Evaluation of an in-house cat scratch disease IgM ELISA to detect Bartonella henselae in a routine laboratory setting.

Cat scratch disease (CSD) is caused by Bartonella henselae infection and is a common cause of regional lymphadenopathy. The diagnosis of CSD largely depends on serology, but detection of B. henselae in an affected lymph node by PCR is also an important diagnostic tool. We evaluated an IgM in-house ELISA protocol and analyzed its performance in routine CSD serology. Serum samples from PCR-positive patients (n = 126), PCR-negative patients (n = 123), and age-matched controls (n = 126) were used for evaluation. The sensitivity of the IgM ELISA was only 56%, showing that the performance of B. henselae serology under routine laboratory settings is low, probably caused by the wide variability in disease duration in patients suspected of CSD whose samples were submitted to our laboratory. Most patients (46%) with a positive IgM response were between 0 and 20 years of age. We conclude that the serodiagnosis of B. henselae is hampered by the low sensitivity and specificity of the assays when used in a routine laboratory setting. For this reason, a negative IgM or PCR result can never exclude CSD, especially with late sample collection.

[A man who contracted hepatitis E in the Netherlands]. / Een man met in Nederland opgelopen hepatitis E.

A 55-year-old man was admitted to our hospital because of malaise, jaundice en cholestatic liver function impairment, 4 days after his return from vacation in Surinam. Serological tests were positive for IgG and IgM antibodies to hepatitis E virus (HEV) and serum PCR was positive, consistent with HEV infection. The infection was acquired in the Netherlands and not abroad, considering the incubation period. The patient recovered spontaneously. HEV infection is rare in the Netherlands and is associated with travel to tropical or subtropical areas. The virus is transmitted by the faecal-oral route through contaminated water or food. Since 2000 there have been cases reported in the Netherlands, without any association with travelling abroad and in which the infection might be related to zoonotic transmission. The diagnosis is primarily based upon serologic tests for the detection of IgM and IgG antibodies to HEV in serum confirmed by immunoblot. It is important that HEV infection is considered in patients with acute hepatitis in whom no other cause can be found for hepatitis, even without any travel history to endemic areas.

Serological testing for Bartonella henselae infections in The Netherlands: clinical evaluation of immunofluorescence assay and ELISA.

Cat-scratch disease (CSD), caused by Bartonella henselae infection, can mimic malignancy and can manifest atypically. Reliable serological testing is therefore of great clinical importance. The diagnostic performance of immunofluorescence assay (IFA) and ELISA was evaluated in a group of Dutch patients with proven CSD (clinical diagnosis confirmed by PCR). Sera of 51 CSD patients and 56 controls (patients with similar symptoms, but who were B. henselae PCR-negative and had an alternative confirmed diagnosis) were tested for anti-B. henselae IgM and IgG by IFA and ELISA. A commercially available IFA test for IgM had a sensitivity of 6%. In-house assays for IgM showed specificities of 93% (IFA) and 91% (ELISA), but with low sensitivities (53% and 65%, respectively). With a specificity of 82% (IFA) and 91% (ELISA), in-house IgG testing showed a significantly higher sensitivity in IFA (67%) than in ELISA (28%, p <0.01). Sensitivity was higher for genotype I (38-75%) than for genotype II (7-67%) infections, but this was only statistically significant for IgG ELISA (p <0.05). In conclusion, detection of IgM against B. henselae by in-house ELISA and IFA was highly specific for the diagnosis of CSD. The high seroprevalence in healthy individuals limits the clinical value of IgG detection for diagnosing CSD. Given the low sensitivity of the serological assays, negative serology does not rule out CSD and warrants further investigation, including PCR. Adding locally isolated (e.g., genotype II) B. henselae strains to future tests might improve the sensitivity.

The use of Bartonella henselae-specific age dependent IgG and IgM in diagnostic models to discriminate diseased from non-diseased in Cat Scratch Disease serology.

Cat Scratch Disease (CSD) is caused by Bartonella henselae infection and is a common cause of regional lymphadenopathy. The diagnosis of CSD largely depends on serology, but is hampered by both low sensitivity and specificity of the applied IgG and IgM assays. Using an in-house ELISA, we detected a significant age-dependent increase in the IgG levels in the general population compared to CSD patients. With this knowledge, we developed diagnostic models to differentiate diseased from non-diseased persons. Evaluation of these models using samples from PCR-positive patients (n=155) and age-matched controls (n=244) showed an important increase in the assay performance if the combination of the IgG and IgM results were taken into account. If the specificity was set at 98% the sensitivity was only 45% and 32% for the IgM and IgG ELISA, respectively but increased to 59% when these results were combined. Also the use of age-dependent factors further improved the clinical relevance of the outcome raising the sensitivity to 64%. Although the sensitivity of the ELISA remains low we conclude that the use of models using the combination of both IgM and IgG test results and age-depending factors can be a useful diagnostic tool in the serodiagnosis of CSD.

Hepatitis E is a cause of unexplained hepatitis in The Netherlands.

BACKGROUND: Hepatitis E virus (HEV) is the major etiologic agent of enterically transmitted viral hepatitis in much of the developing world. Evidence provided in recent years shows that HEV is also prevalent in very low numbers in non-endemic countries. Recently, a cluster of three patients with acute hepatitis E but no history of travel to endemic countries was discovered in the geographical area provided with service by the Public Health Laboratory Groningen and Drenthe, The Netherlands. OBJECTIVE: This lead to the question whether hepatitis E is a cause of unexplained hepatitis in this district. STUDY DESIGN: The prevalence of anti-HEV IgG and IgM among 209 patients with clinical signs of hepatitis, negative test for hepatitis A-C, no history of foreign travel and no other cause of hepatocellular damage was compared with a matched control group of 209 individuals. RESULTS: We found a significant difference in seroprevalence between the two groups for IgG anti-HEV as determined with the Abbot HEV EIA (6.2% versus 0.5%); however this difference could not be confirmed with the Genelabs Diagnostics HEV IgG ELISA (6.7% versus 3.8%). For confirmed cases of IgM anti-HEV we also detected a significant difference between the two groups (3.3% versus 0.5%). Remarkably, the combination of IgG and IgM anti-HEV was only found among hepatitis patients. CONCLUSION: This study provides evidence of locally acquired hepatitis E in The Netherlands. Therefore, in cases of unexplained acute hepatitis, the diagnosis of hepatitis E should be considered even in the absence of foreign travel.

Efficacy of donor vaccination before hematopoietic cell transplantation and recipient vaccination both before and early after transplantation.

Allogeneic hematopoietic cell transplantation is followed by humoral immunodeficiency. We evaluated whether antibody levels can be improved by recipient vaccination on day -1 and 50 and whether the levels can be further improved by donor vaccination on day -20. A total of 85 patients were randomized or assigned to one of the following strategies of immunization with Streptococcus pneumoniae polysaccharides, Haemophilus influenzae polysaccharide-protein conjugate, tetanus toxoid (protein recall antigen) and hepatitis B surface antigen (protein neo-antigen): (1) donor on day -20, recipient on days -1, +50 and +365 (D(-20)R(-1,50,365)); (2) donor nil, recipient on days -1, +50 and +365 (D(N)R(-1,50,365)); or (3) donor nil, recipient on day +365 (D(N)R(365)). For H. influenzae and tetanus, IgG levels after grafting were the highest in the D(-20)R(-1,50,365) patients, intermediate in the D(N)R(-1,50,365) patients and the lowest in the D(N)R(365) patients. For S. pneumoniae and hepatitis B, antibody levels appeared to be similar in all three patient groups. The results suggest that for polysaccharide-protein conjugate antigens or protein recall antigens, recipient immunization on days -1 and 50 improves antibody levels and that donor vaccination on day -20 further improves the levels. In contrast, neither recipient immunization on days -1 and 50 nor donor immunization on day -20 appears to be efficacious for polysaccharide antigens and poorly immunogenic protein neo-antigens.

The effect of clenbuterol supplementation on growth performance and on plasma hormone and metabolite levels of broilers.

In the present experiment, clenbuterol was supplemented (.42 ppm) from Day 1 or from 2 or 4 wk of age until slaughter age (6 wk). The effects on growth performance and on plasma hormone and metabolite profiles were investigated at 2, 4, and 6 wk of age in male and female broilers. There were no consistent or cumulative effects of clenbuterol feeding on growth and feed efficiency. Clenbuterol feeding from Day 1, but not later, depressed subsequent feed intake. Relative abdominal fat pad weight was reduced profoundly and was even more pronounced after prolonged supplementation and for females. Fat content of thigh meat (including skin), but not of breast meat (without skin), was reduced by clenbuterol feeding. No consistent effects of clenbuterol supplementation on plasma thyroid hormones, growth hormone, insulin-like growth factor-I, and corticosterone levels were detected. Very low density lipoprotein (VLDL) concentrations were depressed in male broilers fed clenbuterol for 6 wk, but the plasma triglyceride level did not follow this pattern. There was no evidence for a consistent effect of clenbuterol on lipolysis in vivo, measured by plasma glycerol level. Between 4 and 6 wk of age, plasma VLDL and glycerol levels decreased in females but increased in males. This corresponds to the higher fat deposition in females. However, the most consistent effects were age-related changes in the plasma levels of most hormones and metabolites. From the present study, it seems that clenbuterol acts primarily on fat deposition, the extent being dependent on sex, location of fat, and duration of beta-adrenergic agonist supplementation.

Immunocytochemical demonstration of progesterone and estrogen receptors in feathers and skin of adult hens.

Clinical evidence indicates that ovarian steroids are involved in the control of moulting in the chicken. This immunocytochemical study investigates if feather papillae and growing feathers are target tissues for ovarian steroids. Progesterone (PR) and estrogen (ER) receptors were demonstrated using monoclonal antibodies in feathers and surrounding skin of laying hens. Both receptor types were present in the nuclei of dermal papillae and in the nuclei of the epidermal germinative layer cells of growing and full-grown feathers. In growing feathers most nuclei of the intermediate layer (ramogenic column, rachis, axial plate) were immunostained, but during the final stages of differentiation into barbules, only estrogen receptors remained prominent. Skin adjacent to feathers showed ER and PR receptors in nuclei of cells from epidermis, muscles and arteries. During egg-laying pause, plasma progesterone levels decrease ten-fold and it is supposed that this results in a much greater endocrine efficiency of the remaining estrogen levels which are only reduced by 50% when egg-laying stops. The moult-inhibiting effect of progesterone in laying hens could be due to its well-established downregulation on estrogen receptors and therefore, on the endocrine effect of ER at cellular level in feather papillae. Such may account for the presence of both receptor types on the same feather cells, as observed in the present study.

Bayesian estimation of hepatitis E virus seroprevalence for populations with different exposure levels to swine in The Netherlands.

Hepatitis E virus (HEV) is ubiquitous in pigs worldwide and may be zoonotic. Previous HEV seroprevalence estimates for groups of people working with swine were higher than for control groups. However, discordance among results of anti-HEV assays means that true seroprevalence estimates, i.e. seroprevalence due to previous exposure to HEV, depends on choice of seroassay. We tested blood samples from three subpopulations (49 swine veterinarians, 153 non-swine veterinarians and 644 randomly selected individuals from the general population) with one IgM and two IgG ELISAs, and subsets with IgG and/or IgM Western blots. A Bayesian stochastical model was used to combine results of all assays. The model accounted for imperfection of each assay by estimating sensitivity and specificity, and accounted for dependence between serological assays. As expected, discordance among assay results occurred. Applying the model yielded seroprevalence estimates of approximately 11% for swine veterinarians,approximately 6% for non-swine veterinarians and approximately 2% for the general population. By combining the results of five serological assays in a Bayesian stochastical model we confirmed that exposure to swine or their environment was associated with elevated HEV seroprevalence.

Detection of hepatitis E virus-specific immunoglobulin a in patients infected with hepatitis E virus genotype 1 or 3.

Currently, diagnosis of acute hepatitis E virus (HEV) in patients is primarily based on anti-HEV immunoglobulin M (IgM) detection. However, several investigations suggest the use of HEV-specific IgA for diagnosing acute HEV infections. We evaluated two commercially available assays, an IgA enzyme-linked immunosorbent assay (ELISA) (Diacheck) and an adapted immunoblot protocol (Mikrogen) for IgA detection and compared the performance in genotype 1- and 3-infected patients. The specificity of the IgA assays was high, with no positive reactions in a control group of 18 acute hepatitis patients who were negative for HEV. The sensitivity calculated in nine PCR-positive type 1-infected patients was 100% in both assays but was clearly lower in genotype 3-infected patients (n = 14), with sensitivities of only 67% and 57% for the ELISA and immunoblot assay, respectively. The lower IgA responses detected in genotype 3-infected patients could be caused by the use of only the genotype 1 and 2 antigens in the serological assays. Interestingly in two patients with possible infection through blood transfusion no response or intermediate IgA responses were detected, and this might confirm the parenteral route of transmission. In both the type 1- and type 3-infected patients both the IgA and IgM responses disappeared simultaneously. We conclude that IgA detection is of limited value for the serodiagnosis of acute HEV cases, particularly with genotype 3.

Use of serological assays for diagnosis of hepatitis E virus genotype 1 and 3 infections in a setting of low endemicity.

Because of the occurrence of genotype 3 hepatitis E virus (HEV) in regions of low endemicity, it is important to validate the currently used serological assays for diagnosing infections with viruses belonging to this lineage, since these assays only use antigens derived from genotype 1 and 2 viruses. We evaluated the Genelabs enzyme-linked immunosorbent assay (ELISA) and the RecomBlot from Mikrogen for the detection of HEV-specific immunoglobulin M (IgM) and IgG under conditions of low endemicity. We compared test results of 16 patients with locally acquired genotype 3 HEV, 8 genotype 1 patients, 167 healthy controls from the general population, and 101 cases with hepatitis due to other viral causes. The measured specificities of the ELISA (98%) and the RecomBlot (97%) were comparable to those given by the manufacturer for IgM but were significantly lower for IgG (93% by ELISA and 66% by immunoblotting, versus reported values of 98% for ELISA and 95% for blotting). Antibody levels detected following infections with genotype 3 were lower than those following genotype 1 infections except for those measured in the IgM ELISA. Reactivity to the four antigens used in the immunoblot assay were analyzed and showed differences in the IgM immunoblot reactions between genotype 1 patients and genotype 3 patients. The ORF3 antigen was the most specific antigen. The specificity could be improved by a combined testing regimen with confirmation by immunoblotting of all positive ELISA results and by raising the cutoff of the IgG immunoblot assay without loss of sensitivity. We conclude that a combination of ELISA and immunoblotting is needed for acceptable specificity and sensitivity of HEV assays under conditions of low endemicity.

Swine-like hepatitis E viruses are a cause of unexplained hepatitis in the Netherlands.

Hepatitis E virus (HEV) infections in developed countries are recognized as an imported disease related to travel to endemic regions. However, increasing evidence suggests that HEV infection may also occur in the developed countries and that swine may act as a possible reservoir. To investigate the indigenous transmission of HEV in the Netherlands, sera from 50 blood donors and 1027 sera from patients with acute hepatitis were screened with an ELISA for HEV-specific IgG and IgM. Because the Netherlands is considered a nonendemic region, all positive ELISA results were confirmed by immunoblot to exclude false-positive results. Evidence of recent HEV infection was detected in 0% of the blood donors and 4.4% of the cases, based on combined positive IgM and IgG responses. The serodiagnosis was confirmed by a positive polymerase chain reaction (PCR) in 24 patients with hepatitis (2.3% overall, 51% of confirmed IgM+/IgG+ cases). IgG antibodies alone were detected in 4.2% of patients. We found related sequences to virus strains detected in Dutch pigs (genotype 3, 91-97% homology) in 89% of PCR-confirmed HEV patients. The detection of unique swine-like HEV sequences in 16 indigenous hepatitis patients without a recent travel history suggests that HEV is endemic in the Netherlands. We recommend including HEV tests in unexplained acute hepatitis patients, despite their travel history.

Age and strain differences in plumage renewal during natural and induced moulting in hybrid hens.

1. Replacement of remiges and body-feathers was studied in several flocks of brown and white layers and broiler-breeders under near commercial conditions. 2. Spontaneous moulting of primaries was not circannual in modern laying hybrids; moulting response was more retarded in brown than in white hens. 3. Following induction of moulting by a standard procedure in brown hens, the extent of replacement of remiges increased with increasing age, but even at 81 weeks of age, moult was not completed, but ceased when half of the flock had moulted less than half of the primaries. 4. At comparable age the moulting response was considerably more extensive in broiler-breeders than in layers; moulting was not enhanced in hens that had already started to moult before the induction. 5. Compared to the remiges, the moulting response of the body-feathers was more complete and quicker. However, these results might be influenced by a lower threshold of sensitivity to feather growth-induction of the empty follicles, the cumulative number of which is considerable among the contour-feathers after several months of laying.

A new approach to recording plumage deterioration.

1. Recording methods for the scoring of plumage wear are reviewed. 2. A new technique, distinguishing between feather tracts, the surface to which damage occurs and the degree of degradation to the feathers themselves is elaborated: a 5 step scale for degradation is applied to all appropriate fractions of 6 feather regions. After correcting for the normal feather weight in the respective areas, all 6 area values are summed to constitute an overall index of body plumage wear (IBPW).

Effect of temperature during induced moulting on plumage renewal and subsequent production.

1. Four groups of 18 cages each containing 4 brown laying hybrids aged 79 weeks were formed by two levels of restriction of a moulting diet (lasting for 29 d) being applied at two moulting temperatures (11 degrees C and 29 degrees C, lasting for 56 d). 2. Low temperature-low feeding resulted in greatly retarded growth of remiges, but the final extent was similar in all 4 groups, and reached 4 to 5 new primaries (median value). Body-plumage of hens moulted at 11 degrees C was 25% heavier than of hens moulted at 29 degrees C. 3. Second year production variables (rate of laying, egg mass, efficiency of food utilisation) were greatly influenced by moulting temperature (low moulting temperature performing better), but not by feeding rate. 4. The persistence of the improved food utilisation is related to energetic consequences of improved plumage renewal during moulting at the lower temperature, which can be seen as an acclimatization effect.

Effects of feather wear and temperature on prediction of food intake and residual food consumption.

Heat production, which accounts for 0.6 of gross energy intake, is insufficiently represented in predictions of food intake. Especially when heat production is elevated (for example by lower temperature or poor feathering) the classical predictions based on body weight, body-weight change and egg mass are inadequate. Heat production was reliably estimated as [35.5-environmental temperature (degree C)] x [Defeathering (=%IBPW) + 21]. Including this term (PHP: predicted heat production) in equations predicting food intake significantly increased accuracy of prediction, especially under suboptimal conditions. Within the range of body weights tested (from 1.6 kg in brown layers to 2.8 kg in dwarf broiler breeders), body weight as an independent variable contributed little to the prediction of food intake; especially within strains its effect was better included in the intercept. Significantly reduced absolute values of residual food consumption were obtained over a wide range of conditions by using predictions of food intake based on body-weight change, egg mass, predicted heat production (PHP) and an intercept, instead of body weight, body-weight change, egg mass and an intercept.

Role of ovarian steroids in the control of moult induction in laying fowls.

1. Thirty-five Warren SSL hens were selected on the basis of variation in moulting response following a force-moult at 81 weeks of age. Fourteen hens were from a group which returned to layers mash ad libitum from day 9, while 21 came from a group with dietary restriction prolonged to day 28. Blood samples were taken on days 23 and 36, and hormone concentrations were measured. Moulting was recorded on days 0, 11, 23, 36, 52 and 68. 2. Progesterone (P4) but not oestradiol (E2) inhibited moulting during egg laying and the sharp fall in P4 concentration when laying ceased was a primary factor in the induction of moulting. 3. Thyroxine (T4) concentrations were closely correlated with all criteria of plumage renewal, but not with the onset of moulting. Although T4 was closely correlated with subsequent feather growth it is questionable whether T4 is a primer of moult-induction independent of P4. 4. Feathers which were pulled out required 12 +/- 2 d to be replaced beyond the feather-papillae stage. For remiges this timing was similar regardless of whether hens were moulting or still laying. The timing was similar for remiges and body-feathers in moulting hens, but the reappearance of body-feathers in laying hens was slightly delayed.

Effect of plumage renewal after induced moulting on subsequent laying efficiency and persistence.

1. Brown egg layers and dwarf broiler breeder females were force-moulted by different diets. The relationship between the extent of feather replacement and subsequent laying performance was studied. Some brown hens were subjected to metabolic experiments in order to compare post-moult heat production in relation to moulting success. 2. The extent of moulting had a clear effect on the post-moult heat production, and the differences were still present after 6 months in the second year of laying. 3. The extent of feather renewal during moulting showed high and very significant rank correlations with efficiency of food utilisation during the subsequent laying cycle. These correlations were generally higher than those of other features of the moulting procedure (body-weight-loss, minimum weight, duration of pause in laying). 4. The long-term energetic implications of the extent of moulting play an important role in subsequent performances and the persistence of lay during the following year is related to the extent of feather replacement.

Growth hormone response to TRH in male broiler chickens selected for body weight gain or food conversion and reared at either a moderate or a high ambient temperature.

The aim of the present experiment was to study the growth hormone (GH) response upon thyrotropin releasing hormone (TRH) challenge (2 micrograms/kg body weight) in broiler chickens selected for body weight gain (GL line: fat line) or for feed efficiency (FC line: lean line) reared at either a moderate (33-23 degrees C) or high (33 degrees C) ambient temperature. A higher plasma GH level at 5 min after TRH administration was observed in the high temperature conditioned chickens of both lines. Also at high ambient temperature, an enhanced GH decrease between 15 min and 30 min post-injection and a higher acute elimination rate was calculated compared to moderate ambient temperature. A significantly higher GH secretory response was observed in the leaner FC line chickens, which was probably related to the more pronounced pulsatory GH secretion rate in these chickens. There was no difference in GH acute elimination rate between both lines in both environments. No interactions between line and rearing temperature for these parameters of GH dynamics were observed.

Concentrations of hormones, glucose, triglycerides and free fatty acids in the plasma of broiler chickens selected for weight gain or food conversion.

1. The concentrations of growth hormone (GH), insulin-like growth factor I (IGF-I), thyroxine (T4), triiodothyronine (T3), reverse-triiodothyronine (rT3), triglycerides (Tri), free fatty acids (FFA) and glucose (Glu) were determined at 2, 4 and 6 weeks of age in blood plasma of male and female chickens of broiler lines selected for body weight (GL) or food conversion (FC). 2. Plasma concentrations measured in the same animal over a 24 h or a 2 week interval were not significantly correlated with each other. For different traits measured in the same plasma sample only the correlation between T4 and rT3 differed significantly from zero. 3. All traits were dependent on age. Line and sex effects were significant (P less than 0.05) for GH, T4, Tri, FFA and Glu. Additionally, line significantly influenced the plasma T3/T4 ratio and sex influenced plasma rT3. Interactions between line, sex and/or age were seldom significant. 4. Within line and sex, GH (at 6 weeks of age) and T3 (at 4 weeks of age) were negatively, and IGF-I and Tri (both at 6 weeks of age) positively correlated with the amount of abdominal fat at 6 weeks of age. No significant correlation between body weight at 2, 4 or 6 weeks of age and any of the plasma traits was found.