Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders.

INTRODUCTION: In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas. MATERIALS AND METHODS: Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent. RESULTS AND DISCUSSION: High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females). CONCLUSIONS: Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

Abundance modelling of invasive and indigenous Culicoides species in Spain.

In this paper we present a novel methodology applied in Spain to model spatial abundance patterns of potential vectors of disease at a medium spatial resolution of 5 x 5 km using a countrywide database with abundance data for five Culicoides species, random regression Forest modelling and a spatial dataset of ground measured and remotely sensed eco-climatic and environmental predictor variables. First the probability of occurrence was computed. In a second step a direct regression between the probability of occurrence and trap abundance was established to verify the linearity of the relationship. Finally the probability of occurrence was used in combination with the set of predictor variables to model abundance. In each case the variable importance of the predictors was used to biologically interpret results and to compare both model outputs, and model performance was assessed using four different accuracy measures. Results are shown for C. imicola, C. newsteadii, C. pulicaris group, C. punctatus and C. obsoletus group. In each case the probability of occurrence is a good predictor of abundance at the used spatial resolution of 5 x 5 km. In addition, the C. imicola and C. obsoletus group are highly driven by summer rainfall. The spatial pattern is inverse between the two species, indicating that the lower and upper thresholds are different. C. pulicaris group is mainly driven by temperature. The patterns for C. newsteadii and C. punctatus are less clear. It is concluded that the proposed methodology can be used as an input to transmission-infection-recovery (TIR) models and R0 models. The methodology will become available to the general public as part of the VECMAP™ software.

The influence of peptide impurity profiles on functional tissue-organ bath response: the 11-mer peptide INSL6[151-161] case.

Bioactive peptides have great pharmaceutical potential as nutraceuticals, diagnostics, and therapeutic drugs in several clinical areas. Thus, the search for novel lead peptides with a biological function has attracted renewed interest. Crude peptide material (i.e., ~70% purity) of INSL6[151-161] (NH2-FRSLFWGNHSQ-COOH) was found to trigger a contractile response in guinea pig ileum longitudinal smooth muscle preparations using tissue-organ baths. However, the purified peptide (i.e., ≥ 95% purity) had no effect on this model. Further investigation with crude materials from other suppliers, with purities ranging between 50% and 80%, indicated that the crude products gave a false-positive functional tissue-organ bath conclusion. These observations question the functionality conclusions when using crude-purity peptide materials; during the initial research or discovery phase, peptide quality is generally neglected, possibly leading to misinterpretation of biological results due to by-products from peptide synthesis and, thus, wrong fail/pass decisions. Therefore, we strongly recommend appropriate quality control testing before using any peptides for initial biomedical research or discovery purposes.

Assessing biosecurity practices, movements and densities of poultry sites across Belgium, resulting in different farm risk-groups for infectious disease introduction and spread.

The existing diversity within poultry systems affects the potential risk of infectious disease introduction and spread. Population data on the level of biosecurity and between-farm contacts is scarce, despite its importance for identifying possible routes of disease transmission. A study was carried out in Belgium to investigate and differentiate professional and hobby poultry sites based on their biosecurity levels and farm movements. Questionnaire data from a total of 37 professional poultry farms, 19 hatcheries and 286 hobby poultry sites were analyzed using a combination of a linear scoring system, a Categorical principal component analysis (CATPCA) and a Two-Step cluster analysis (TSCA). In general, the level of biosecurity was lower in hobby poultry flocks, mainly due to the poor confinement against the outdoor environment and the poor infrastructural hygiene. Most Belgian professional poultry farms and hatcheries had an acceptable level of adoption of standard biosecurity practices, however less attention was given to the way transportation vehicles and employers were brought onto farms and professional visitors welcomed. Considerable variation in the movements and in the structure of the networks arising from these movements was found. Movement frequencies were higher at professional farms compared to hobby farms. Results showed that multiple category farming systems had the highest total movement frequencies. Monthly frequencies of professional visits often exceeded those of poultry and egg movements. Professional and hobby poultry sites were also connected, but movements of poultry and eggs were found only to occur from professional to hobby sites. However, hobby poultry keepers were personally purchasing the poultry and eggs on the professional poultry sites. Six groups of poultry sites were differentiated, which are interpreted as very low to very high risk groups, based on the potential of infectious disease introduction and spread.

Antiserum against the conserved nine amino acid N-terminal peptide of influenza A virus matrix protein 2 is not immunoprotective.

The recent emergence and rapid spread of the pandemic H1N1 swine influenza virus reminded us once again of the need for a universal influenza vaccine that can elicit heterosubtypic protection. Here, we show the superior immunogenicity and immunoprotective capacity of the full-length matrix protein 2 ectodomain (M2e) peptide coupled to keyhole limpet haemocyanin (KLH) compared with the N-terminal 9 aa residues of M2e (SP1). Immunization with M2e-KLH protected mice against a lethal challenge with influenza A virus and significantly reduced weight loss and lung virus titres. In addition, passive transfer of serum raised in rabbits against M2e-KLH protected mice against a lethal influenza virus challenge, whereas serum from rabbits immunized with SP1-KLH did not. Nevertheless, immunofluorescence staining revealed that rabbit serum raised against SP1-KLH bound specifically to infected Madin-Darby canine kidney cells. We conclude that the peptide SP1 contains an immunogenic epitope that is not sufficient for immunoprotection.

Occurrence and molecular characterization of Cryptosporidium and Giardia in captive wild ruminants in Belgium.

Both Cryptosporidium and Giardia are frequently found in the stool of domestic ruminants, especially young animals. Wild ruminants are also host to these protozoa, but the prevalence of these parasites in both free-ranging and captive nondomesticated ruminants needs to be further investigated. Moreover, the role of wild ruminants serving as reservoirs for these zoonotic parasites remains unclear. Therefore, a cross-sectional survey was conducted to estimate the occurrence of Cryptosporidium and Giardia in captive wild ruminants younger than 6 mo and to determine the potential of these animals to serve as reservoirs for these zoonotic parasites. A total of 67 captive wild ruminants belonging to 21 different animal species at the Antwerp Zoo (Belgium), along with 82 American bison (Bison bison) on a commercial breeding farm, were sampled for the detection of Cryptosporidium and Giardia, using a commercial immunofluoresence assay (Merifluor Cryptosporidium/Giardia IFA). The Cryptosporidium prevalence was 7.5% in the Antwerp Zoo animals and 3.7% in the bison from the breeding farm. All but two of the Cryptosporidium-positive animals were younger than 1 mo of age. Molecular characterization by amplification of the 70-kDa heat-shock protein and the 18S ribosomal DNA gene identified Cryptosporidium parvum in four animals of the Antwerp Zoo. The prevalence of Giardia was 8.9% in the Antwerp Zoo animals and 23.2% in the bison calves. Most Giardia-positive animals were older than 1 mo of age. Molecular characterization on the beta-giardin gene and the triose phosphate isomerase gene identified Giardia duodenalis assemblage A in the Antwerp Zoo and both G. duodenalis assemblage A and assemblage E in the bison calves. These findings indicate that both protozoan parasites are prevalent in captive wild ruminants and that these animals can serve as a potential reservoir for zoonotic transmission.

Evaluation of three strategic parasite control programs in captive wild ruminants.

Parasite control in wild ruminants is based mainly on anthelmintics. The objective of the present study was to evaluate three parasite control programs in seven herds of captive wild ruminants in 3 consecutive years. In the first year, a biannual spring-summer treatment regime with fenbendazole at 7.5 mg/kg body weight p.o. for 3 days was applied. The next year, an early-season treatment program with three administrations of fenbendazole at the same dosage at 3-wk intervals was used. In the third year, an early-season treatment program with ivermectin (0.2 mg/kg p.o. for 3 days), applied three times at 5-wk intervals, was evaluated. Effectiveness of these control programs was assessed by fecal egg counts and by scores of body condition and fecal consistency at weekly intervals. Involved animal species were Arabian oryx (Oryx leucoryx), scimitar-horned oryx (Oryx dammah), slender-horned gazelle (Gazella leptoceros), Soay sheep (Ovis aries soay), ibex (Capra ibex), red deer (Cervus elaphus hippelaphus), and Nelson's elk (Cervus elaphus nelsoni). With the spring-summer regime, fecal egg counts remained low during the first 5 mo, but from September onward, they slowly increased to significant levels in all seven herds. The early-season program with fenbendazole resulted in fecal egg counts near zero during the entire year in four herds but a significant egg shedding from August onward was noted in Arabian oryx, scimitar-horned oryx, and Soay sheep. The early-season program with ivermectin resulted in very low to zero egg shedding in gazelle, adult Soay sheep, ibex, red deer, and Nelson's elk during the entire grazing season, but failed to prevent high shedding in October in Arabian oryx and scimitar-horned oryx. High contamination of grassy pastures with infective larvae/eggs in the previous year or before the first treatment may explain failure of the treatment regimes in some herds.

A 12-month survey of the gastro-intestinal helminths of antelopes, gazelles and giraffids kept at two zoos in Belgium.

Faecal egg count patterns and clinical signs associated with gastro-intestinal (GI) nematodes of 107 zoo ruminants were monitored at fortnightly intervals for 1 year. The ruminants in this study were kept under different husbandry conditions at two sites of the Royal Zoological Society of Antwerp, the Antwerp Zoo and the Animal Park Planckendael. Artiodactylids involved were Arabian oryx (Oryx leucoryx), scimitar-horned oryx (Oryx dammah), bongos (Tragelaphus euryceros isaaci), sitatungas (Tragelaphus spekii gratus), common eland (Taurotragus oryx), impala (Aepyceros melampus), slender-horned gazelles, (Gazella leptoceros), blue wildebeest (Connochaetes taurinus taurinus), Kordofan giraffes (Giraffe camelopardalis antiquorum) and okapi (Okapia johnstoni). Nematode eggs were recovered from 586 of 1606 (36.5%) individual faecal samples, using flotation techniques. Infection levels were distinctly low at Antwerp Zoo, probably due to zero grazing and daily dung removal. At Planckendael, the herds of Arabian oryx, scimitar-horned oryx and slender-horned gazelles showed markedly higher egg counts than the other herds, with more than 10% of the faecal egg counts having more than 100 eggs per gram (epg) and maximum faecal egg counts of 600, 750 and 1350 epg, respectively. Faecal egg counts increased during the mid-grazing season (July) and peaked at the end of the grazing season (October). No clinical signs, such as loss of faecal consistency, could be correlated with faecal egg counts (P > 0.05). With the exception of significantly more Nematodirus spp. eggs that were present in juvenile eland, no differences in faecal egg counts could be found between the sexes and different age groups. Abomasa and intestines of 17 animals that died during the survey were available for total worm counts. In one Arabian oryx, four slender-horned gazelles and one sitatunga low burdens ranging from 200 to 14,300 were found. Nematode species recovered were Camelostrongylus mentulatus from the abomasa and Trichostrongylus retortaeformis, Nematodirus fillicollis, Capillaria spp. and Trichuris spp. from the intestines. Our findings suggest different nematode infection levels between herds, which are mainly due to husbandry conditions but to a lesser extent to species- or individual susceptibility. Identification of ungulates that are highly infected and knowledge of the seasonal variation of their helminths can contribute greatly to a well-adjusted species-specific management and helminth control program.

A 12-month survey of gastrointestinal helminth infections of cervids kept in two zoos in Belgium.

Infections with helminths are a major health issue in captive and wild deer. In this study, fecal egg count patterns and clinical signs associated with gastrointestinal nematodes were assessed for 12 mo in nine cervid herds kept under different husbandry conditions at two sites. At site 1, an urban zoo, fecal egg counts remained low and no clinical signs of parasitic gastroenteritis were seen in the herds of fallow deer (Dama dama), Dybowski's deer (Cervus nippon dybowski), pudu (Pudu pudu), and reindeer (Rangifer tarandus tarandus). Helminth infection at this site may have been successfully prevented by daily dung removal of the small sandy-soil enclosures, and applying routine anthelmintic treatment was not justified. At site 2, a wild animal park, involved species were red deer (Cervus elaphus hippelaphus), Nelson's elk (Cervus elaphus nelsoni), Père David's deer (Elaphurus davidianus), European elk (Alces alces alces), and reindeer (Rangifer tarandus tarandus). Nematode eggs were frequently encountered in herds of red deer, Nelson's elk, and European elk, which were kept on larger, grassy enclosures that were irregularly cleaned. The trimodal pattern of fecal egg counts in herds from the wild animal park, consisting of a small spring rise in June, a peak in October, and a small rise in February, indicates that infective larvae on pastures are the main source of infection. In addition, routine anthelmintic treatment with fenbendazole in April and July limited egg shedding, but reinfection rapidly occurred. In two European elk and one reindeer, increasing fecal egg counts were associated with loss of fecal consistency and reduced appetite. Three genera and three species of nematodes were recovered at necropsy of one red deer and three Nelson's elk: Spiculopteragia spiculoptera, Trichostrongylus spp., Nematodirus filicollis, Capillaria spp., Oesophagostomum radiatum, and Trichuris spp., with total worm counts between 950 and 8,700.