Marianne M. Sloet van Oldruitenborgh-Oosterbaan

Schmallenberg Virus in Culicoides spp. Biting Midges, the Netherlands, 2011

To determine which species of Culicoides biting midges carry Schmallenberg virus (SBV), we assayed midges collected in the Netherlands during autumn 2011. SBV RNA was found in C. scoticus, C. obsoletus sensu stricto, and C. chiopterus. The high proportion of infected midges might explain the rapid spread of SBV throughout Europe.

High fat intake lowers hepatic fatty acid synthesis and raises fatty acid oxidation in aerobic muscle in Shetland ponies

The metabolic effects of feeding soyabean oil instead of an isoenergetic amount of maize starch plus glucose were studied in ponies. Twelve adult Shetland ponies were given a control diet (15 g fat/kg DM) or a high-fat diet (118 g fat/kg DM) according to a parallel design. The diets were fed for 45 d. Plasma triacylglycerol (TAG) concentrations decreased by 55 % following fat supplementation. Fat feeding also reduced glycogen concentrations significantly by up to 65 % in masseter, gluteus and semitendinosus muscles (P < 0.05 and P < 0.01 and P < 0.01 respectively). The high-fat diet significantly increased the TAG content of semitendinosus muscle by 80 % (P < 0.05). Hepatic acetyl-CoA carboxylase and fatty acid synthase activities were 53 % (P < 0.01) and 56 % (P < 0.01) lower respectively in the high-fat group, but diacylglycerol acyltransferase activity was unaffected. Although carnitine palmitoyltransferase-I (CPT-I) activity in liver mitochondria was not influenced, fat supplementation did render CPT-I less sensitive to inhibition by malonyl-CoA. There was no significant effect of diet on the activity of phosphofructokinase in the different muscles. The activity of citrate synthase was raised significantly (by 25 %; P < 0.05) in the masseter muscle of fat-fed ponies, as was CPT-I activity (by 46 %; P < 0.01). We conclude that fat feeding enhances both the transport of fatty acids through the mitochondrial inner membrane and the oxidative capacity of highly-aerobic muscles. The higher oxidative ability together with the depressed rate of de novo fatty acid synthesis in liver may contribute to the dietary fat-induced decrease in plasma TAG concentrations in equines.

Intradermal testing of horses with and without insect bite hypersensitivity in the Netherlands using an extract of native Culicoides species.

Intradermal tests using a Culicoides nubeculosus extract have proven unreliable for diagnosis of equine insect bite hypersensitivity (IBH) in the Netherlands. The aim of this study was to evaluate the diagnostic value of an extract derived from the Dutch species C. obsoletus and C. pulicaris. Thirteen pairs of horses were tested, each pair consisting of one horse with IBH and the other a healthy control. Each horse was injected intradermally with 0.1 mL of three concentrations of Culicoides whole body extract (1 : 1000 w/v, 1 : 10,000 w/v and 1 : 25,000 w/v), histamine solution (0.01 mg/mL, positive control) and phosphate-buffered saline (negative control). Skin responses were evaluated after 30 min and at 1, 4 and 24 h. At all time points the absolute wheal diameter elicited by Culicoides extract 1 : 1000 w/v was significantly larger (P < 0.01) in the IBH horses than in the control horses. Using the 1 : 10,000 w/v extract the difference was significant at 1, 4 and 24 h and using the 1 : 25,000 w/v extract only at 24 h. The relative wheal diameter was greater in IBH than in control horses at all concentrations and time points except at 0.5 h for the 1 : 10,000 w/v and 1 : 25,000 w/v concentrations. At the 1 : 1000 w/v concentration, the sensitivity and specificity was 92% using the relative wheal diameter. These results indicate that intradermal testing using 1 : 1000 w/v concentration Culicoides extracts relevant to the locality provides useful support for a clinical diagnosis of equine insect hypersensitivity.

Cloning and expression of candidate allergens from Culicoides obsoletus for diagnosis of insect bite hypersensitivity in horses.

Insect bite hypersensitivity (IBH) is an IgE-mediated (Type I) hypersensitivity reaction induced by allergens from biting midges of the Culicoides spp. The aim of the present study was to identify, clone and express recombinant allergens from C. obsoletus, the main species found feeding on horses in the Netherlands, by sequence homology searches on the C. obsoletus specific RNA database, with previously described allergens from C. nubeculosus and C. sonorensis. BLAST searches with these described allergens resulted in similarity hits with 7 genes coding for C. obsoletus allergens. These allergens were expressed as hexahistidine tagged recombinant proteins in E. coli. Allergens were termed Cul o 1-Cul o 7. A maltase (Cul o 1) plus Cul s 1 (maltase of C. sonorensis) were additionally expressed in insect cells using the baculovirus expression system to compare homologous allergens from different species produced with different expression systems in diagnostic in vitro and in vivo tests. We demonstrate that IBH affected horses in the Netherlands show higher IgE levels to Cul o 1 than to Cul s 1, as determined by an IgE ELISA. Furthermore, we show that Cul o 1 produced in E. coli is at least as suitable for in vitro diagnosis of IBH affected horses as Cul o 1 produced in the baculovirus/insect cell expression system. The resulting proteins were evaluated for their ability to discriminate IBH affected and healthy horses by ELISA and intradermal testing. The frequency of positive test results by ELISA within IBH affected horses ranged from 38% to 67% for the different allergens. When results of IgE-binding to Cul o 1-Cul o 7 were combined the test had a sensitivity of 92% and specificity of 85%. The capability of the allergens to induce Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test. The results show that E. coli expressed recombinant allergens from C. obsoletus are valuable tools to determine the allergen specific sensitisation profile (component resolved diagnosis) in horses with IBH in countries were C. obsoletus is the most abundant species and may facilitate in the development of future immunotherapy.

Experimental infection with neuropathogenic equid herpesvirus type 1 (EHV-1) in adult horses.

Equid herpesvirus type 1 (EHV-1)-associated myeloencephalopathy (EHM) may follow an infection with the virus in horses. This study tested three hypotheses: (1) a large inhaled dose of a neuropathogenic EHV-1 strain would induce a cell-associated viraemia in all infected horses; (2) neurological disease will only occur in viraemic horses, and (3) the cerebrospinal fluid (CSF) composition following EHV-1 viraemia will be an indicator for EHM. Four EHV-1 seronegative horses were inoculated with EHV-1 by inhalation. Three developed clinical signs of neurological disease, which were mild in two horses and lacking typical EHM histopathological findings, but moderately severe in the third horse. This latter animal was the only one found to be viraemic, with xanthochromic CSF and spinal cord histopathology findings characteristic of EHM. This study showed that cell-associated viraemia was not guaranteed, despite a large-dose inoculation with EHV-1, yet viraemia was probably a pre-requisite for subsequent development of EHM. The histopathological changes used to confirm EHM may be predicted from CSF analysis.

Culicoides obsoletus extract relevant for diagnostics of insect bite hypersensitivity in horses.

Insect bite hypersensitivity (IBH) is an allergic dermatitis in horses caused by the bites of Culicoides species. The aim of the present study was to evaluate the applicability of whole body extracts of C. obsoletus (the main species found feeding on horses in the Netherlands), C. nubeculosus (rarely found in The Netherlands) and C. sonorensis (typical for North America) for diagnosis of IBH in horses in The Netherlands. Blood and serum samples of 10 clinically confirmed IBH affected and 10 healthy control horses were used to evaluate the IgE titers (ELISA) against the Culicoides whole body extracts of the three Culicoides species. Basophil degranulation was assessed by histamine release test (HRT) after stimulation with these extracts at 5, 0.5 and 0.05 μg/ml. IBH affected horses had significantly higher IgE titers against C. obsoletus than against C. nubeculosus and C. sonorensis. Furthermore, C. obsoletus induced significantly higher histamine release in whole blood of IBH affected horses compared to the other extracts at 0.5 μg/ml. Western blot data revealed IgE binding to many proteins in C. obsoletus extract. This interaction was absent or weak in C. nubeculosus and C. sonorensis extracts for IBH affected horses. Results on individual level indicate that the HRT is more sensitive than ELISA in diagnosing IBH. However, ELISA is more practical as a routine test, therefore the ELISA was further evaluated using C. obsoletus extract on 103 IBH affected and 100 healthy horses, which resulted in a test sensitivity and specificity of 93.2% and 90.0%, respectively. The IgE ELISA readings enabled the analysis of the predicted probability of being IBH affected. From an optical density 450nm value of 0.33 onwards, the probability of IBH affected was more than 0.9. The results presented in this paper show that the use of native Culicoides spp. that feed on horse, is important for improved diagnosis and that the described ELISA based on C. obsoletus can be used routinely to diagnose IBH in countries where this species is the main Culicoides feeding on horses.

Lipid metabolism in equines fed a fat-rich diet

The hypothesis tested was that dietary fat, when compared with an isoenergetic amount of non-structural carbohydrates, stimulates lipolysis in adipose tissue and also stimulates the fatty-acid oxidative capacity in skeletal muscle from horses. Six adult horses were fed a high-fat, glucose or starch containing diet according to a 3 x 3 Latin square design with feeding periods of three weeks. The diets were formulated so that the intake of soybean oil versus either glucose or corn starch were the only variables. In accordance with previous work, whole plasma triacylglycerol (TAG) concentration decreased significantly by 58% following fat supplementation. This fat effect was accompanied by a 247% increase in lipoprotein lipase (LPL) activity in post-heparin plasma. The dietary variables did neither significantly affect the basal in vitro lipolytic rate nor the lipolytic rate after adding noradrenaline. There was no significant diet effect on the activities of hexokinase and phosphofructokinase as indicators of glycolytic flux and citrate synthase and 3-hydroxy-acyl-CoA dehydrogenase as indicators of fatty-acid oxidative capacity. The concentrations of muscle glycogen and TAG were not affected by fat supplementation. It is concluded that our hypothesis is not supported by the present results.

Iron Deficiency in Stabled Dutch Warmblood Foals

Forty-three Dutch Warmblood foals were divided at random into 3 rearing groups immediately after birth: a box-rest group, a box-rest with exercise group, and a pasture group. All stabled foals (box-rest and exercise groups) were fed freshly cut grass harvested from the same pastures on which the pasture group foals were grazing. Blood samples were obtained monthly for CBC and biochemical analyses. At 1-3 months of age, the foals at pasture were active but the foals in the box-rest and exercise groups were listless. Mean hemoglobin concentrations, PCVs, blood iron concentrations, and saturation of total iron binding capacity were significantly lower (P < .05) in the box-rest group (11.3+/-1.2 g/dL, 33+/-3%, 123+/-67 microg/dL, and 18+/-9%) and the exercise group (11.6+/-1.5 g/dL, 34+/-4%, 101+/-61 microg/dL, and 15+/-10%) compared with the pasture group (14.0+/-0.8 g/dL, 42+/-3%, 212+/-67 microg/dL, and 32+/-10%). Oral supplementation of iron to all foals resulted in significant increases in the values of these variables in the box-rest group and exercise group at 4-5 months of age, and the stabled foals were as active as the foals at pasture. In the pasture group, no significant changes occurred. Management practices clearly influence the iron state in young growing foals from 1 to 3 months of age, and although not a frequent cause of anemia in the horse, an absolute deficit of body iron may occur in stabled foals fed freshly cut grass.

Bluetongue, Schmallenberg - what is next? Culicoides-borne viral diseases in the 21st Century.

In the past decade, two pathogens transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), bluetongue virus and Schmallenberg virus, have caused serious economic losses to the European livestock industry, most notably affecting sheep and cattle. These outbreaks of arboviral disease have highlighted large knowledge gaps on the biology and ecology of indigenous Culicoides species. With these research gaps in mind, and as a means of assessing what potential disease outbreaks to expect in the future, an international workshop was held in May 2013 at Wageningen University, The Netherlands. It brought together research groups from Belgium, France, Germany, Spain, Switzerland, United Kingdom and The Netherlands, with diverse backgrounds in vector ecology, epidemiology, entomology, virology, animal health, modelling, and genetics. Here, we report on the key findings of this workshop.

Culicoides species associated with sheep in the Netherlands and the effect of a permethrin insecticide.

Culicoides spp. act as vectors for a number of viral diseases of animals including bluetongue in sheep. The aims of this study were to determine: (1) which Culicoides spp. are associated with sheep in The Netherlands; (2) the time of the day when they are most active; and (3) the effect of treatment of animals with a permethrin insecticide. Two pairs of sheep were each housed within mosquito tents of either one or two layers of netting and all trapped Culicoides spp. were identified microscopically. For the permethrin insecticide study, one of two pairs of sheep was treated with 3.6% permethrin and all animals were housed in tents of similar design. Of the 6210 midges captured, 54.1% were identified as C. chiopterus and 42.7% as C. obsoletus. C. imicola was not identified. The average insect feeding rate was 35-40% and midge activity was greatest around sunset. Permethrin treatment reduced the number of midges captured by 50% and also resulted in a decrease in the percentage of midges that had fed. The findings provide useful information on the behaviour and distribution of Culicoides spp. that will facilitate the development of appropriate control strategies to minimise the risk of insect-vector borne virus diseases such as bluetongue.