Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union.

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Relative quantification and detection of different types of infectious bursal disease virus in bursa of Fabricius and cloacal swabs using real time RT-PCR SYBR green technology.

In present study, different types of infectious bursal disease virus (IBDV), virulent strain DK01, classic strain F52/70 and vaccine strain D78 were quantified and detected in infected bursa of Fabricius (BF) and cloacal swabs using quantitative real time RT-PCR with SYBR green dye. For selection of a suitable internal control gene, real time PCR parameters were evaluated for three candidate genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA and beta-actin to IBDVs. Based on this beta-actin was selected as an internal control for quantification of IBDVs in BF. All BF samples with D78, DK01 or F52/70 inoculation were detected as virus positive at day 1 post inoculation (p.i.). The D78 viral load peaked at day 4 and day 8 p.i., while the DK01 and F52/70 viral load showed relatively high levels at day 2 p.i. In cloacal swabs, viruses detectable were at day 2 p.i. for DK01 and F52/70, day 8 p.i. for D78. Importantly, the primers set were specific as the D78 primer set gave no amplification of F52/70 and DK01 and the DK01 primer set gave no amplification of D78, thus DK01 and D78 could be quantified simultaneously in dually infected chickens by use of these two set of primers. The method described here is robust and may sever as a useful tool with high capacity for diagnostics as well as in viral pathogenesis studies.

New avian influenza A virus subtype combination H5N7 identified in Danish mallard ducks.

During the past years increasing incidences of influenza A zoonosis have made it of uppermost importance to possess methods for rapid and precise identification and characterisation of influenza A viruses. We present here a convenient one-step RT-PCR method that will amplify full-length haemagglutinin (HA) and neuraminidase (NA) directly from clinical samples and from all known subtypes of influenza A. We applied the method on samples collected in September 2003 from a Danish flock of mallards with general health problems and by this a previously undescribed influenza A subtype combination, H5N7, was identified. The HA gene showed great sequence similarity to the highly pathogenic avian influenza A virus (HPAIV) A/Chicken/Italy/312/97 (H5N2); however, the cleavage site sequence between HA1 and HA2 had a motif typical for low pathogenic avian influenza viruses (LPAIV). The full-length NA sequence was most closely related to the HPAIV A/Chicken/Netherlands/01/03 (H7N7) that infected chickens and humans in the Netherlands in 2003. Ten persons with direct or indirect contact with the Danish mallard ducks showed signs of influenza-like illness 2-3 days following the killing of the ducks, but no evidence of influence infections was detected. To our knowledge this is the first report of an H5N7 influenza A virus.

Growth hormone stimulates bone formation and strength of cortical bone in aged rats.

The influence of growth hormone on bone formation, mechanical strength, and composition has been investigated in femur middiaphyseal cortical bone from 2-year-old male rats. The rats were given biosynthetic human growth hormone (bhGH) at 2.7 mg/kg/day in two daily injections for 20, 40, or 80 days, and all animals were killed 80 days after the start of bhGH administration. Control animals were given saline. All animals were labeled with tetracycline on days 41 and 69. Only in the bhGH-80-day group was subperiosteal tetracycline double labeling seen all around the femur diaphysis, and this pattern was found in all animals of the group. Double labeling subperiosteally at the posteromedial aspect was found in all animals of the experiment, but compared with the control group, a 400% and an 800% increase in mineral apposition rate was seen in the bhGH-40-day and bhGH-80-day groups, respectively. Light microscopy and polarization microscopy showed that this newly deposited bone was organized in the same concentric lammellae and had the same direction of the collagen fibers when compared with the surrounding bone formed before the start of bhGH injections. The cortical bone cross-sectional area was increased in the bhGH-40-day and bhGH-80-day groups. At the endosteum, scattered labeling was found in animals from all groups, and no differences in medullary cross-sectional areas were seen. The mechanical analysis revealed an increased mechanical strength of the whole diaphyseal bone after bhGH administration. When the data were corrected for dimensions of the diaphyseal bone, no differences in intrinsic mechanical properties of the bone tissue were found. No differences in apparent density of dry defatted bone, ash, and collagen were seen, whereas apparent density of dry defatted bone minus ash was decreased in all groups given bhGH. Correspondingly, a slight increase in ash concentrations of the bhGH-injected animals was seen. bhGH administration also increased the body weight, muscle mass, and total serum IGF-I and thyroxine concentrations.

Human parathyroid hormone (1-34) and (1-84) increase the mechanical strength and thickness of cortical bone in rats.

An anabolic effect on bone of intermittent parathyroid hormone (PTH) treatment has been found in patients with osteoporosis and also in experimental animals. Controversies exist, however, about whether the positive effect on the trabecular bone balance occurs at the expense of the cortical bone. We examined the biomechanical quality of cortical bone after intermittent treatment with different doses of PTH and, furthermore, compared the effects of PTH-(1-34) and PTH-(1-84). Groups of rats were treated with biosynthetic human PTH-(1-34) or PTH-(1-84), 1.1, 3.3, 10, or 30 nmol/kg/day for 30 days. No changes in the body weights and no changes in the lengths of the femora were observed after the PTH treatments. The biomechanical properties were analyzed by means of a materials-testing machine. A dose-related increase in the bending strength and stiffness of the femora was found, and this increase in mechanical strength corresponds with a 9-12% increase in the cross-sectional area of the femoral diaphyses. The deflection capability and energy absorption were not influenced by any of the PTH treatments. No differences were found between the effects of PTH-(1-34) or PTH-(1-84) on the biomechanical properties of the femora. Consequently, intermittent treatment with biosynthetic PTH-(1-34) or PTH-(1-84) increased the formation of cortical bone, and the biomechanical competence of the femora was found to be preserved.

A dose-response study of the effects of biosynthetic human growth hormone on formation and strength of granulation tissue.

The dose-response relationship between biosynthetic human GH (b-hGH) and biomechanical properties (maximum stress, strain at maximum stress, relative failure energy, and maximum stiffness) and collagen deposition of granulation tissue in sc implanted cellulose sponges were investigated after 7 days of implantation in female rats. GH was administered in doses of 0.5, 2.0, and 8.0 mg/kg body weight/day. In the first part of the experiment, treatment with b-hGH started 7 days before implantation of the sponges, in the second part at the day of implantation, and for the 2.0 mg also 2 days before implantation. When b-hGH treatment was started 7 days before implantation, collagen deposition, maximum stress, and maximum stiffness were increased in the sponges from all the hormone-treated groups. In the group treated with 2.0 mg b-hGH/kg body weight.day, also an increase in failure energy was found. When b-hGH treatment was started at the day of implantation or 2 days before implantation, no differences in collagen deposition and biomechanical parameters were found. B-hGH treatment resulted in an increased weight gain in all three groups in the period before implantation, but only 8.0 mg b-hGH/kg body weight resulted in an increased weight gain during the following implantation period. The groups starting hormone treatment at the day of implantation showed an increased weight gain during the implantation period. The study shows that mechanical strength and collagen formation in sc implanted cellulose sponges in rats are increased by b-hGH when treatment is started 7 days before implantation with a maximum increase at a dose of 2.0 mg b-hGH/kg body weight.day.

The effects of phenytoin (diphenylhydantoin) on the extrathyroidal turnover of thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine, and 3',5'-diiodothyronine in man.

The extrathyroidal metabolism of T4, T3, rT3, and 3',5'-diiodothyronine (3',5'-T2) was studied before and after treatment with 350 mg phenytoin (DPH) daily for 14 days in six hypothyroid patients receiving constant L-T4 replacement. The total and free serum concentrations of the four iodothyronines were reduced by approximately 30% during DPH treatment, whereas the free fractions in serum were unaltered. Concomitantly, serum TSH increased 137% (P less than 0.02). The production rate (PR) of T4 decreased 16% (P less than 0.005), indicating decreased intestinal absorption (bioavailability) of oral L-T4 during DPH treatment. The fractional rate of 5'-deiodination of T4 to T3 increased from 27% to 31% (P less than 0.05), whereas the rate of 5-deiodination of T4 to rT3 decreased from 45% to 25% (P less than 0.05). The urinary excretion of free and conjugated T4 was 2.3% of the T4 PR and was unaffected by DPH. Thus, the amount of T4 metabolized through nondeiodinative pathways apart from urinary excretion increased from 25% to 44% (P less than 0.05). The apparent distribution volume (Vd) of T4 increased (P less than 0.05), whereas the pool size was unchanged. The PR of T3 did not change during DPH treatment, nor did the mean transit time or the cellular clearance. The rT3 PR was reduced by 54% (P less than 0.02) during DPH treatment. Concomitantly, the transit time increased 10-fold (P less than 0.05), whereas Vd and pool size increased 5-fold (P less than 0.01 and P less than 0.05, respectively). The turnover of 3',5'-T2, in contrast to that of the other iodothyronines, did not change significantly during DPH treatment. T3 formation from T4 was measured in liver microsomal fractions from rats treated for 8 days with DPH and was almost identical to that in untreated animals. The data demonstrate that DPH in therapeutic concentrations did not affect serum protein binding of the iodothyronines. DPH reduced the intestinal absorption of T4 and increased the nondeiodinative metabolism of T4. The resulting decrease in total and free serum T4 and T3 was associated with an increase in serum TSH, demonstrating reduced negative feedback on the pituitary. Our data do not support the assumption that DPH induces increased hepatic deiodinating enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanical properties and biochemical composition of rat cortical femur and tibia after long-term treatment with biosynthetic human growth hormone.

The influence of biosynthetic human growth hormone (b-hGH) on female rat cortical femur and tibia was studied after administration of hormone doses of 0.16, 1.10, or 8.33 mg/kg body weight/day for 90 days. The mechanical properties, dimensions, real density, ash weight, and the mineral and collagen concentrations of the bones were measured. In both femur and tibia a positive linear relation was found between the dose of hormone and ultimate load, ultimate stiffness, energy absorption at ultimate load, load at failure, energy absorption at failure, and deflection at failure. In the femur a positive correlation between dose and deflection at ultimate load was also found. After normalizing the mechanical data for the dimensions of the bones, no differences were found in the hormone treated groups compared to placebo, except for the elastic modulus (Young's modulus), which was decreased in the femur in the group given 8.33 mg b-hGH. The mineral and collagen concentration were unaffected in both femur and tibia, whereas the real density was decreased in the femur. The growth-hormone-induced changes in the mechanical properties seem to be caused mainly by increased dimensions of the bones.

Increased mechanical strength of healing rat tibial fractures treated with biosynthetic human growth hormone.

The effects of biosynthetic human growth hormone on the biomechanical properties of healing tibial fractures and intact bones in the rat were studied after 20 and 40 days of healing. Growth hormone, 2.0 mg per kg per day, was given subcutaneously in two daily doses. Control animals were injected with a corresponding volume of saline. After 20 days of fracture healing, there were no differences in mechanical properties between the healing fractures and intact bones. After 40 days, the ultimate load and maximum stiffness of the fractures of the b-hGH injected animals had increased to more than 400% of the corresponding values of the saline injected animals, and ultimate stress and energy absorption at ultimate load had increased to 270% compared with the saline injected animals. Ultimate load, stiffness, and energy absorption of the intact bones increased in the b-hGH injected animals, but no differences were found in ultimate stress values or normalized energy, indicating that the changes in the intact bones were quantitative phenomena.

Tissue ingrowth into titanium and hydroxyapatite-coated implants during stable and unstable mechanical conditions.

Lack of initial mechanical stability of cementless prostheses may be responsible for fibrous tissue fixation of prosthetic components to bone. To study the influence of micromovements on bony ingrowth into titanium alloy (Ti) and hydroxyapatite (HA)-coated implants, a loaded unstable device producing movements of 500 microns during each gait cycle was developed. Mechanically stable implants served as controls. The implants were inserted into the weight-bearing regions of all four femoral condyles in each of seven mature dogs. Histological analysis after 4 weeks of implantation showed a fibrous tissue membrane surrounding both Ti and HA-coated implants subjected to micromovements, whereas variable amounts of bony ingrowth were obtained in mechanically stable implants. The pushout test showed that the shear strength of unstable Ti and HA implants was significantly reduced as compared with the corresponding mechanically stable implants (p less than 0.01). However, shear strength values of unstable HA-coated implants were significantly greater than those of unstable Ti implants (p less than 0.01) and comparable to those of stable Ti implants. The greatest shear strength was obtained with stable HA-coated implants, which was threefold stronger as compared with the stable Ti implants (p less than 0.001). Quantitative determination of bony ingrowth agreed with the mechanical test except for the stronger anchorage of unstable HA implants as compared with unstable Ti implants, where no difference in bony ingrowth was found. Unstable HA-coated implants were surrounded by a fibrous membrane containing islands of fibrocartilage with higher collagen concentration, whereas fibrous connective tissue with lower collagen concentration was predominant around unstable Ti implants. In conclusion, micromovements between bone and implant inhibited bony ingrowth and led to the development of a fibrous membrane. The presence of fibrocartilage and a higher collagen concentration in the fibrous membrane may be responsible for the increased shear strength of unstable HA implants. Mechanically stable implants with HA coating had the strongest anchorage and the greatest amount of bony ingrowth.

The use of serotype 1- and serotype 3-specific polymerase chain reaction for the detection of Marek's disease virus in chickens.

A serotype 1- and serotype 3-specific detection of Marek's disease virus (MDV) by polymerase chain reaction (PCR) was developed. The sensitivity of the method when applied to cell culture grown virus was comparable with that of cultivation. The method was applied to various tissue samples from chickens experimentally inoculated with serotype 1 or serotype 3 MDV.The serotype 1 strains CVI988 and RB-1B could be detected in feather follicle epithelium up to 56 and 84 days post-inoculation (p.i.), respectively, while the MDV-3 serotype was detected until 42 days p.i. The purpose of this study was to develop and evaluate a reliable and easy-to-handle method for surveillance of the occurrence of MDV in chicken flocks. We emphasize the development of a method, which can be applied to types of samples conveniently collected in the field, e.g. feather tips and blood samples. In addition, the PCR was applied to samples collected from four commercial table egg layer flocks of young stock or pullets vaccinated with either serotype 1 (CVI988) or serotype 3 (HVT) vaccine. These flocks had various clinical signs of Marek's disease. MDV-1 was detected in buffy-coat cells, spleen, liver, skin, feather tips and ovaries. The detection of MDV in feather tips appeared to be as sensitive as co-cultivation of buffy-coat cells, although an inhibiting factor was observed in extracts from feather tips of non-white chickens. This inhibition could be overcome in most extracts by applying a bovine serum albumen pretreatment. The PCR proved to be a convenient tool for the monitoring of MDV in the poultry population, and feather tips were the most convenient and sensitive samples.

Serum levels of chicken mannan-binding lectin (MBL) during virus infections; indication that chicken MBL is an acute phase reactant.

Mannan-binding lectin (MBL) is a serum collectin which is believed to be an opsonin of the innate immune defence against various microorganisms. MBL is a minor acute phase reactant in man. We investigated the concentration of serum MBL in chickens infected with infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). The concentration of serum MBL increased about twofold (from approximately 6 to 12 microg/ml) due to these viral infections. The concentration peaked 3-7 days after infection with IBV, and 3-5 days after ILTV infection, depending on the ILTV strain used. The increased levels returned to normal values 6-10 days after infection. The results indicated that MBL is a minor acute phase reactant in chickens.

Pathogenesis of H7 influenza A viruses isolated from ostriches in the homologous host infected experimentally.

Infections of ostriches with avian influenza A viruses are generally associated with clinical disease, but the occasional high mortality in young birds does not appear to be related directly to virus pathotype. In this study we investigated the pathogenesis of two H7 viruses for 11-wk-old ostriches inoculated intranasally, and clinical symptoms, virus excretion, and immune response were studied. One of the viruses (A/Ostrich/Italy/1038/00) was highly pathogenic for chickens, whereas the other (A/Ostrich/South Africa/1609/91) was of low pathogenicity for chickens. Clinical signs in ostriches receiving virulent virus were slight depression and hemorrhagic diarrhea, while the group receiving avirulent virus was clinically normal except for green diarrhea. Both viruses were transmitted to in-contact sentinel birds housed with the infected groups 3 days postinfection. Postmortem examination of the birds infected (including the sentinel bird) with virus highly pathogenic for chickens were grossly normal except for localized pneumonic lesions. The results of the study are presented and discussed.

Major histocompatibility complex-linked immune response of young chickens vaccinated with an attenuated live infectious bursal disease virus vaccine followed by an infection.

The influence of the MHC on infectious bursal disease virus (IBDV) vaccine response in chickens was investigated in three different chicken lines containing four different MHC haplotypes. Two MHC haplotypes were present in all three lines with one haplotype (B19) shared between the lines. Line 1 further contains the BW1 haplotype isolated from a Red Jungle Fowl. Line 131 further contains the B131 haplotype isolated from a meat-type chicken. Finally, Line 21 further contains the international B21 haplotype. The chickens were vaccinated with live attenuated commercial IBDV vaccine at 3 wk of age, followed by a challenge with virulent IBDV at 6 wk of age. In this study, we found a notable MHC haplotype effect on the specific antibody response against IBDV, as measured by ELISA. The BW1 haplotype was found to have a significantly higher serum antibody titer against IBDV (7,872) than haplotypes B19 (mean 5,243), B21 (5,570), and B131 (5,333) at 8 d postinfection. However, a virus-neutralizing antibody test did not reflect this result. Nevertheless, the MHC haplotype-associated protective immunity was further supported by the bursa of Fabricius (bursa) recovery from the disease, as measured by histological scorings of the bursa. Chickens carrying the BW1 haplotype had a significantly lower bursa lesion score (1.7) than the haplotypes B19 (mean 3.8), B21 (3.6), and B131 (4.3) 8 d postinfection. Furthermore, multiple line effects were found in other variables when comparing Day 6 with Day 8. Body weight, relative weights of the bursa and the spleen, percentage and relative number of MHC II molecules on MHC II-positive lymphocytes, percentage and relative number of CD4 molecules on CD4-positive lymphocytes, and the specific antibody response all differed significantly among lines. Line 1, with Red Jungle Fowl genes, was clearly differentiated from the other two investigated lines. These results suggest an MHC II restricted T-cell dependent secondary antibody response against IBDV.

Serum levels of mannan-binding lectin in chickens prior to and during experimental infection with avian infectious bronchitis virus.

Mannan-binding lectin (MBL) is a glycoprotein and a member of the C-type lectin super family, the collectin family, and the acute phase protein family. The MBL exerts its function by directly binding to microbial surfaces through its carbohydrate recognition domains, followed by direct opsonization or complement activation via MBL-associated serine proteases (MASP)-1 and -2. Thus, MBL plays a major role in the first-line innate defense against pathogens. We investigated the MBL concentrations in serum during experimental infectious bronchitis virus (IBV) infections in chickens. The results showed that the acute phase MBL response to infection with IBV was, to a degree (P < 0.0068), dependent on whether the chickens were inoculated after 12 h of rest (dark) or after 12 h of activity (light). The acute phase response in chickens challenged after 12 h of activity peaked after 4.6 d with an increase of 24%, whereas the acute phase response in chickens challenged after 12 h of rest peaked after 3.1 d with an increase of 51%. The specific antibody titer against IBV was also tested, and a difference (P < 0.0091) between the two experimental groups was found with peak titer values of 6,816 and 4,349. However, the highest value was found in chickens inoculated after 12 h of activity. Thus, an inverse relation exists between the MBL response and the IBV specific antibody response. The ability of MBL to activate the complement cascade was tested in a heterologous system by deposition of human C4 on the chicken MBL/MASP complex. The complement activation was directly associated with the concentration of MBL in serum, indicating neutralization of the virus before the humoral antibody response took over.

Similarity of avian paramyxovirus serotype 1 isolates of low virulence for chickens obtained from contaminated poultry vaccines and from poultry flocks.

At present Denmark has the status of a 'non-vaccinating' country for Newcastle disease and its poultry population should therefore be free of antibodies to avian paramyxovirus 1 (APMV-1). Three live avian vaccines against infectious bronchitis, avian encephalomyelitis, and chick anaemia which had been found to be contaminated with APMV-1 viruses of low virulence for chickens were examined. The vaccines were produced by the same company and the affected batches had been used in Denmark in 1996/97. Furthermore, APMV-1 isolates of low virulence were obtained from three commercial broiler breeder flocks, one of which had been vaccinated with two of the contaminated vaccines. The flocks belonged to the same hatchery organisation. A comparison of viral F0 gene sequences and typing of virus isolates with a panel of monoclonal antibodies showed that the vaccine and field isolates were identical.

Strains of avian paramyxovirus type 1 of low pathogenicity for chickens isolated from poultry and wild birds in Denmark.

Twenty-one strains of avian paramyxovirus type 1 of low virulence for chickens were isolated in Denmark between 1996 and the beginning of 2003. The low virulence of the strains was demonstrated by sequencing the fusion (F) gene at the cleavage site motif and in some cases by determining the intracerebral pathogenicity index in day-old chicks. By using a panel of monoclonal antibodies it was shown that the isolates belonged to four different antigenic groups (five C2 isolates, six E isolates, six H isolates and four G/Q isolates). They were placed in three distinguishable genetic groups by phylogenetic analysis of a partial sequence of the F gene. The origin of the six E isolates was probably contaminated vaccines; the other viruses were isolated from wild birds and from poultry which probably came into contact with wild birds.

The incidence of rotavirus infection in children from two selected study areas in Zimbabwe.

Five hundred and twenty faecal samples or rectal swabs, collected over one year from children mainly under 24 months, were tested for the presence of human rotavirus (HRV) in enzyme linked immunosorbent assays (ELISA). Fifty-three (23.6 pc) of 225 diarrhoea samples and 25 (8.5 pc) of 295 control samples were HRV positive. The association between diarrhoea and detection of HRV in stools was statistically significant with an overall odds ratio of diarrhoea patients of 3.32 (0.001 greater than p). No significant difference in the incidence of HRV infection between populations at a communal farming location and a high density suburb community was proved. The highest HRV incidence was found during the dry cool season in diarrhoea patients between four and 24 months of age. Overall proportions of 16.7 pc and 28.6 pc HRV positive in groups of 0-3 and 4-6 months old diarrhoea patients respectively was remarkably high, suggesting influence of close human contact, crowding in residences and insufficient sanitary facilities on the transmission of the HRV. Approximately 10 pc of 4-18 months old control patients were HRV positive.

The detection of rotavirus antigen in faeces of asymptomatic children from two different communities in Zimbabwe.

A study of the existence in Zimbabwe of Rotavirus asymptomatic infection was conducted. Rectal swabs were collected from children aged between one month and two years, who were attending health clinics at Chiweshe Hospital (rural area) and Rujeko (a Harare high density suburb) in Zimbabwe. These infants were tested for the presence of Rotavirus antigen, using the ELISA test. Out of 292 specimens collected during a period of one year, 6.9 pc were found positive for Rotavirus antigen although none of the infants had symptoms of Rotavirus infection. Our results show no significant difference (x2 = .357, P = .55) between the prevalence of Rotavirus in children, 1-12 months old from Rujeko and Chiweshe. Likewise, there was no significant difference (x2 = 1.52, P = .281) in the 13-14 months children. It was however shown that a significant difference (x2 = 9.28, P = 0.0096) appeared between winter months versus dry or wet months. We, therefore, conclude that Rotavirus antigen is prevalent in asymptomatic children, and that it is more frequent during winter months.