Foci report on indigenous Dermacentor reticulatus populations in Belgium and a preliminary study of associated babesiosis pathogens.

The occurrence of autochthonous clinical cases of canine and equine babesiosis in Belgium during the last two decades suggests that the vector of the pathogens responsible for these diseases, Dermacentor reticulatus (Ixodida: Ixodidae), may be present in this country. Consequently, evidence for the presence of this tick species in different locations within Belgium was investigated. Four different locations were monitored by flagging in 2010; these included the locations at which D. reticulatus was previously found on a dog in 2009 and on two red deer in 2007. Two different species of tick were identified, Ixodes ricinus (Ixodida: Ixodidae) and D. reticulatus. A total of 282 D. reticulatus adult ticks (98 males, 184 females) were collected from the four sites. Ticks were found mainly from early March until the end of May and a peak in activity was apparent in March. A Babesia spp. (Piroplasmida: Babesiidae) genus-specific polymerase chain reaction test based on the amplification of a fragment of the 18S rRNA gene was used to investigate the potential presence of Babesia spp. All DNA extracts isolated from the total tick samples yielded negative results. Additional studies to accurately determine the distribution and vectorial capacity of this important tick species in Belgium are warranted.

Pathogenicity and virulence of the present hantaviruses in Bosnia and Herzegovina: the impact on renal function.

Dobrava (DOBV) and Puumala (PUUV) viruses are endemic throughout the Balkans and cause haemorrhagic fever with renal syndrome (HFRS). The aim of this study was to assess the impact of two different hantaviruses on renal function in HFRS patients during the acute stage of illness. We also aimed to assess the DOBV and PUUV distribution between symptomatic HFRS patients and asymptomatic hantavirus antibody-positive subjects. The study included 264 symptomatic HFRS patients and 63 asymptomatic hantavirus antibody-positive healthy subjects. In our study, 131 (49.6%) HFRS patients were regarded as PUUV- and 69 (26.1%) as DOBV-infected patients, while in 64 (24.2%) of HFRS patients that showed all clinical and biochemical signs of HFRS, the causal hantavirus could not be determined with commercially available tests. DOBV-infected patients were associated with more requirements for haemodialysis treatment, lower diuresis and higher serum creatinine and urea values compared to PUUV-infected patients. PUUV was significantly predominant in asymptomatic hantavirus antibody-positive subjects (69.8%) compared to HFRS patients. DOBV was present in 17.5% of asymptomatic subjects and, interestingly, the preferential hantavirus serotype could not be determined in 12.7% of the asymptomatic antibody-positive subjects.

Hantavirus outbreak in Western Europe: reservoir host infection dynamics related to human disease patterns.

Within Europe, Puumala virus (PUUV) is the causal agent of nephropathia epidemica (NE) in humans, a zoonotic disease with increasing significance in recent years. In a region of Belgium with a historically high incidence of NE, bank voles (the PUUV reservoir hosts), were monitored for PUUV IgG antibody prevalence in nine study sites before, during, and after the highest NE outbreak recorded in Belgium in 2005. We found that the highest numbers of PUUV IgG-positive voles coincided with the peak of NE cases at the regional level, indicating that a PUUV epizootic in bank voles directly led to the NE outbreak in humans. On a local scale, PUUV infection in voles was patchy and not correlated to NE incidence before the epizootic. However, during the epizootic period PUUV infection spread in the vole populations and was significantly correlated to local NE incidence. Initially, local bank-vole numbers were positively associated with local PUUV infection risk in voles, but this was no longer the case after the homogeneous spreading of PUUV during the PUUV outbreak.

A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.

Situation of hantavirus infections and haemorrhagic fever with renal syndrome in European countries as of December 2006.

Hantavirus infections are widely distributed in Europe with the exception of the far north and the Mediterranean regions. The underlying causes of varying epidemiological patterns differ among regions: in western and central Europe epidemics of haemorrhagic fever with renal syndrome (HFRS) caused by hantavirus infections follow mast years with increased seed production by oak and beech trees followed by increased rodent reproduction. In the northern regions, hantavirus infections and HFRS epidemics occur in three to four year cycles and are thought to be driven by prey - predator interactions. Hantavirus infections and HFRS seem to be on the increase in Europe, partly because of better diagnostics, partly perhaps due to environmental changes. Unfortunately, hantavirus infections are still heavily under-diagnosed in many European countries. Here we report the results of a survey conducted in 2007 amongst the member laboratories of the European Network for diagnostics of Imported Viral Diseases (ENIVD).

Haemorrhagic Fever with Renal Syndrome: an analysis of the outbreaks in Belgium, France, Germany, the Netherlands and Luxembourg in 2005.

This article aims to describe the Haemorrhagic Fever with Renal Syndrome (HFRS) situation in 2005 in five neighbouring countries (Belgium, France, Germany, the Netherlands and Luxembourg) and define the most affected areas. The 2005 HFRS outbreaks in these countries were the most significant in the region since 1990, with a total of 1,114 confirmed cases. The main feature of the epidemic was the extension of the known endemic area in several of the affected countries, with the involvement of urban areas for the first time. A significant increase in the number of cases was noted for the first time in the province of Liège in Belgium and in the Jura department in France.

Thermal survival characteristics of cell subpopulations isolated from a heterogeneous human colon tumor.

Responses of a heterogeneous human colon adenocarcinoma model tumor system to in vitro hyperthermic treatment at various temperatures have been studied. This model tumor system consists of an original tumor line (DLD-1) obtained from surgical biopsy, and two derivative subpopulations termed clones A and D. These 3 tumor cell populations differ in many properties, including karyotype and DNA content, production of specific antigens, and sensitivities to other cytotoxic agents such as chemotherapeutic drugs and X-irradiation. In these experiments, exponentially growing tumor cells were exposed to hyperthermia (42.2, 42.5, 43.0, 44.0, or 45.0 degrees) for graded time periods. A single-hit, multitarget equation was used to express the dependence of survival on time at a given temperature, and values for extrapolation numbers, quasi-threshold time (min), and T0 (mean lethal time; min) were obtained for the initial regions of survival. At the lower temperatures of 42.2 and 42.5 degrees, biphasic survival curves were obtained for all three tumor lines and, as a consequence, a second mean lethal time (T0,f) was also determined for the final thermal-resistant portion of the survival curves. Using the T0 values as an index of relative resistance, values at 42.2 and 42.5 degrees indicated that, in this temperature region, the parent (DLD-1) line was the most sensitive, the clone A line showed intermediate sensitivity, and the clone D line was the most resistant. In the thermally resistant portion of the survival curve, T0 values indicated that the clone A subpopulation was the most sensitive, the DLD-1 line showed intermediate sensitivity, and the clone D tumor subpopulation remained the most resistant. At the higher temperatures of 43, 44, and 45 degrees, in which thermotolerance is not observed during heat treatment, values for T0 indicated the parent (DLD-1) tumor line was still the most sensitive tumor line, and the clone A and clone D lines showed approximately equal resistance. These data indicate that significant differences may exist among subpopulations of heterogeneous tumors in their survival responses to hyperthermia.

Quality assurance for the diagnostics of viral diseases to enhance the emergency preparedness in Europe.

The threat posed by emerging and re-emerging communicable diseases and, more recently, by the intentional release of infectious agents in a susceptible population, has been receiving considerable attention at the national and international levels. Public health efforts to strengthen disease detection, surveillance and control have been intensified. However, clinicians and clinical microbiology laboratories play an important role in the early detection of disease, the identification of the putative agent, and notification of the appropriate authorities. To be effective in this role, laboratories must be specially prepared to handle viral agents safely, and need, among other things, the appropriate rapid and sensitive diagnostic tests. In 1998 the European Network for Diagnostics of "Imported" Viral Diseases (ENIVD) was established. ENIVD presently comprises, as permanent members, 44 expert laboratories in 21 European Union (EU) member states and 4 non-EU countries and is one of the networks on infectious diseases funded by the European Commission. ENIVD fulfils many of the important tasks required for the surveillance and control of imported, rare and emerging viral infections such as the exchange of expertise and the organisation of external quality assurance (EQA) programmes, both of which are needed to improve diagnostics. Here, we summarise the data generated by recent EQA activities focussed on the diagnostics of infections with hantavirus, dengue virus, filovirus, Lassa virus, orthopox virus and the SARS-coronavirus (SARS-CoV). These were carried out between 1999 and 2004 and involved 93 laboratories from 41 countries, including laboratories from additional countries outside of Europe. Particularly the EU-candidate countries and Eastern neighbouring countries will be invited to join the network in the near future. A public website is available at http://www.enivd.de.

Quality assurance for the diagnostics of viral diseases to enhance the emergency preparedness in Europe.

The threat posed by emerging and re-emerging communicable diseases and, more recently, by the intentional release of infectious agents in a susceptible population, has been receiving considerable attention at the national and international levels. Public health efforts to strengthen disease detection, surveillance and control have been intensified. However, clinicians and clinical microbiology laboratories play an important role in the early detection of disease, the identification of the putative agent, and notification of the appropriate authorities. To be effective in this role, laboratories must be specially prepared to handle viral agents safely, and need, among other things, the appropriate rapid and sensitive diagnostic tests. In 1998 the European Network for Diagnostics of 'Imported' Viral Diseases (ENIVD) was established. ENIVD presently comprises, as permanent members, 44 expert laboratories in 21 European Union (EU) member states and 4 non-EU countries and is one of the networks on infectious diseases funded by the European Commission. ENIVD fulfils many of the important tasks required for the surveillance and control of imported, rare and emerging viral infections such as the exchange of expertise and the organisation of external quality assurance (EQA) programmes, both of which are needed to improve diagnostics. Here, we summarise the data generated by recent EQA activities focussed on the diagnostics of infections with hantavirus, dengue virus, filovirus, Lassa virus, orthopox virus and the SARS-coronavirus (SARS-CoV). These were carried out between 1999 and 2004 and involved 93 laboratories from 41 countries, including laboratories from additional countries outside of Europe. Particularly the EU-candidate countries and Eastern neighbouring countries will be invited to join the network in the near future. A public website is available at http://www.enivd.de.

A retrospective serological survey on human babesiosis in Belgium.

In Europe, most clinical babesiosis cases in humans have been attributed to Babesia divergens and Babesia sp. EU1. Babesia microti infection of humans occurs mainly in the United States; although a case of autochthonous B. microti infection and serological evidence of infection have been reported in Europe. The Indirect Fluorescent Antibody Test was used to screen sera from 199 anonymous Belgian patients with history of tick bite and clinical symptoms compatible with a tick-borne disease. The serological screen detected positive reactivity in 9% (n = 18), 33.2% (n = 66), and 39.7% (n = 79) of the samples against B. microti, B. divergens, and Babesia sp. EU1, respectively. Thus, evidence of contact among three potentially zoonotic species of Babesia and humans has been confirmed in Belgium. Preventive action and development of better diagnostic tools should help in prevention of clinical cases and to clarify the true burden of such infection for individuals and public health.

The Presence of Borrelia miyamotoi, A Relapsing Fever Spirochaete, in Questing Ixodes ricinus in Belgium and in The Netherlands.

Borrelia miyamotoi is a tick-borne bacterium that may cause relapsing fever in humans. As this pathogen has been discovered in Europe only recently, only little is known about its local impact on human health and its spatial distribution. In this study, we show the results of PCR screenings for B. miyamotoi in flagged Ixodes ricinus from Belgium and the Netherlands. B. miyamotoi was detected in nine of thirteen, and three of five locations from the Netherlands and Belgium, respectively. These outcomes indicate that B. miyamotoi is more spread than previously thought. The mean infection rate B. miyamotoi was 1.14% for Belgium and 3.84% for the Netherlands.

Incidence of hantavirus infections in Belgium.

Over the last two decades, and from the moment that serological detection was possible, human hantavirus infections have been documented in most European countries. This paper summarises the available data on hantavirus cases in Belgium. These data enable the demonstration of the existence of a 3-year epidemic cycle in Belgium, which is apparently linked to rodent population dynamics.

Spatial and temporal dynamics of Puumala hantavirus infection in red bank vole (Clethrionomys glareolus) populations in Belgium.

Dynamics of hantavirus infection and population densities in rodents were investigated from 1996 to 1999 in southern Belgium. Evidence of Puumala infection was restricted to Clethrionomys glareolus. Although the serotype was not determined, antibodies against hantavirus were also found in eight Apodemus sylvaticus. In fall 1996, the seroprevalence in C. glareolus was high (20.1%, 37 of 184) and the infection was widely distributed in the area studied whereas a focal occurrence of positive rodents and lower seroprevalence rates were recorded in spring 1997 (14.3%, six of 42), fall 1997 (6. 6%, 11 of 166), spring 1998 (6.4%, three of 47) and fall 1998 (6.7%, 11 of 165). A pullulation of rodents was observed in spring 1999 and was associated with a markedly higher seroprevalence in C. glareolus (47.7%, 189 of 396). In all seasons, infection rates in adults were higher than in juveniles and subadults. No significant difference of prevalence was recorded between males and females. In two trapping sites, the temporary disappearance of positive animals after a crash in rodent populations suggests that a threshold in density is necessary for the maintenance of the enzootic cycle.

Genetic characterization of Puumala hantavirus strains from Belgium: evidence for a distinct phylogenetic lineage.

Puumala hantavirus (PUUV) sequences were recovered from red bank voles (Clethrionomys glareolus) trapped between 1996 and 1998 in four localities of southern Belgium: Thuin, Montbliart, Momignies and Couvin. In addition, three PUUV isolates originating from bank voles trapped in the 1980s in southern (Montbliart) and northern (Turnhout) Belgium were genetically characterized. Analysis of the complete S and partial M segment sequences showed that the Belgian PUUV strains constitute a genetic lineage, distinct from other known PUUV lineages from Europe and Japan. This lineage also includes a wild strain (Cg-Erft) originating from a neighbouring area of Germany. Within the Belgian lineage, geographical clustering of genetic variants was observed. In the Montbliart site, the range of diversity between the most temporally distant strains (from 1986 and 1996-1998) was higher than between those from 1996 and 1998, suggesting slight genetic drift via accumulation of neutral or quasi-neutral substitutions with time.

Ontogeny of cholecystokinin receptors in the human striatum.

The distribution of cholecystokinin binding sites was studied by receptor autoradiography in the human striatum at midgestation, birth and adulthood. In the adult, cholecystokinin receptors are inhomogeneously distributed with patches of reduced labeling. In the caudate nucleus but not in the putamen these patches match the striosomal organization as revealed by acetylcholinesterase staining. At midgestation, patches of high density of cholecystokinin receptors are in register with the dopamine D1 receptor-enriched striosomes. At birth, this striosomal organization has already evolved into the adult pattern of higher matrix level in contrast to the striosomal pattern of acetylcholinesterase staining.

Distribution of hantavirus foci in Belgium.

During 1999 and 2000, we performed rodent captures on 15 sites all over Belgium to evaluate the presence of hantaviruses in local rodent populations. Viral antibody and RNA detection was performed by ELISA/focus reduction neutralisation test and RT-PCR, respectively. We found hantavirus-positive rodents on 13 out of 15 trapping sites and 3 rodent species were found positive for hantavirus infection. Apart from Puumala virus that was carried by Clethrionomys glareolus, 2 additional rodent species, Microtus arvalis and Apodemus sylvaticus, were found antibody- and/or RNA-positive.

Human Granulocytic Ehrlichiosis in Belgium: an underestimated cause of disease.

OBJECTIVES: Human Granulocytic Ehrlichiosis (HGE) is a recently discovered zoonosis and, in Europe, not always included in laboratory testing when a patient presents with a history of tick bite. The available serology results indicate that HGE should be included in the screening panel when a tick-borne disease is suspected. METHODS: Serological methods were applied; i.e. indirect immunofluorescence and Western Blot analysis. Sixty-five serum samples from 47 patients were analysed, of six patients sequential samples were available. RESULTS: 33.8% of the submitted samples were found positive in indirect immunofluorescence, Western Blot confirmed 46.1% of these positive samples. CONCLUSIONS: Although the causative agent and the vector for HGE, Ixodes ticks, are present in Belgium, serology for HGE is seldom solicited. Ehrlichiosis is apparently not always considered as a plausible or possible cause for illness, even when the patient presents with a history of tick bite. We present here a, true be it, incomplete picture of the present situation in Belgium, but nevertheless indicating that it is warranted to test patients with a history of tick bite not only for Lyme disease, but also for HGE.

Intercalary flexor tendon grafts. A morphological study of intrasynovial and extrasynovial donor tendons.

Intercalary intrasynovial and extrasynovial flexor tendon graft donors were placed within the synovial sheaths of the medial and lateral forepaw digits of 22 dogs and treated with early controlled passive mobilization. Specimens were studied by light and transmission electron microscopy at 10 and 21 days. Early repair in the extrasynovial tendons occurred by an ingrowth of connective tissue from the digital sheath. Adhesions obliterated the gliding surface and occupied the space between the tendon's gliding surface and surrounding tissues. There was no epitenon response noted in the extrasynovial tendon grafts. While there was considerable new collagen fibril formation within the repair site at the ultrastructural level, there was a lack of longitudinal remodeling. In contrast, the intrasynovial tendon grafts showed early healing, with minimal adhesion formation, by a proliferation and migration of cells from the epitenon. These cells showed greater cellular activity and collagen production at 10 and 21 days compared to cells in extrasynovial tendons at the same intervals. The findings of this study suggest that the use of intrasynovial autogenous tendon graft donors, coupled with early controlled motion, stimulates an intrinsic repair process in both the tendon stump and autogeneous tendon graft. These findings differ significantly from the experimental findings in which extrasynovial, paratenon-covered grafts are used.

Diagnosis of human granulocytic anaplasmosis in Belgium by combining molecular and serological methods.

We report here one new, hospitalized case of Anaplasma phagocytophilum in Belgium. The clinical presentation of anaplasmosis, its treatment and the molecular and serological relevant laboratory methods are briefly developed.