The dynamics of Salmonella occurrence in commercial laying hen flocks throughout a laying period.

Contaminated eggs and egg products have been recognized for many years as an important source of Salmonella infections in humans in the European Union and in the United States. Longitudinal studies can help to increase our knowledge about the dynamics of the occurrence of Salmonella in the course of a laying period. The total of 41 laying hen flocks-18 in Belgium, six in Denmark and 17 in Germany-were followed during an entire laying period. Samples taken from the empty cleaned and disinfected poultry houses were all negative for Salmonella. After hens arrived on the farms, five pooled faecal samples, one pooled dust sample and 40 cloacal swabs (Belgium and Germany) or 40 swabs from fresh droppings (Denmark) were taken four times from 18 flocks, three times from 21 flocks and two times from two flocks in the course of the laying period. Ten flocks (two Belgian and eight German flocks) tested up to three times positive for Salmonella. Forty-three out of 50 positive samples contained Salmonella Enteritidis phage type 4 (29 isolates) or phage type 8 (14 isolates). The probability of subsequent Salmonella-positive findings increased significantly in Salmonella-positive flocks (P<0.05, odds ratio = 6.4). However, the probability of finding Salmonella did not depend on the time of sampling in the laying period or the season.

Origin of the chicken splenic reticular cells influences the effect of the infectious bursal disease virus on the extracellular matrix.

The effects of infectious bursal disease virus (IBDV) (strain F52/70) infection were studied by immunohistochemical methods on the splenic extracellular matrix (ECM). The major fibrillar components of the ECM, the type I and type III collagens and the main ECM organizing glycoproteins (laminin, tenascin and fibronectin) were monitored up to 11 days post-infection (d.p.i.). By 3 d.p.i., the collagens that form the basic scaffold of the antigen-trapping region of the spleen are destroyed, which is followed by deterioration of the glycoproteins. The ECM in the red pulp and the other regions of the white pulp (periarteriolar lymphatic sheath and germinal centre) seem to be normal. The reason for the significantly different pathological alterations in the ECM between the two regions of the spleen may be explained by the origin of the reticular cells. The reticular cells in the antigen-trapping zone and other splenic regions are of haemopoietic and mesenchymal origins, respectively. Possibly, the reticular cells of the haemopoietic origin are more susceptible for the IBDV infection than the mesenchymal ones. Development of the antigen-trapping, B-cell-dependent zone of the splenic white pulp precedes that of the periarteriolar lymphatic sheath and germinal centre, which suggests that this region may contribute to B-cell maturation. Damage of the ECM in the antigen-trapping zones results in impairment of tissue organization, which may contribute to the permanent immunosuppression.

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.

Detection of vvIBDV in vaccinated SPF chickens.

The purpose of our experiment was to investigate, if apparently healthy, vaccinated chickens may be involved in maintaining and spreading infectious bursal disease virus (IBDV) in poultry environments. We aimed at simultaneous detection and identification of very virulent field strain IBDV (vvIBDV) as well as vaccine strain IBDV in experimentally infected chickens. Two groups of specific pathogen free (SPF) chickens were vaccinated using the intermediate infectious bursal disease (IBD) vaccine D78. Group 1 was vaccinated at the age of one week and group 2 at the age of three weeks. Both groups were challenged with vvl BDV at the age of four weeks. A third, vaccinated, non-challenged group served as negative control. No clinical symptoms were observed in any of these groups. The chickens were euthanised and submitted to autopsy and sample preparation in groups of three at fixed intervals from the age of 28 to 44 days. Gross pathological lesions were not observed. Lymphoid tissues from the bursa of Fabricius, bone marrow, spleen and thymus in addition to cloacal- and bursal swaps were analysed by one-step reverse transcription polymerase chain reaction (RT-PCR). Positive results were confirmed by two-step strain specific duplex (DPX) RT-PCR. The vaccine strain was detected in bursa tissues from all groups, while the challenge strain was detected in few bursal as well as non-bursal tissue samples. The results indicate a possibility of replication of vvlBDV in vaccinated chickens.

Evaluation of a serological Salmonella mix-ELISA for poultry used in a national surveillance programme.

A Mix-ELISA using lipopolysaccharide antigens from Salmonella enterica serotype Enteritidis and Typhimurium was evaluated using samples collected over time in the Danish salmonella surveillance programme for poultry. Serological samples (n = 42,813) taken from broiler-breeder flocks after a year of bacteriological monitoring with negative results were used for calculating the flock and individual test specificities, which were 0.997 and 0.999, respectively. Layer flocks from the table egg sector were used for calculation of positive predictive values. In the survey, flocks were examined for salmonella by Mix-ELISA and by faecal culture, and in case of a positive result in either of these a repeated, serological testing was performed, and 60 animals were organ-cultured. If one of these samplings was positive, the flock was declared salmonella infected. In a period of 3 months, 35 flocks were found to be positive in the routine samples. Of these, 32 were serologically positive, 2 both serologically and faecally positive and 1 flock only faecally positive. For flocks serologically positive in the surveillance programme, a positive-predictive value of 0.62 for organ culture positivity was found, and while considering serological follow-up samples, the value was 0.95.