flying foxes, and by extension, spillover to horses and humans. However, the impact of Hendra virus infection on relevant physiologic biomarkers in flying foxes has not been measured. Over 12 mo in eastern Australia, we captured and sampled 446 individual black flying foxes ( Pteropus alecto ), a putative primary reservoir host species, and measured a suite of hematologic, plasma biochemistry, and urinary biomarkers. All mean hematologic and biochemical values in both Hendra virus-positive and virus-negative cohorts were within the published reference ranges for black flying foxes. We found no association between Hendra virus infection (as indicated by PCR detection of Hendra virus RNA) and biomarkers for nutritional stress, reproductive stress, or extreme metabolic demand. However, we identified associations between several other biomarkers and Hendra virus infection, which may partly elucidate the physiologic effects of Hendra virus infection in flying foxes. Our findings highlight the need for critical evaluation of putative risk factors for infection in flying foxes and provide insights for future epidemiologic studies of Hendra virus and related viruses in the Pteropus species.

Increased expression of ATP binding cassette transporter genes following exposure of Haemonchus contortus larvae to a high concentration of monepantel in vitro.

BACKGROUND: There is some evidence that ATP binding cassette (ABC) transporters play a role in resistance to anthelmintics, particularly against macrocyclic lactones. Some anthelmintics, including ivermectin (IVM), have been shown to induce transcription of multiple ABC transporters in nematodes; however, the effects of monepantel (MPL) on transcription of these transporter genes has not been studied. METHODS: Larvae of two MPL-susceptible isolates of Haemonchus contortus were exposed to MPL at two concentrations (2.5 and 250 µg/ml) for periods of 3, 6 and 24 h. Transcription levels of sixteen ABC transporter genes were measured at the end of the incubation periods. The consequences of MPL exposure were examined by measuring rhodamine-123 efflux from the larvae, and their sensitivity to subsequent treatment with IVM or levamisole. RESULTS: Multiple ABC transporter genes showed significantly higher transcription in both worm isolates following exposure to MPL at 250 µg/ml for 3, 6 or 24 h, particularly the P-glycoprotein (P-gp) genes pgp-11, pgp-12 and pgp-14. Of these, only pgp-11 maintained the elevated levels 24 h after the end of the drug exposure period. In contrast, there was only a single instance of low-level upregulation as a result of exposure to MPL at 2.5 µg/ml. Larvae exposed to MPL at 250 µg/ml showed an increased efflux of rhodamine-123 and a proportion of the larval population showed an ability to subsequently tolerate higher concentrations of IVM in migration assays. There was no increased tolerance to IVM following pre-exposure to MPL at 2.5 µg/ml. CONCLUSIONS: Exposure of H. contortus larvae to 250 µg/ml MPL results in increased transcription of multiple transporter genes and increased R-123 efflux. The subsequent ability of a proportion of the larvae to tolerate IVM suggests a protective role of ABC transporters across different chemical entities. However, these observations were only made at a concentration of MPL well above that experienced by parasitic life stages in vivo, and hence their significance remains unclear.

Synergism between ivermectin and the tyrosine kinase/P-glycoprotein inhibitor crizotinib against Haemonchus contortus larvae in vitro.

Anthelmintic resistance is a major problem in parasitic nematodes of livestock worldwide. One means to counter resistance is to use synergists that specifically inhibit resistance mechanisms in order to restore the toxicity, and hence preserve the usefulness, of currently available anthelmintics. P-glycoproteins (P-gps) eliminate a wide variety of structurally unrelated xenobiotics from cells, and have been implicated in anthelmintic resistance. Crizotinib is a tyrosine kinase inhibitor under development as a cancer therapeutic. The compound also inhibits P-gps, and has been shown to reverse multidrug resistance in cancer cells. We were therefore interested in determining if the compound was able to increase the sensitivity of Haemonchus contortus larvae to ivermectin, as measured by in vitro larval development and migration assays with a drug-resistant and a -susceptible isolate. In migration assays, co-administration of crizotinib increased the toxicity of ivermectin to resistant larvae (up to 5.7-fold decrease in ivermectin IC50), and rendered the resistant larvae equally or more sensitive to ivermectin than the susceptible isolate. On the other hand, co-administration of crizotinib had no effect on ivermectin sensitivity in the susceptible isolate. In development assays, significant increases in the sensitivity of both the resistant (up to 1.9-fold) and susceptible (up to 1.6-fold) larvae to ivermectin were observed, although the magnitude of the observed synergism was less than seen in migration assays, and the resistant larvae retained significant levels of ivermectin resistance. By highlighting the ability of the P-gp inhibitor crizotinib to increase the sensitivity of H. contortus larvae to ivermectin, this study provides further evidence that P-gp inhibitors are potential tools for modulating the efficacy of anthelmintics. In addition, the differences in the outcomes of the two assays, with 'resistance-breaking' effects being much more marked in migration assays, suggest that some life-stage-specific aspects may exist in the interaction of ivermectin with P-gps in the two worm isolates.

Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae.

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate.

Temporal Variation in Physiological Biomarkers in Black Flying-Foxes (Pteropus alecto), Australia.

Bats of the genus Pteropus (Pteropodidae) are recognised as the natural host of multiple emerging pathogenic viruses of animal and human health significance, including henipaviruses, lyssaviruses and ebolaviruses. Some studies have suggested that physiological and ecological factors may be associated with Hendra virus infection in flying-foxes in Australia; however, it is essential to understand the normal range and seasonal variability of physiological biomarkers before seeking physiological associations with infection status. We aimed to measure a suite of physiological biomarkers in P. alecto over time to identify any seasonal fluctuations and to examine possible associations with life-cycle and environmental stressors. We sampled 839 adult P. alecto in the Australian state of Queensland over a 12-month period. The adjusted population means of every assessed hematologic and biochemical parameter were within the reported reference range on every sampling occasion. However, within this range, we identified significant temporal variation in these parameters, in urinary parameters and body condition, which primarily reflected the normal annual life cycle. We found no evident effect of remarkable physiological demands or nutritional stress, and no indication of clinical disease driving any parameter values outside the normal species reference range. Our findings identify underlying temporal physiological changes at the population level that inform epidemiological studies and assessment of putative physiological risk factors driving Hendra virus infection in P. alecto. More broadly, the findings add to the knowledge of Pteropus populations in terms of their relative resistance and resilience to emerging infectious disease.

Characterisation of the circulating acellular proteome of healthy sheep using LC-MS/MS-based proteomics analysis of serum.

BACKGROUND: Unlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states. The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum. METHODS: Serum samples from healthy sheep were subjected to shotgun proteomic analysis using nano liquid chromatography nano electrospray ionisation tandem mass spectrometry (nanoLC-nanoESI-MS/MS) on a quadrupole time-of-flight instrument (TripleTOF® 5600+, SCIEX). Proteins were identified using ProteinPilot™ (SCIEX) and Mascot (Matrix Science) software based on a minimum of two unmodified highly scoring unique peptides per protein at a false discovery rate (FDR) of 1% software by searching a subset of the Universal Protein Resource Knowledgebase (UniProtKB) database (http://www.uniprot.org). PeptideShaker (CompOmics, VIB-UGent) searches were used to validate protein identifications from ProteinPilot™ and Mascot. RESULTS: ProteinPilot™ and Mascot identified 245 and 379 protein groups (IDs), respectively, and PeptideShaker validated 133 protein IDs from the entire dataset. Since Mascot software is considered the industry standard and identified the most proteins, these were analysed using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification tool revealing the association of 349 genes with 127 protein pathway hits. These data are available via ProteomeXchange with identifier PXD004989. CONCLUSIONS: These results demonstrated for the first time the feasibility of characterising the ovine circulating acellular proteome using nanoLC-nanoESI-MS/MS. This peptide spectral data contributes to a protein library that can be used to identify a wide range of proteins in ovine serum.

Susceptibility of Adult Cat Fleas (Siphonaptera: Pulicidae) to Insecticides and Status of Insecticide Resistance Mutations at the Rdl and Knockdown Resistance Loci.

The susceptibility of 12 field-collected isolates and 4 laboratory strains of cat fleas, Ctenocephalides felis was determined by topical application of some of the insecticides used as on-animal therapies to control them. In the tested field-collected flea isolates the LD50 values for fipronil and imidacloprid ranged from 0.09 to 0.35 ng/flea and 0.02 to 0.19 ng/flea, respectively, and were consistent with baseline figures published previously. The extent of variation in response to four pyrethroid insecticides differed between compounds with the LD50 values for deltamethrin ranging from 2.3 to 28.2 ng/flea, etofenprox ranging from 26.7 to 86.7 ng/flea, permethrin ranging from 17.5 to 85.6 ng/flea, and d-phenothrin ranging from 14.5 to 130 ng/flea. A comparison with earlier data for permethrin and deltamethrin implied a level of pyrethroid resistance in all isolates and strains. LD50 values for tetrachlorvinphos ranged from 20.0 to 420.0 ng/flea. The rdl mutation (conferring target-site resistance to cyclodiene insecticides) was present in most field-collected and laboratory strains, but had no discernible effect on responses to fipronil, which acts on the same receptor protein as cyclodienes. The kdr and skdr mutations conferring target-site resistance to pyrethroids but segregated in opposition to one another, precluding the formation of genotypes homozygous for both mutations.

Effects of third generation P-glycoprotein inhibitors on the sensitivity of drug-resistant and -susceptible isolates of Haemonchus contortus to anthelmintics in vitro.

P-glycoproteins (P-gps) play an important role in the sensitivity of nematodes to anthelmintic drugs. They have been implicated in a number of anthelmintic resistances, particularly for macrocyclic lactone drugs. Hence, inhibition of nematode P-gps has been suggested as a means of reversing some types of anthelmintic resistance. The present study aimed to investigate the ability of the most-recently developed group of P-gp inhibitors (the so-called 'third generation' of inhibitors) including tariquidar, zosuquidar and elacridar, to increase the sensitivity of Haemonchus contortus larvae to various anthelmintics (ivermectin, levamisole and thiabendazole) in vitro. We compared these compounds to some older P-gp inhibitors (e.g. verapamil and valspodar). Larval migration and development assays were used to measure the sensitivity of larvae to anthelmintics alone, or in combination with P-gp inhibitors. Significant increases in sensitivity to ivermectin were observed with zosuquidar and tariquidar in larval migration assays (synergism ratios up to 6-fold). Several of the inhibitors increased the sensitivity of both the drug-resistant and -susceptible isolates (e.g. tariquidar with ivermectin in migration assays, zosuquidar with ivermectin in larval development assays), while others had significant effects on the resistant isolate only (e.g. zosuquidar with ivermectin in migration assays, verapamil with ivermectin in development assays). This suggests that some of the inhibitors interact with P-gps representing intrinsic pathways present across nematode populations with quite different drug sensitivities, while other inhibitors interact with P-gps of significance only to resistant nematodes, and hence most likely representing an acquired resistance mechanism. The study highlights the potential of the third generation of P-gp inhibitors for increasing the sensitivity of nematodes to anthelmintics.

In vitro levamisole selection pressure on larval stages of Haemonchus contortus over nine generations gives rise to drug resistance and target site gene expression changes specific to the early larval stages only.

There is some evidence that resistance to levamisole and pyrantel in trichostrongylid nematodes is due to changes in the composition of nicotinic acetylcholine receptors (nAChRs) which represent the drug target site. Altered expression patterns of genes coding for nAChR subunits, as well as the presence of truncated versions of several subunits, have been implicated in observed resistances. The studies have mostly compared target sites in worm isolates of very different genetic background, and hence the ability to associate the molecular changes with drug sensitivity alone have been clouded to some extent. The present study aimed to circumvent this issue by following target site gene expression pattern changes as resistance developed in Haemonchus contortus worms under laboratory selection pressure with levamisole. We applied drug selection pressure to early stage larvae in vitro over nine generations, and monitored changes in larval and adult drug sensitivities and target site gene expression patterns. High level resistance developed in larvae, with resistance factors of 94-fold and 1350-fold at the IC50 and IC95, respectively, in larval development assays after nine generations of selection. There was some cross-resistance to bephenium (70-fold increase in IC95). The expression of all the putative subunit components of levamisole-sensitive nAChRs, as well as a number of ancillary protein genes, particularly Hco-unc-29.1 and -ric-3, were significantly decreased (up to 5.5-fold) in the resistant larvae at generation nine compared to the starting population. However, adult worms did not show any resistance to levamisole, and showed an inverse pattern of gene expression changes, with many target site genes showing increased expression compared to the starting population. A comparison of the larval/adult drug sensitivity data with the known relationships for field-derived isolates indicated that the adults of our selected population should have been highly resistant to the drug if the larval/adult sensitivity relationships were in accordance with previous field isolates. Hence, our selected worms showed a life-stage drug sensitivity pattern quite different to that seen in the field. The present study has highlighted an association between drug target site changes and resistance to levamisole in H. contortus larvae. However, it has also highlighted the artificial nature of the larval selection method with levamisole, as the resistance phenotype and the associated molecular changes were only observed in the drug-pressured life stage. The study therefore reinforces the need for caution in extrapolating larval-based laboratory selection outcomes to field resistances.