Strongyle egg shedding and egg reappearance periods in horses with pituitary pars intermedia dysfunction.

Pituitary pars intermedia dysfunction (PPID) is the most common endocrine disorder of older horses. Immune dysfunction in horses with PPID could increase susceptibility to infectious diseases, including strongyle infections; however, few data are available. The aim of this study was to determine if horses with PPID had increased strongyle faecal egg counts (FEC) compared with control horses, over a fourteen-week period in Victoria, Australia. Clinical signs and plasma adrenocorticotropic hormone (ACTH) concentrations were used to categorise horses into PPID (n=14) or control (n=31) groups. Faecal samples were collected for FEC determination prior to anthelmintic treatment, and fortnightly post-treatment for each horse. Generalised linear mixed modelling, using a gamma distribution, was used to compare differences between groups in the repeated measures study. The confounding variable of age was controlled for as a fixed effect. Following anthelmintic treatment, mean FEC was greater for the PPID group compared to the control group on day 56 (405 ± 756 eggs per gram [EPG] vs 40 ± 85 EPG, p=0.05) and day 70 (753 ±1598 EPG vs 82 ±141 EPG, p=0.04). There were no differences in mean FEC between groups on days 84 and 98. Cumulative FEC (day 14 to day 98) was significantly greater for the PPID horses than control horses (2118 ± 4016 EPG vs 798 ± 768 EPG, p<0.0001). Group egg reappearance period was shorter for PPID horses (day 56 post-anthelmintic treatment) compared to control horses (day 70) and 30% of the PPID horses reached a FEC threshold of >200 EPG on day 42, compared to 0% of control horses (p=0.02). These results suggest that the rate of a re-established patent infection between groups could be different due to a comprised immune response in PPID horses or differences in the host-parasite relationship regarding encysted stage larvae. However, despite differences between groups, some horses with PPID consistently had no detectable or low FEC (<200 EPG) during the study period. These findings highlight the importance of individual FEC monitoring to determine if anthelmintic treatment is required, in line with sustainable parasite management practices.

Putting the cart before the horse: mixed-methods participatory investigation of working equid harnessing practices in three selected towns of the Oromia national regional state in Ethiopia.

BACKGROUND: Millions of working equids provide socio-economic support for many low-income communities worldwide. With the prevalence of harness-related wounds reported as higher than 60%, this study aims to describe the equipment used by working equids in three locations of the Ethiopian national regional state of Oromia (Fiche, Bishoftu and Shashamene), and the attitudes and practices of equid owners, users and harness makers regarding work equipment. This mixed-methods study consists of cross-sectional surveying of working equids used for taxi or transport of goods or water, as well as cart-driver questionnaires and focus groups (FG) with working equid stakeholders. Activities conducted with FG included participatory ranking of equipment attributes and equipment drawing exercises. Indicators of equipment design and assembly, as well as cart-driver attitudes and practices were described quantitatively. Associations between equipment characteristics and species, work-type and cart-driver indicators were investigated through univariable logistic regression models, whereas focus group discussions were transcribed and analysed thematically. RESULTS: In total, 368 working equid surveys and cart-driver questionnaires were completed and 87 participants took part in nine FG. Equipment composition and characteristics differed considerably from ideal animal draught and harnessing principles described in the literature, with none of the observed harnesses adhering to all principles and thus not considered fully adequate. Various harness compositions were used, with only saddles and breast collars present in all. Donkey equipment had fewer components than that of horses, such as swingle trees (OR 0.02; 95% CI 0.01-0.06; p < 0.001) or girths (OR 0.09; 95%CI 0.02-0.4; p = 0.002). Horse equipment was more likely to have functional elements such as breeching (OR 7.8; 95% CI 2.9-20.9; p < 0.001). Of all equipment attributes, FG participants ranked cost, design and impact on animal wellbeing as having the highest importance. Thematic analysis identified motivations and priorities regarding equipment; awareness and knowledge of design and function; barriers to using ideal equipment; and consequences of equipment design and practices as key themes. CONCLUSIONS: Various weaknesses of design, assembly and use of equid work equipment were identified. Promoting behavioural change through awareness and training could lead to a sustainable improvement of work equipment quality, access, and prevention of equipment-related problems.

Worm control practices used by Thoroughbred horse managers in Australia: A national survey.

This study assessed worm control practices used by Australian Thoroughbred farm managers with an online questionnaire survey. The questionnaire comprised 52 questions (close-ended: 44; open-ended: 8) about farm demography and general husbandry practices, farm managers' knowledge of gastrointestinal nematodes (GIN) and their importance, diagnosis, worm control strategies and anthelmintics, anthelmintic resistance (AR) and grazing management. Following the pilot survey, the link for the questionnaire survey was sent to all (n = 657) registered members of the Thoroughbred Breeders Australia on 12th April 2020. The response rate for the questionnaire was 18.5% (122 of 675). The farm managers reported a good understanding of GIN and their importance in different age groups of horses as most respondents (70% of 122) perceived worm-related illness to be more important in young (i.e., foals, weanlings and yearlings) than adult (> 3 years old) horses. Although most respondents (93%, 113 of 122) used anthelmintics prophylactically to control GIN, only 15% (18 of 122) observed worm-related illness in their horses. Just under 40% of respondents were performing faecal egg counts, with less than 20% using the results of faecal egg counts to guide deworming decisions. The interval-based deworming strategy was the most common method (≥55% of 122 respondents) to control GIN in all age groups of horses. Macrocyclic lactones were the first choice of anthelmintics for all age groups of horses. Although the majority of respondents (88%, 107 of 122) perceived resistance in GIN against commonly used anthelmintics as an important issue in managing worms in horses, only 29% assessed the efficacy of anthelmintics and 91% (111 of 122) were unaware of AR on their properties. Grazing management practices, such as manure removal, were more frequently performed on smaller paddocks (<0.20 ha: 58%) than on larger paddocks (>0.20 ha: 18%). Multiple correspondence analyses showed that the likelihood of suboptimal worm control practices on small farms (n = ≤50 horses) was greater than that of medium (n = 51-100) and large (n = >100) farms. This study provides insights into the demography of Thoroughbred farms in Australia, husbandry practices used by stud managers and their knowledge about worms, control options and AR concerns, thereby paving the way for taking any initiatives to address the problem of AR in GIN of Australian Thoroughbred horses.

Understanding temporal and spatial distribution of intestinal nematodes of horses using faecal egg counts and DNA metabarcoding.

This study reports the spatial and temporal distribution of ascarid and strongylid nematodes in Thoroughbred horses by age category across different climatic zones in Australia over an 18-month period. Faecal samples (n = 2046) from individual horses were analysed using the modified McMaster technique for faecal egg counts (FECs). Strongylids were identified using PCR-directed next-generation sequencing of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA. Yearlings had the highest prevalence (82%) of strongyle eggs followed by weanlings (79%), foals (58%), wet mares (49%) and dry mares (46%). For Parascaris spp., foals had the highest prevalence (35%) followed by weanlings (21%) and yearlings (10%). The highest mean FECs for Parascaris spp. were observed in foals (525 eggs per gram [EPG] of faeces) while those for strongyles were in yearlings (962 EPG). Among horses that were classified as adults at the time of sampling, 77% (860 of 1119) of mares were low (i.e., <250 EPG) strongyle egg-shedders. Mean strongyle FEC counts were highest in the Mediterranean (818 EPG) followed by summer (599 EPG), winter (442 EPG), and non-seasonal (413 EPG) rainfall zones. Twenty-six nematode species were detected, with Cylicostephanus longibursatus (26.5%), Cylicocyclus nassatus (23.7%) and Coronocyclus coronatus (20.5%) being the most frequently detected species. Their richness and relative abundance varied with horse age, season and climatic zone. In addition, Strongylus equinus and Triodontophorus spp. (T. brevicauda and T. serratus) were also detected. This comprehensive study elucidates spatial (climatic zone) and temporal (i.e., seasonal) trends in prevalence and burdens of intestinal nematodes in Australian horses using non-invasive conventional and molecular methods. The information presented in this study is crucial for developing integrated management strategies to control horse parasites in farmed horses.

Chlamydia psittaci: an emerging cause of equine abortion and fatal neonatal illness in south-eastern Australia.

While well known as a zoonotic pathogen of birds, Chlamydia psittaci is less well recognised as a cause of abortion in mares and severe infection in foals. However, in the past decade it has been causing epizootics of both in south-eastern Australia, as Charles El-Hage and James Gilkerson explain.

A national survey of anthelmintic resistance in ascarid and strongylid nematodes in Australian Thoroughbred horses.

This study quantified the extent of anthelmintic resistance (AR) in ascarid and strongylid nematodes against commonly used anthelmintics in Australian Thoroughbred horses. Faecal egg count reduction tests (FECRTs, n = 86) and egg reappearance period (ERP) tests were conducted on 22 farms across Australia. Faecal egg counts (FECs) were determined using the modified McMaster technique, and percent faecal egg count reduction (%FECR) was calculated using the Bayesian hierarchical model and hybrid Frequentist/Bayesian analysis method. The results were interpreted using old (published in 1992) and new (2023) research guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP). The species composition of strongylid nematodes was detected utilising a DNA-metabarcoding method using pre- and post-treatment samples. Resistance was observed in strongylid nematodes to commonly used single-active and combination anthelmintics, including ivermectin (IVM %FECR range: 82%-92%; 95% lower credible interval (LCI) range: 80%-90%), abamectin (ABM: 73%-92%; 65%-88%), moxidectin (MOX: 89%-91%; 84%-89%), oxfendazole (OFZ: 0%-56%; 0%-31%) and its combination with pyrantel (OFZ + PYR: 0%-82%; 0%-78%). Resistance in Parascaris spp. was observed to IVM (10%-43%; 0%-36%), ABM (0%; 0%) and MOX (0%; 0%). When the new thresholds recommended by the WAAVP were used, AR was detected in six additional FECRTs for strongylids and three more tests for Parascaris spp., introducing resistance to OFZ and OFZ + PYR in the latter. Shortened ERPs (4-6 weeks) of strongylids were observed in 31 FECRTs in which AR was not detected at 2 weeks post-treatment for all the anthelmintics tested. Among cyathostomins, Cylicocyclus nassatus, Cylicostephanus longibursatus and Coronocyclus coronatus were the most prevalent species at 2 weeks post-treatment, whereas the main species appearing at five weeks following treatments with macrocyclic lactones were Cylicocyclus nassatus, Cylicostephanus longibursatus and Cylicocyclus ashworthi. After treatment with OFZ + PYR, the latter three, plus Coronocyclus coronatus and Cyathostomum catinatum, were detected at 5 weeks post-treatment. Overall, the study highlights the prevalence of AR in both ascarids and strongylid nematodes against commonly used anthelmintic products to control worms in Australian horses. The results indicate that ML combination products provided acceptable efficacy at 2 weeks. However, ERP calculations suggest that products work less effectively than previously measured. It is suggested to regularly monitor the efficacy of the anthelmintics and consider changing the worm control practices to better manage worms and AR in Australian horses.

Assessment of worm control practices recommended by equine veterinarians in Australia.

This study aimed to assess Australian veterinarians' knowledge, perceptions and treatment strategies for worm control in horses with an online questionnaire. The questionnaire comprised 64 questions covering various aspects of: (i) veterinary practice; (ii) the veterinarian's knowledge of gastrointestinal nematodes (GINs) and the importance of parasites in different age groups of horses; (iii) the diagnosis and control of worms; (iv) anthelmintics and anthelmintic resistance (AR); (v) grazing management; and (vi) the means of communication and the discussion between veterinarians and their clients regarding worm control. Following a pilot survey, a link for the questionnaire survey was sent to all (n = 1,148) registered members of Equine Veterinarians Australia in April 2020. The response rate for the questionnaire was 10% (118 of 1,148). The findings of this study illustrate veterinarians' good understanding of aspects of equine parasites, including control. However, respondents mainly recommended frequent, interval-based prophylactic deworming in young horses, and only 40% (96 of 239) diagnosed GIN infections based on faecal egg count (FEC) results in all age groups of horses. Furthermore, only 27% (88 of 330) of the respondents made deworming decisions based on FECs. Most of the respondents recommended macrocyclic lactones (MLs) for all age groups of horses (71%, 481 of 677), and the most frequently used method to calculate the dose of anthelmintics was by estimating the weight of animals visually (53%, 63 of 118). Although the majority of respondents (97%, 115 of 118) perceived AR to be a critical issue in managing worms in horses, 58% (67 of 118) of them were unaware of the status of AR on their clients' properties. Forty-two percent (50 of 118) of the respondents perceived the presence of AR in worms, including pinworms (16%), strongylins (15%), species of Draschia and Habronema (6%), Strongyloides westeri (2%) and tapeworms (1%). Twenty-seven percent (32 of 118) of the respondents rarely discussed equine worm control practices with their clients. This study provides insights into the perception and worm control practices recommended by Australian veterinarians to manage equine parasites. The findings highlight the importance of continued education and awareness of AR, and the use of non-chemical methods as well as consideration of the legislation of prescription-only use of anthelmintics based on FECs to achieve sustainable control of GINs in Australian horses.

Genomic diversity and natural recombination of equid gammaherpesvirus 5 isolates.

BACKGROUND: Equid gammaherpesvirus 5 (EHV5) is closely related to equid gammaherpesvirus 2 (EHV2). Detection of EHV5 is frequent in horse populations worldwide, but it is often without a clear and significant clinical impact. Infection in horses can often present as subclinical disease; however, it has been associated with respiratory disease, including equine multinodular pulmonary fibrosis (EMPF). Genetic heterogeneity within small regions of the EHV5 glycoprotein B (gB) sequences have been reported and multiple genotypes of this virus have been identified within individual horses, but full genome sequence data for these viruses is limited. The primary focus of this study was to assess the genomic diversity and natural recombination among EHV5 isolates. RESULTS: The genome size of EHV5 prototype strain and the five EHV5 isolates cultured for this study, including four isolates from the same horse, ranged from 181,929 to 183,428 base pairs (bp), with the sizes of terminal repeat regions varying from 0 to 10 bp. The nucleotide sequence identity between the six EHV5 genomes ranged from 95.5 to 99.1%, and the estimated average nucleotide diversity between isolates was 1%. Individual genes displayed varying levels of nucleotide diversity that ranged from 0 to 19%. The analysis of nonsynonymous substitution (Ka > 0.025) revealed high diversity in eight genes. Genome analysis using RDP4 and SplitsTree programs detected evidence of past recombination events between EHV5 isolates. CONCLUSION: Genomic diversity and recombination hotspots were identified among EHV5 strains. Recombination can drive genetic diversity, particularly in viruses that have a low rate of nucleotide substitutions. Therefore, the results from this study suggest that recombination is an important contributing factor to EHV5 genomic diversity. The findings from this study provide additional insights into the genetic heterogeneity of the EHV5 genome.

Prevalence and diversity of ascarid and strongylid nematodes in Australian Thoroughbred horses using next-generation sequencing and bioinformatic tools.

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n = 1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10-14%) and 72% (70-74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251-500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses.

Equine Psittacosis and the Emergence of Chlamydia psittaci as an Equine Abortigenic Pathogen in Southeastern Australia: A Retrospective Data Analysis.

Chlamydia psittaci is an important zoonotic pathogen. Although primarily a pathogen of birds, from which infection can spillover into humans and other mammalian hosts, the importance of C. psittaci as a cause of equine reproductive loss and the risk of infection to humans in contact with infected horses are increasingly being recognised in Australia and elsewhere. Despite the risks to both human and equine health, C. psittaci infection in horses is incompletely understood. This study aimed to update and summarise cases of equine psittacosis in Australia in the period 2018-2022, thus addressing a knowledge gap relating to recent cases in this country. These cases were identified from the examination of records held by state and federal veterinary authorities and from a review of published cases. A total of 31 cases were identified. Spatial and temporal trends were identified, with cases being more prevalent in winter and spring and geographically restricted to Victoria and New South Wales. The results show that cases of equine reproductive loss due to C. psittaci are consistent and ongoing and demonstrate the importance of routinely considering C. psittaci in diagnostic investigations. The need for ongoing study to better understand this important zoonotic pathogen is evident.

Egg reappearance periods of anthelmintics against equine cyathostomins: The state of play revisited.

Cyathostomins are the most common and highly prevalent parasites of horses worldwide. Historically, the control of cyathostomins has mainly relied on the routine use of anthelmintic products. Increasing reports on anthelmintic resistance (AR) in cyathostomins are concerning. A potential method proposed for detecting emerging AR in cyathostomins has been estimating the egg reappearance period (ERP). This paper reviews the data available for the ERP of cyathostomins against the three major classes of anthelmintics, macrocyclic lactones, tetrahydropyrimidines, and benzimidazoles. Published peer-reviewed original research articles were obtained from three databases (PubMed, CAB Direct and Web of Science) and were evaluated for their inclusion in a systematic review. Subsets of articles were then subjected to a review of ERP data. A total of 54 (of 134) studies published between 1972 and 2022 met the criteria for inclusion in the systematic review. Until the beginning of 2022, there was no agreed definition of the ERP; eight definitions of ERP were identified in the literature, complicating the comparison between studies. Additionally, potential risk factors for the shortening of the ERP, including previous anthelmintic use and climate, were frequently not described. Reports of shortened ERP for moxidectin and ivermectin are frequent: 20 studies that used comparable ERP definitions reported shortened moxidectin and ivermectin ERPs of 35 and 28 days, respectively. It is unclear whether the ERPs of these anthelmintics reduced to such levels are due to the development of AR or some biological factors related to horses, cyathostomin species, and/or the environment. The ERPs for other anthelmintics, such as fenbendazole and pyrantel, were frequently not reported due to established resistance against these drugs. Future research in horses is required to understand the mechanism(s) behind the shortening of ERP for cyathostomins. Based on this systematic review, we propose recommendations for future ERP studies.

Molecular diagnostics for gastrointestinal helminths in equids: Past, present and future.

This review is aimed to (i) appraise the literature on the use of molecular techniques for the detection, quantification and differentiation of gastrointestinal helminths (GIH) of equids, (ii) identify the knowledge gaps and, (iii) discuss diagnostic prospects in equine parasitology. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we retrieved 54 studies (horses: 50/54; donkeys and zebras: 4/54) from four databases. Polymerase chain reaction (PCR) was employed in all of the studies whereas PCR amplicons were sequenced in only 18 of them. Other techniques used (including modifications of PCR) were reverse line blot, quantitative (q)PCR, restriction fragment length polymorphism, nested-PCR, PCR-directed next-generation sequencing, Southern blotting, single strand conformation polymorphism, PCR-enzyme linked immunosorbent assay, matrix-assisted laser desorption/ionisation-time of flight and random amplification of polymorphic DNA. Most of the studies (53/54) used nuclear ribosomal RNA (including the internal transcribed spacers, intergenic spacer, 5.8 S, 18 S, 28 S and 12 S) as target loci while cytochrome c oxidase subunit 1 and random genomic regions were targeted in only three and one studies, respectively. Overall, to date, the majority of molecular studies have focused on the diagnosis and identification of GIHs of equids (i.e. species of Anoplocephala, Craterostomum, cyathostomins, Oesophagodontus, Parascaris, Strongylus, Strongyloides and Triodontophorus), with a recent shift towards investigations on anthelmintic resistance and the use of high-throughput nemabiome metabarcoding. With the increasing reports of anthelmintic resistance in equid GIHs, it is crucial to develop and apply techniques such as advanced metabarcoding for surveillance of parasite populations in order to gain detailed insights into their diversity and sustainable control. To the best of our knowledge, this is the first systematic review that evaluates molecular investigations published on the diagnosis and quantification of equid GIHs and provides useful insights into important knowledge gaps and future research directions in equid molecular parasitology.

Prevalence and Factors Associated with Working Equid Lameness in Low- and Middle-Income Countries: A Systematic Review and Meta-Analysis.

Lameness is an important concern in working equids of low- and middle-income communities (LMICs) with significant One Welfare implications. This study aims to determine the prevalence and influencing factors of lameness in working equids of LMICs. A systematic review and meta-analysis were performed to investigate pooled outcome prevalence using a random intercept regression model. Subgroup and sensitivity analysis were performed through meta-regression. A meta-analysis of study factors for lameness prevalence was performed. Sixty-four studies were included in the review. The pooled prevalence of lameness was 29.9% (n = 42, 95% CI 17-47%), while the pooled prevalence of gait abnormality was 62.9% (n = 12; 95% CI 31-87%). When considering both outcomes together, the pooled prevalence was 38.4% (n = 46; 95% CI 23-57%) with a significant (p = 0.02) difference between lameness (29.5%; 95% CI 16-48%) and gait abnormality (78.8%; 95% CI 40-95%). Species, country income level, gait assessed, and risk of bias did not significantly affect the pooled prevalence. Lower body condition scores, unresponsive attitudes, and old age were the most frequently reported factors positively associated with lameness-related outcomes. Working 7 days per week was positively associated with lameness. The standardization of outcome terminology, grading systems, and study factor categorization is recommended to enable more accurate interpretation and comparison between studies.

Whole genome sequence analysis of equid gammaherpesvirus -2 field isolates reveals high levels of genomic diversity and recombination.

BACKGROUND: Equid gammaherpesvirus 2 (EHV2) is a gammaherpesvirus with a widespread distribution in horse populations globally. Although its pathogenic significance can be unclear in most cases of infection, EHV2 infection can cause upper respiratory tract disease in foals. Co-infection of different strains of EHV2 in an individual horse is common. Small regions of the EHV2 genome have shown considerable genetic heterogeneity. This could suggest genomic recombination between different strains of EHV2, similar to the extensive recombination networks that have been demonstrated for some alphaherpesviruses. This study examined natural recombination and genome diversity of EHV2 field isolates. RESULTS: Whole genome sequencing analysis of 18 EHV2 isolates, along with analysis of two publicly available EHV2 genomes, revealed variation in genomes sizes (from 173.7 to 184.8 kbp), guanine plus cytosine content (from 56.7 to 57.8%) and the size of the terminal repeat regions (from 17,196 to 17,551 bp). The nucleotide sequence identity between the genomes ranged from 86.2 to 99.7%. The estimated average inter-strain nucleotide diversity between the 20 EHV2 genomes was 2.9%. Individual gene sequences showed varying levels of nucleotide diversity and ranged between 0 and 38.1%. The ratio of nonsynonymous substitutions, Ka, to synonymous substitutions, Ks, (Ka/Ks) suggests that over 50% of EHV2 genes are undergoing diversifying selection. Recombination analyses of the 20 EHV2 genome sequences using the recombination detection program (RDP4) and SplitsTree revealed evidence of viral recombination. CONCLUSIONS: Analysis of the 18 new EHV2 genomes alongside the 2 previously sequenced genomes revealed a high degree of genetic diversity and extensive recombination networks. Herpesvirus genome diversification and virus evolution can be driven by recombination, and our findings are consistent with recombination being a key mechanism by which EHV2 genomes may vary and evolve.

Assessment of Humoral and Long-Term Cell-Mediated Immune Responses to Recombinant Canarypox-Vectored Equine Influenza Virus Vaccination in Horses Using Conventional and Accelerated Regimens Respectively.

During Australia's first and only outbreak of equine influenza (EI), which was restricted to two northeastern states, horses were strategically vaccinated with a recombinant canarypox-vectored vaccine (rCP-EIV; ProteqFlu™, Merial P/L). The vaccine encoded for haemagglutinin (HA) belonging to two equine influenza viruses (EIVs), including an American and Eurasian lineage subtype that predated the EIV responsible for the outbreak (A/equine/Sydney/07). Racehorses in Victoria (a southern state that remained free of EI) were vaccinated prophylactically. Although the vaccine encoded for (HA) belonged to two EIVs of distinct strains of the field virus, clinical protection was reported in vaccinated horses. Our aim is to assess the extent of humoral immunity in one group of vaccinated horses and interferon-gamma ((EIV)-IFN-γ)) production in the peripheral blood mononuclear cells (PBMCs) of a second population of vaccinated horses. Twelve racehorses at work were monitored for haemagglutination inhibition antibodies to three antigenically distinct equine influenza viruses (EIVs) The EIV antigens included two H3N8 subtypes: A/equine/Sydney/07) A/equine/Newmarket/95 (a European lineage strain) and an H7N7 subtype (A/equine/Prague1956). Cell-mediated immune responses of: seven racehorses following an accelerated vaccination schedule, two horses vaccinated using a conventional regimen, and six unvaccinated horses were evaluated by determining (EIV)-IFN-γ levels. Antibody responses following vaccination with ProteqFlu™ were cross-reactive in nature, with responses to both H3N8 EIV strains. Although (EIV)IFN-γ was clearly detected following the in vitro re-stimulation of PBMC, there was no significant difference between the different groups of horses. Results of this study support reports of clinical protection of Australian horses following vaccination with Proteq-Flu™ with objective evidence of humoral cross-reactivity to the outbreak viral strain A/equine/Sydney/07.

Cyathostomin resistance to moxidectin and combinations of anthelmintics in Australian horses.

BACKGROUND: Cyathostomins are the most important and common parasitic nematodes of horses, with > 50 species known to occur worldwide. The frequent and indiscriminate use of anthelmintics has resulted in the development of anthelmintic resistance (AR) in horse nematodes. In this study we assessed the efficacy of commonly used anthelmintics against cyathostomins in Australian thoroughbred horses. METHODS: Two drug efficacy trials per farm were conducted on two thoroughbred horse farms in the state of Victoria, Australia. In the first trial, the horses on Farm A were treated with single and combinations of anthelmintics, including oxfendazole (OFZ), abamectin (ABM), abamectin and morantel (ABM + MOR), moxidectin (MOX) and oxfendazole and pyrantel (OFZ + PYR), at the recommended doses, whereas the horses on Farm B only received MOX, at the recommended dose. The faecal egg count reduction test (FECRT) was used to determine the efficacy and egg reappearance period (ERP) of anthelmintics. Based on the results of the first trial, the efficacies of MOX and a combination of ABM + MOR were reassessed to confirm their activities against cyathostomins. RESULTS: Of the five anthelmintic products tested on Farm A, resistance against OFZ, ABM and OFZ + PYR was found, with efficacies of - 41% (- 195% lower confidence limit [LCL]), 73% (60% LCL) and 82% (66% LCL) at 2 weeks post-treatment, respectively. The FECRT showed high efficacies of MOX and ABM + MOR (100%) at 2 week post-treatment and shortened ERPs for these anthelmintics (ABM + MOR: 4 weeks; MOX: 5 weeks). Resistance to MOX was found on Farm B, with a reduced efficacy of 90% (70% LCL) and 89% (82% LCL) at 2 weeks post-treatment in trials one and two, respectively. CONCLUSIONS: This study provides the first evidence of MOX- and multidrug-resistant (ABM and combinations of anthelmintics) cyathostomins in Australia and indicates the need for continuous surveillance of the efficacy of currently effective anthelmintics and large-scale investigations to assess the ERP for various anthelmintics.

Association of Equine Herpesvirus 5 with Mild Respiratory Disease in a Survey of EHV1, -2, -4 and -5 in 407 Australian Horses.

Equine herpesviruses (EHVs) are common respiratory pathogens in horses; whilst the alphaherpesviruses are better understood, the clinical importance of the gammaherpesviruses remains undetermined. This study aimed to determine the prevalence of, and any association between, equine respiratory herpesviruses EHV1, -2, -4 and -5 infection in horses with and without clinical signs of respiratory disease. Nasal swabs were collected from 407 horses in Victoria and included clinically normal horses that had been screened for regulatory purposes. Samples were collected from horses during Australia's equine influenza outbreak in 2007; however, horses in Victoria required testing for proof of freedom from EIV. All horses tested in Victoria were negative for EIV, hence archived swabs were available to screen for other pathogens such as EHVs. Quantitative PCR techniques were used to detect EHVs. Of the 407 horses sampled, 249 (61%) were clinically normal, 120 (29%) presented with clinical signs consistent with mild respiratory disease and 38 (9%) horses had an unknown clinical history. Of the three horses detected shedding EHV1, and the five shedding EHV4, only one was noted to have clinical signs referable to respiratory disease. The proportion of EHV5-infected horses in the diseased group (85/120, 70.8%) was significantly greater than those not showing signs of disease (137/249, 55%). The odds of EHV5-positive horses demonstrating clinical signs of respiratory disease were twice that of EHV5-negative horses (OR 1.98, 95% CI 1.25 to 3.16). No quantitative difference between mean loads of EHV shedding between diseased and non-diseased horses was detected. The clinical significance of respiratory gammaherpesvirus infections in horses remains to be determined; however, this survey adds to the mounting body of evidence associating EHV5 with equine respiratory disease.

Metagenomic investigation of potential abortigenic pathogens in foetal tissues from Australian horses.

BACKGROUND: Abortion in horses leads to economic and welfare losses to the equine industry. Most cases of equine abortions are sporadic, and the cause is often unknown. This study aimed to detect potential abortigenic pathogens in equine abortion cases in Australia using metagenomic deep sequencing methods. RESULTS: After sequencing and analysis, a total of 68 and 86 phyla were detected in the material originating from 49 equine abortion samples and 8 samples from normal deliveries, respectively. Most phyla were present in both groups, with the exception of Chlamydiae that were only present in abortion samples. Around 2886 genera were present in the abortion samples and samples from normal deliveries at a cut off value of 0.001% of relative abundance. Significant differences in species diversity between aborted and normal tissues was observed. Several potential abortigenic pathogens were identified at a high level of relative abundance in a number of the abortion cases, including Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Streptococcus equi subspecies zooepidemicus, Pantoea agglomerans, Acinetobacter lwoffii, Acinetobacter calcoaceticus and Chlamydia psittaci. CONCLUSIONS: This work revealed the presence of several potentially abortigenic pathogens in aborted specimens. No novel potential abortigenic agents were detected. The ability to screen samples for multiple pathogens that may not have been specifically targeted broadens the frontiers of diagnostic potential. The future use of metagenomic approaches for diagnostic purposes is likely to be facilitated by further improvements in deep sequencing technologies.

Molecular detection of Strongyloides sp. in Australian Thoroughbred foals.

BACKGROUND: Strongyloides westeri is found in the small intestine of young horses, mainly in foals up to about 16 weeks of age. The main source of infection for foals is through transmammary transmission, and foals can develop acute diarrhoea, weakness, dermatitis and respiratory signs. The epidemiology of S. westeri in Australia is largely unknown. Further, molecular techniques have never been employed for detection of S. westeri in horses. This pilot study aimed to assess the utility of a molecular phylogenetic method for the detection of S. westeri in the faeces of foals. METHODS: Faecal samples were collected from a foal of less than 2 months of age, and eggs of Strongyloides sp. were detected using the modified McMaster technique. DNA was extracted from purified eggs, and a partial fragment of the small subunit of the nuclear ribosomal DNA (18S) was characterised using polymerase chain reaction, DNA sequencing and phylogenetic methods. RESULTS: Microscopic examination of faeces revealed small ellipsoidal eggs typical of Strongyloides sp. The 18S sequence generated by PCR in this study revealed 98.4% identity with that of a reference sequence of S. westeri available from GenBank. Phylogenetic analyses revealed a polyphyletic clustering of S. westeri sequences. CONCLUSION: This is the first study reporting the detection of DNA of Strongyloides sp. in faeces of a foal using a molecular phylogenetic approach targeting the variable region of 18S rDNA. It is anticipated that this study will allow future molecular epidemiological studies on S. westeri in horses.