Oocyst shedding patterns of Eimeria species and their association with management and performance at ten rose veal starter farms in the Netherlands.

Coccidiosis at rose veal starter farms is often diagnosed however, this was the first study performed considering this topic on this type of rearing unit. The objective of this study was to identify Eimeria species faecal shedding patterns at ten Dutch rose veal starter farms during rearing. Further objectives were to investigate associations with (gut) health, production and management decisions. Faecal samples from twelve randomly selected calves per farm were collected weekly during 9 consecutive weeks. Thereafter samples were pooled in a predetermined composition of six. These calves were clinically evaluated every sample visit and weighed at the first and last visit. Laboratory tests included a flotation test of the samples. If this yielded Eimeria oocysts, both oocysts per gram feces (OPG) and species differentiation were established using a modified McMaster method. Management parameters and technical herd results were identified after finalizing the study period using a questionnaire. Studied patterns in oocyst shedding included the pool's cumulated OPG, maximum OPG level and the number of sample days (SD) that OPG exceeded a confirmed level. Statistical analysis included univariate and multivariate analysis. Associated (p < 0.10) OPG patterns considering rumen fill, faecal consistency and average daily weight gain (ADG) were tested using a model with herd included as random effect. RESULTS: on all ten farms E. alabamensis, E.bovis and E. zuernii besides non-pathogenic species were identified, often as mixed infections. Peak OPG occurred predominately at SD 21 and 28 (E. alabamensis and E. bovis), and at SD 42 (E. zuernii). In 16 pools, OPG levels ≥500 for E. bovis or E. zuernii, were found. Significant correlation (p < 0.0001) showed between the log10 OPG of pathogenic Eimeria and of all Eimeria species. Multivariate analysis showed significant correlation between cumulative faecal consistency scores and cumulative log10 OPG of pathogenic Eimeria species (ß = 0.16; p = 0.008). Pools exceeding 750 OPG for E. bovis showed 93 g lower ADG and pools experiencing ≥2 SD with >1000 OPG for E. alabamensis 141 g lower ADG. From the questionnaire we identified lower cumulated OPG of all Eimeria species except for E. bovis at farms where the units were cleaned before arrival of the calves. CONCLUSION: As a rule, on Dutch rose veal starter farms, mixed Eimeria infections occur, but shedding patterns differ between farms. Clinical and growth performance is related to OPG patterns found. Cleaning units before arrival of calves lowers oocyst shedding during the rearing period.

The lipopolysaccharide model for the experimental induction of transient lameness and synovitis in Standardbred horses.

An established lipopolysaccharide (LPS) model previously described in Warmbloods, was inconsistent in Standardbred horses, where lameness was not detected despite the presence of synovitis. The present study aimed to determine the dose of LPS from E. coli O55:B5 required to induce mild to moderate lameness following middle carpal joint injection in Standardbred horses and to quantitate the induced lameness over time, with and without anti-inflammatory pre-treatment. In a baseline trial, eight healthy, clinically sound Standardbred horses were used in a rule-based dose-escalation design trial, starting at a dose of 10 endotoxin units (EU). Lameness at trot was evaluated visually and quantitatively (using an inertial-sensor system and pressure plate analysis). Synovial fluid aspirates were analysed for total nucleated cell counts, total protein and prostaglandin E2 (PGE2). Following 2 months wash-out, the effective LPS-dose determined in the baseline trial was used to evaluate the effect of anti-inflammatory treatment. A mixed model for repeated measures with horse as random effect was used for analysis. After injection of 10 EU LPS, the desired degree of lameness was observed in the baseline trial, with maximal lameness at post-injection hour (PIH) 4, followed by a rapid decline and return to baseline by PIH 48. No lameness was observed following pre-treatment with meloxicam. In synovial fluid, PGE2 was significantly higher at PIH 8 and PIH 24 in the baseline trial compared with following meloxicam pre-treatment. In conclusion, injection of the middle carpal joint with 10 EU LPS consistently induces a transient lameness and synovitis in Standardbred horses.

Dynamic evaluation of toe-heel and medio-lateral load distribution and hoof landing patterns in sound, unshod Standardbred horses with toed-in, toed-out and normal hoof conformation.

Dynamic load distribution and landing patterns play an important role in equine orthopaedics. The aim of this study was to analyse dynamic load distribution and hoof landing patterns of sound Standardbreds. Twenty-four sound, unshod Standardbreds were walked and trotted over a pressure plate to obtain the peak vertical force (PVF), vertical impulse (VI) and stance time (ST) of both forelimbs. Asymmetry indices between both forelimbs were calculated and the dynamic medio-lateral and toe-heel load distribution of the forelimbs were compared between normal, toed-in and toed-out horses. The hoof landing patterns were determined on the first loaded zone. Linear mixed models and Chi-square analysis were used for statistical analysis (α = 0.05). At walk and trot, there were no significant differences in asymmetry of PVF, VI and ST between normal (n = 9/24 horses), toed-in (n = 6/24 horses), and toed-out horses (n = 9/24 horses). In both forelimbs, a flat landing (39.6% and 70.8% at walk and trot, respectively) predominated over all hoof conformations. No significant association between hoof conformation and landing pattern was observed (P > 0.05). Toed-out horses had significantly higher loading of the medial zone at the end of the stance phase at walk than normal horses (P = 0.002). No other statistically significant differences in load distribution were observed (P > 0.05). In conclusion, flat landing was more common in Standardbred horses than previously described in Warmbloods, emphasising the need for breed-specific clinical reference data and biomechanical studies. Moreover, mild deviations in hoof conformation did not necessarily affect load distribution.

Dietary supplementation of micro-encapsulated sodium butyrate in healthy horses: effect on gut histology and immunohistochemistry parameters.

BACKGROUND: As colic and intestinal disorders are a major concern in horses, the aim of the present study was to investigate the effect of dietary supplementation of butyrate, known to have a diverse array of beneficial effects on intestinal health. The effect of micro-encapsulated sodium butyrate supplementation on gut histology and immunohistochemistry parameters was studied in 14 healthy warmblood horses destined for slaughter in two separate periods. Horses were fed a low fiber - high starch diet, designed to induce subsequent starch overflow in the large intestine, aiming to create a mild challenge for large intestinal health. Treatment included supplementation with either micro-encapsulated sodium butyrate (Excential Butycoat®, Orffa, Werkendam, the Netherlands) or placebo (containing only coating material). The horses were fed for 20 consecutive days at a dosage of 0.4 g/kg BW (body weight). At day 21, the horses were slaughtered and intestinal samples were collected for determination of gut pH, villus length, crypt depth and area % of CD3+ and CD20+ cells. RESULTS: Horses on the butyrate supplemented diet had significantly reduced crypt depths in the right dorsal colon compared to placebo-fed horses (P < 0.001). However, a treatment x period interaction (P = 0.002) was discovered regarding this parameter, which could not be explained by the authors. Further investigation into the number of KI67+ cells in the RDC crypts did not reveal any significant differences between treatments (P = 0.650), indicating that the reduction in crypt depth in butyrate-fed horses could not be explained by a significant difference in cellular proliferation. Intestinal pH, villus length and expression of intestinal CD3+ and CD20+ cells were not significantly affected by treatment at any intestinal level. CONCLUSIONS: Our data indicate that supplementation of micro-encapsulated sodium butyrate to the equine diet did not influence gut histology (with the exception of a decrease found in the crypts of the RDC) or immunohistochemistry parameters in healthy horses. Further research is warranted to investigate the impact of butyrate supplementation in horses with intestinal disease.

Domesticated equine species and their derived hybrids differ in their fecal microbiota.

BACKGROUND: Compared to horses and ponies, donkeys have increased degradation of dietary fiber. The longer total mean retention time of feed in the donkey gut has been proposed to be the basis of this, because of the increased time available for feed to be acted upon by enzymes and the gut microbiota. However, differences in terms of microbial concentrations and/or community composition in the hindgut may also underpin the increased degradation of fiber in donkeys. Therefore, a study was conducted to assess if differences existed between the fecal microbiota of pony, donkey and hybrids derived from them (i.e. pony × donkey) when fed the same forage diet. RESULTS: Fecal community composition of prokaryotes and anaerobic fungi significantly differed between equine types. The relative abundance of two bacterial genera was significantly higher in donkey compared to both pony and pony x donkey: Lachnoclostridium 10 and 'probable genus 10' from the Lachnospiraceae family. The relative abundance of Piromyces was significantly lower in donkey compared to pony × donkey, with pony not significantly differing from either of the other equine types. In contrast, the uncultivated genus SK3 was only found in donkey (4 of the 8 animals). The number of anaerobic fungal OTUs was also significantly higher in donkey than in the other two equine types, with no significant differences found between pony and pony × donkey. Equine types did not significantly differ with respect to prokaryotic alpha diversity, fecal dry matter content or fecal concentrations of bacteria, archaea and anaerobic fungi. CONCLUSIONS: Donkey fecal microbiota differed from that of both pony and pony × donkey. These differences related to a higher relative abundance and diversity of taxa with known, or speculated, roles in plant material degradation. These findings are consistent with the previously reported increased fiber degradation in donkeys compared to ponies, and suggest that the hindgut microbiota plays a role. This offers novel opportunities for pony and pony × donkey to extract more energy from dietary fiber via microbial mediated strategies. This could potentially decrease the need for energy dense feeds which are a risk factor for gut-mediated disease.

Multi-kingdom characterization of the core equine fecal microbiota based on multiple equine (sub)species.

BACKGROUND: Equine gut microbiology studies to date have primarily focused on horses and ponies, which represent only one of the eight extant equine species. This is despite asses and mules comprising almost half of the world's domesticated equines, and donkeys being superior to horses/ponies in their ability to degrade dietary fiber. Limited attention has also been given to commensal anaerobic fungi and archaea even though anaerobic fungi are potent fiber degrading organisms, the activity of which is enhanced by methanogenic archaea. Therefore, the objective of this study was to broaden the current knowledge of bacterial, anaerobic fungal and archaeal diversity of the equine fecal microbiota to multiple species of equines. Core taxa shared by all the equine fecal samples (n = 70) were determined and an overview given of the microbiota across different equine types (horse, donkey, horse × donkey and zebra). RESULTS: Equine type was associated with differences in both fecal microbial concentrations and community composition. Donkey was generally most distinct from the other equine types, with horse and zebra not differing. Despite this, a common bacterial core of eight OTUs (out of 2070) and 16 genus level groupings (out of 231) was found in all the fecal samples. This bacterial core represented a much larger proportion of the equine fecal microbiota than previously reported, primarily due to the detection of predominant core taxa belonging to the phyla Kiritimatiellaeota (formerly Verrucomicrobia subdivision 5) and Spirochaetes. The majority of the core bacterial taxa lack cultured representation. Archaea and anaerobic fungi were present in all animals, however, no core taxon was detected for either despite several taxa being prevalent and predominant. CONCLUSIONS: Whilst differences were observed between equine types, a core fecal microbiota existed across all the equines. This core was composed primarily of a few predominant bacterial taxa, the majority of which are novel and lack cultured representation. The lack of microbial cultures representing the predominant taxa needs to be addressed, as their availability is essential to gain fundamental knowledge of the microbial functions that underpin the equine hindgut ecosystem.

Evaluation of titanium dioxide and chromic oxide as digestibility markers in ponies fed alfalfa hay in relation to marker dosing frequency.

In equines, Cr2O3 is widely accepted as an indigestible marker, but there are health concerns regarding the carcinogenic properties of Cr2O3. Recently, TiO2 has been suggested to be an alternative digestibility marker in equines. However, a comparison between Cr2O3 and TiO2 has not been made in equines. Six Welsh pony geldings (initial BW: 254±3 kg; 7 years of age) fed chopped alfalfa hay were used to evaluate the use of TiO2 (Ti) and Cr2O3 (Cr) as markers for calculating apparent digestibility and to investigate the effect of frequency of marker administration on the measurement of digestibility values. Diets contained 4.65 kg dry matter (DM) chopped alfalfa hay supplemented with minerals, vitamins, TiO2 (3.3 g Ti/day) and Cr2O3 (3.2 g Cr/day). Ponies were dosed with either 3.3 g Ti and 3.2 g Cr once daily (DF1) or with 1.65 g Ti and 1.60 g Cr twice daily (DF2). After adaptation to the diets and procedures for 14 days, voluntary voided faeces were collected quantitatively over 7 days and analysed for moisture, ash, Ti and Cr. Apparent total tract DM digestibility (DMD) and organic matter digestibility (OMD) were calculated using the total faecal collection (TFC) and marker method (Ti and Cr). The overall mean cumulative faecal recovery of Cr and Ti (as % of intake) were 102.0% and 96.6%, respectively. Mean daily faecal recoveries of Cr as well as of Ti were not different (P=0.323; P=0.808, respectively) between treatments. Overall daily faecal recovery of Cr differed (P=0.019) from 100% when the marker was dosed once daily, whereas overall daily faecal recovery was similar to 100% for both administration frequencies when Ti was used as a marker. For both markers, the coefficient of variation of the mean faecal marker recovery between horses was lower when the markers were administrated twice per day. Across treatments, cumulative DMD and OMD estimated with Ti were similar (P=0.345; P=0.418, respectively) compared with those values determined by TFC method. When Cr was used, the calculated cumulative DMD tended (P=0.097) to be greater compared with those estimated with TFC, and cumulative OMD values were overestimated (P=0.013). Orally supplemented Ti recovery in the faeces of ponies fed chopped alfalfa hay with Ti administered once or twice daily was close to 100%, making it the preferred marker for digestibility trials in equines.

Equine parasite control and the role of national legislation - A multinational questionnaire survey.

Experts recommend a change in the control of equine parasites across the world in order to adopt a surveillance-based approach utilizing parasite faecal egg counts (FEC). Several European countries have implemented prescription-only restrictions of anthelmintic usage by law, which is in stark contrast to US, where all anthelmintic products continue to be available over the counter. This study aimed to describe and compare equine parasite control strategies employed in Germany, Austria, the Netherlands, US, and Denmark. An invitation to participate in an online questionnaire survey was published on a large equine news website in each of the participating countries. The main focus of the study was on usage of FEC and anthelmintic treatment intensity in three different equine age groups; foals, 1-3 year-olds and >3 years old. A total of 3092 respondents participated in the study. Danish respondents used significantly more faecal analyses in their parasite control strategies than participants from the other four countries (p < 0.0001). Similarly, Danish participants administered significantly fewer anthelmintic treatments per horse per year (p < 0.0001) independent of the age of the horse, and had been using a selective treatment strategy for a significantly longer time period than their counterparts in other countries (p < 0.0001). Only minor differences were found between respondents from the other four countries. This is remarkable as both Austria and Germany have had prescription-only restrictions for 3-4 decades. Yet, their parasite control strategies were more similar to those employed by American respondents. The Netherlands had only recently introduced prescription-only restrictions by the time this survey was conducted which can explain why Dutch respondents were also similar to their American counterparts. Taken together, this study illustrates substantially different worm control strategies practiced in Denmark, and this difference cannot be explained by legislation alone.

Pitfalls and opportunities of teaching veterinary parasitology within an integrated curriculum.

The Faculty of Veterinary Medicine at Utrecht University has seen three major curriculum changes, in 1995, 2001 and 2007. The last change was made because of the European change to a Bachelor-Master system. Almost each time teaching hours tagged for veterinary parasitology have been reduced to currently a minimum of between 46 and 51.5 h, which is much less than the WAAVP-recommended minimum of 70-90 h. This results in a challenge to maintain a qualitatively adequate veterinary parasitology program in a curriculum. Following a brief historic account of previous curricula and implemented curriculum changes, experiences, limitations and opportunities are discussed, including the potential of introducing new teaching materials based, for example, on digital technologies and gaming.

Effect of exercise on apparent total tract digestibility of nutrients and faecal recovery of ADL and TiO2 in ponies.

Exercise and physical training are known to affect gastrointestinal function and digestibility in horses and can lead to inaccurate estimates of nutrient and energy digestibility when markers are used. The effect of exercise on apparent nutrient digestibility and faecal recoveries of ADL and TiO2 was studied in six Welsh pony geldings subjected to either a low- (LI) or high-intensity (HI) exercise regime according to a cross-over design. Ponies performing LI exercise were walked once per day for 45 min in a horse walker (5 km/h) for 47 consecutive days. Ponies submitted to HI exercise were gradually trained for the same 47 days according a standardized protocol. Throughout the experiment, the ponies received a fixed level of feed and the daily rations consisted of 4.7 kg DM of grass hay and 0.95 kg DM of concentrate. The diet was supplemented with minerals, vitamins and TiO2 (3.0 g Ti/day). Total tract digestibility of DM, organic matter (OM), CP, crude fat, NDF, ADF, starch, sugar and energy was determined with the total faeces collection (TFC) method. In addition, DM and OM digestibility was estimated using internal ADL and the externally supplemented Ti as markers. Urine was collected on the final 2 days of each experimental period. Exercise did not affect apparent digestibility of CP, crude fat, starch and sugar. Digestibility of DM (DMD), OM (OMD), ADF and NDF tended to be lower and DE was decreased when ponies received the HI exercise regime. For all treatments combined, mean faecal recoveries of ADL and Ti were 87.8±1.7% and 99.3±1.7%, respectively. Ti was not detected in the urine, indicating that intestinal integrity was maintained with exercise. Dry matter digestibility estimated with the TFC, ADL and Ti for ponies subjected to LI exercise were 66.3%, 60.3% and 64.8%, respectively, while DMD for HI ponies were 64.2%, 60.3% and 65.2%, respectively. In conclusion, physical exercise has an influence on the GE digestibility of the feed in ponies provided with equivalent levels of feed intake. In addition, the two markers used for estimating apparent DMD and OMD indicate that externally supplemented Ti is a suitable marker to determine digestibility of nutrients in horses performing exercise unlike dietary ADL.

Farming characteristics and self-reported health outcomes of Irish farmers.

Background: Irish farmers represent a 'high-risk' group for non-communicable diseases, which, arguably, pose a greater occupational health challenge for farmers. To date, there has been little exploration of the farming characteristics associated with farmers' poor health outcomes. Aims: To examine the relationship between farming and male farmers' self-reported health outcomes and to compare the study findings to national health studies to explore which factors specifically are associated with Irish farmers' poorer health outcomes relative to the general population. Methods: This cross-sectional survey research used self-reported quantitative data on the health outcomes and health behaviours of male farmers from the South-East of Ireland. Data were entered into SPSS and descriptive and binary regression techniques were used for data analysis. Results: There were 314 participants (99% response rate). Age, full-time farming and dairy farming significantly impacted self-reported health outcomes and health behaviours. There was a high prevalence of self-reported arthritis compared with the national average of Irish males. 'Younger' farmers (<45 years) were more likely to engage in harmful health behaviours such as smoking and 'binge-drinking' one or more times per week. Conclusions: This study identified self-reported patterns of risky lifestyle behaviours among particular subgroups of Irish farmers for whom targeted health interventions are warranted. Interventions are particularly important for younger farmers who may see themselves as invincible and impregnable to ill-health.

Ultrasonographic measurements of localized fat accumulation in Shetland pony mares fed a normal v. a high energy diet for 2 years.

Health risks associated with obesity are more likely a factor of the localization of fat excess, rather than of elevated BW per se. The aim of this randomized controlled clinical trial was to determine the effect of a long-term high energy diet on BW, fat accumulation and localization. Eight Shetland pony mares, 3 to 7 years old, were randomly divided into a control and a high energy (HE) diet group fed either maintenance or double maintenance energy requirements (200% net energy (NE)) for two consecutive summers, with a low energy diet in the winter in between. Body condition score (BCS) did not differ between the groups at the onset of the study (control 5.6±0.75 v. HE 6.3±0.5). From 12 weeks after starting the diet, ultrasonography of five different locations (retroperitoneal, axillary, withers, intercostal and rump) for adipose deposition, BCS and BW were measured monthly during the period that ponies received different diets. Statistical analysis was performed using a linear mixed-effects model with post hoc Bonferroni testing. P values <0.05 were considered significant. At week 12 after the onset of the diet, fat thickness in the HE group was significantly greater than in the control group. During the monitoring period, the HE group showed a significant increase in mean (±SE) BW (+52%, 265±13.94kg) and BCS (+70%; to 9.0±0.4), while the control group was unchanged (BW 160±13.98 kg; BCS 3.8±0.4). At all locations, the fat depth in the HE group increased significantly, with the highest increase noted for retroperitoneal deposits. The conclusions were that a 200% NE diet induced subcutaneous and retroperitoneal fat accumulation, with the greatest increase in intra-abdominal deposits. The moderate increase of the subcutaneous fat depth followed by a plateau phase suggests the existence of a limit of adipose tissue expandability, as in man.

Presence and species identity of rumen flukes in cattle and sheep in the Netherlands.

The purpose of the study was to gain knowledge about the prevalence and identity of rumen flukes (RF) in cattle and sheep in the Netherlands. Routine faecal examinations of diagnostic submissions between May 2009 and September 2014 showed a mean annual herd or flock RF prevalence of 15.8% for cattle and 8.0% for sheep. Prevalence in cattle was higher after 2012 than before, which may reflect a change in detection method as well as an increase in true prevalence. During November and December 2014, an abattoir survey was conducted to allow for scoring of rumen fluke burden and to obtain specimens for molecular species characterization. Over 8 visits to 5 abattoirs in areas deemed to pose a high risk for trematode infection, 116 cows and 41 sheep from 27 herds and 10 flocks were examined. Prevalence of RF was higher in beef cattle than in dairy cattle and higher in cattle than in sheep. Median fluke burden was >100 specimens per animal for most positive animals. Using a semi-quantitative RF density score as a gold standard, sensitivity and specificity of a modified quantitative Dorsman egg counting method were estimated at 82.6% and 83.3%, respectively. Of 14 collected adult rumen flukes, twelve (8 bovine and 4 ovine specimens) were identified as Calicophoron daubneyi. The other two, of bovine origin, were identified as Paramphistomum leydeni, which was unexpected as in other European countries all recently collected rumen flukes in both cattle and sheep were identified as C. daubneyi. The findings implicate that multiple rumen fluke species, intermediate host species and transmission cycles may play a role in rumen fluke infections in the Netherlands.

Evaluation of methodological aspects of digestibility measurements in ponies fed different haylage to concentrate ratios.

Methodological aspects of digestibility measurements were studied in four Welsh pony geldings consuming haylage-based diets with increasing proportions of a pelleted concentrate according to a 4×4 Latin square design experiment. Ponies were fed four experimental, iso-energetic (net energy (NE) basis) diets (i.e. 22 MJ NE/day) with increasing proportions of a pelleted concentrate (C) in relation to haylage (H). The absolute amounts of diet dry matter fed per day were 4.48 kg of H (100H), 3.36 and 0.73 kg of H and C (75H25C), 2.24 and 1.45 kg of H and C (50H50C) and 1.12 and 2.17 kg of H and C (25H75C). Diets were supplemented with minerals, vitamins and TiO2 (3.7 g Ti/day). Voluntary voided faeces were quantitatively collected daily during 10 consecutive days and analysed for moisture, ash, ADL, acid-insoluble ash (AIA) and Ti. A minimum faeces collection period of 6 consecutive days, along with a 14-day period to adapt the animals to the diets and become accustomed to the collection procedure, is recommended to obtain accurate estimations on dry matter digestibility and organic matter digestibility (OMD) in equids fed haylage-based diets supplemented with concentrate. In addition, the recovery of AIA, ADL and Ti was determined and evaluated. Mean faecal recovery over 10 consecutive days across diets for AIA, ADL and Ti was 124.9% (SEM 2.9), 108.7% (SEM 2.0) and 97.5% (SEM 0.9), respectively. Cumulative faecal recovery of AIA significantly differed between treatments, indicating that AIA is inadequate to estimate the OMD in equines. In addition, evaluation of the CV of mean cumulative faecal recoveries obtained by AIA, ADL and Ti showed greater variations in faecal excretion of AIA (9.1) and ADL (7.4) than Ti (3.7). The accuracy of prediction of OMD was higher with the use of Ti than ADL. The use of Ti is preferred as a marker in digestibility trials in equines fed haylage-based diets supplemented with increasing amounts of pelleted concentrate.

The preventive effects of two nutraceuticals on experimentally induced acute synovitis.

BACKGROUND: Nutraceuticals are often used in the management of equine osteoarthritis, but scientific evidence of their efficacy is lacking. OBJECTIVES: To study the preventive effects of two new nutraceuticals after the experimental induction of synovitis in comparison with positive and negative control treatments. STUDY DESIGN: Blinded, controlled, randomised experiment. METHODS: Twenty-four healthy Standardbred horses were randomly allocated to supplement AT (multi-ingredient, 28 days), supplement HP (collagen hydrolysate, 60 days), meloxicam (4 days) or placebo (60 days). Synovitis was induced in the right intercarpal joint by intra-articular injection of 0.5 ng lipopolysaccharide (LPS) of Escherichia coli while treatments were continued. Blood and synovial fluid were sampled before treatment, immediately prior to LPS injection, and at 8, 24 and 48 h post-injection. Synovial fluid samples were analysed for total nucleated cell count (TNCC), total protein (TP) and selected biomarkers (prostaglandin E2 [PGE2 ], interleukin-6 [IL-6], glycosaminoglycans [GAGs], type II collagen synthesis [CPII], matrix metalloproteinase [MMP]). Lameness was scored by visual examination and pressure plate analysis immediately prior to LPS injection, and at 8, 24 and 48 h post-injection. Clinical examinations were performed before treatment, immediately prior to LPS injection, at 2, 4 and 6 h post-injection, and then twice per day during the test period. RESULTS: Before treatment and intra-articular challenge, there were no statistically significant differences among the treatment groups for any of the parameters. After intra-articular challenge, the placebo group showed significantly higher synovial fluid TP, TNCC and PGE2 compared with the meloxicam group, although the model did not induce a relevant amount of lameness. Both nutraceuticals resulted in significantly lower synovial fluid TP, TNCC and PGE2 compared with placebo. No statistical differences in IL-6, GAGs, CPII or MMPs were observed among treatment groups. No adverse effects were observed. MAIN LIMITATIONS: Despite evidence of synovitis, lameness was too mild to detect. CONCLUSIONS: The preventive administration of these nutraceuticals showed anti-inflammatory effects in this validated synovitis model. Therefore, further studies of their clinical applicability are warranted.

Species composition of larvae cultured after anthelmintic treatment indicates reduced moxidectin susceptibility of immature Cylicocyclus species in horses.

For the control of cyathostomins in horses, the macrocyclic lactones (MLs), moxidectin (MOX) and ivermectin (IVM) are the most commonly used anthelmintics. However, reduced activity, observed as shortening of the egg reappearance period (ERP) has been described. Shortening of the ERP may be caused by a decreased susceptibility of immature worms for MLs. Alternatively, immature worms may develop faster into egg producing adults as a result of repeated ML treatments. The species composition of the larval cultures obtained shortly after ML and pyrantel (PYR) treatment can confirm the hypothesis of decreased ML susceptibility, as this is often class-specific, whereas faster development would also occur after treatment with anthelmintics with a different mode of action. From 3 farms with a known history of shortened ERP, 8 horses per farm were selected and divided into 2 groups. The MOX-PYR-MOX group was treated twice with MOX (day 0 and 126) and once with PYR (day 84) and the IVM-PYR-IVM group was treated twice with IVM (day 0 and 98) and once with PYR (day 56). Cultured infective larvae (L3s) were counted and differentiated with the reverse line blot on pooled samples. Per cyathostomin species, the number of larvae per gram was calculated. The efficacy of all ML treatments was 100% and a shortened ERP was found on all 3 farms. The species composition of the larval cultures after ML treatment did not differ significantly from that after PYR treatment in the IVM-PYR-IVM group, but it did differ in the MOX-PYR-MOX group. The larval cultures obtained after MOX treatment consisted mostly of Cylicocyclus nassatus, while after PYR treatment Cylicostephanus longibursatus was the most abundant species. In the cultures from 42days after MOX treatment 6 cyathostomin species from 3 genera were found on the farm with the lowest activity (farm 1), while on the farm with the highest activity (farm 3) only 3 species from one genus were found in the same number of examined L3s. The high numbers of L3s of Cylicocyclus species 42days after MOX treatment and the low numbers 42days after PYR treatment can be explained by reduced susceptibility of the immature worms to MOX, but not by a faster development. In conclusion, shortening of the ERP following MOX treatment is most likely a process in which an increasing number of immature worms from an increasing number of species is becoming less susceptible to the active compound.

Semi-quantitative differentiation of cyathostomin larval cultures by reverse line blot.

Cyathostomins are the most prevalent horse nematodes worldwide and over 50 species are described. The eggs and the infective larvae (L3) can easily be obtained or cultured from infected horses, but cannot be differentiated morphologically at species level. A reverse line blot (RLB) method based on the hybridization of a PCR fragment with a species specific probe, has previously been developed for the differentiation of individual eggs and/or L3s, but is too labor intensive for large scale studies. In the present study a RLB method on multiple pooled L3s for the semi-quantitative differentiation of cyathostomin larval cultures was developed and validated. First, the probability of the presence of a certain species within a pool was calculated as function of the frequency and the number of L3s within a pool. Ten L3s per pool were found to be optimal. Next, the probability, the chance of occurrence was calculated when 4 pools per culture were used. The probability distributions for 0, 1, 2, 3 or 4 positive pools were transformed into the corresponding median frequency of the cumulative probability: 0.014, 0.04, 0.08, 0.16 and 0.59, respectively. Based on these calculated probabilities, RLB on 10 L3s per pool and 4 pools per sample was validated by estimating the cross-hybridization, precision and accuracy in 3 groups of horses. First, absence of cross-hybridization was confirmed by differentiation of the same L3s (160 L3s from the 4 horses from group 1) in the RLB on individual as well as on pooled L3s. Cross-hybridization was excluded for 9 of the most common cyathostomins. Next, the precision and accuracy were determined by the differentiation of 10 replicates of 3 cultures from 3 horses from group 2 (1200 L3s). The coefficient of variation (CV) was between 0 and 0.90 and the accuracy was between 0.42 and 1.73. A Monte Carlo simulation based on the observed scores and associated probability distributions gave similar results as the use of a fixed median frequency. The LPGs obtained from 276 larval culture counts from a larger cohort (23 horses, group 3) were not significantly different from the LPGs obtained from summation of the LPG per species found by RLB on pooled L3s. The RLB on pooled L3s was found therefore an useful semi-quantitative method for the differentiation of the most common cyathostomin L3, with a workload of approximately one tenth of that of the RLB on individual L3s.