Sampling of graylag geese
On April 3rd and 4th, 2003, 119 fresh faecal droppings were collected from eight different locations surrounding two adjacent lakes in Klepp municipality in Rogaland county. Immediately after sampling they were frozen at -70°C, and transported frozen to the laboratory where they were kept at -70°C, until virus cultivation or RNA isolation prior to RT-PCR.
Between August 1st and 4th, 2003, cloacal swabs were taken from 100 graylag geese carcasses on the island of Smøla. Based on body sizes and gonads, they were estimated to be 68 adults (31♂, 15♀, 22 unknown) and 32 juveniles (12♂, 11♀), 9 unknown (hatching earlier the same year). The samples were taken from within a few hours up to one day after the bird had been shot during hunting. The carcasses were swabbed in the cloacae. One of the swabs was transferred to Amies agar gel with charcoal, code 114C, from Copan Diagnostics Inc. (California, USA) for later bacteriological examinations. A further two cotton swabs were placed in test tubes containing virus transport medium, one in an individual tube (single sample) and the other as a pool of five samples from different birds in the same tube (pooled sample). All samples were kept on ice until they reached the laboratory on the last day of sampling.
Sampling of feral pigeons and mallards
In the city of Stavanger, 100 pigeons (55 adults and 45 juveniles, hatched the same year) were caught by hand. They were swabbed in the cloacae using the same type of Amies gel swabs as used for the geese. On June 21st and 22nd, 2003, 62 birds were sampled, and the swabs were kept refrigerated at 2–8°C until June 23rd before being sent by mail to the laboratory where they were received the next day for examination. The remaining 32 samples were taken on June 29th, sent by mail to the laboratory on June 30th, and received on July 1st.
In the city of Oslo, 100 pigeons were caught by net (9) or cage traps (91) at 11 different locations, and delivered alive to the laboratory on 11 separate occasions between June 24th and September 3rd, 2003. Of these, 45 were classified by evaluation of the gonads as adults (25♂, 20♀) and 55 as juveniles (30♂, 25♀). The birds were euthanised by a blow to the head, and routine necropsy was carried out. Several samples were collected from each bird, including 3 cm of the colon for bacteriology, and two cloacal swab samples for virological investigations. Moreover, five mallards were caught by hand in Oslo, between June 25th and October 13th, 2003. Two were juveniles (2♀) and three adults (2♂, 1♀). Samples were obtained from the birds in the same way as with the graylag geese on Smøla (three cloacal swabs), and the swabs were kept on ice for up to two hours before they were frozen at -70°C.
Permission to capture mallards and pigeons for live sampling and to euthanise pigeons was given by the Directorate for Nature Management (ref 2003/3992 ARTS-VI-ID).
Virological examinations
Faecal material from the graylag geese from Rogaland, and the pooled swab samples from the other bird populations (except for the pigeons from Stavanger), were inoculated in embryonated hen eggs. Mortality of embryos was registered daily for one week, after which the embryos in the eggs, if no mortality was observed, were killed by chilling [27]. The allantoic fluid was collected, and a hemagglutination (HA) test was performed. The allantois from the eggs where embryonic death was observed within a week was used for a second passage in embryonated eggs, in addition to the performance of an HA test.
RNA isolation was performed on the same geese faecal samples and the individual cotton swabs (single samples), prior to testing by RT-PCR. All the samples from the mallards were frozen and the inoculation of embryonated eggs was carried out after the RT-PCR had been performed on two of the samples. RNA isolation was carried out on a 140 μl sample, using the QIAamp Viral RNA mini kit according to the manufacturer's instructions (Qiagen, the Netherlands). A RT-PCR, for the detection of avian paramyxovirus 1, was performed on the samples using primer sequences (MSF1: 5'-GAC CGC TGA CCA CGA GGT TA-3' and MSF2: 5'-AGT CGG AGG ATG TTG GCA GC-3') obtained from the Weybridge Reference Laboratory, spanning the cleavage site of the fusion protein (Ian Brown, Personal Communication). A RT-PCR, for the detection of influenza A virus, was performed using general influenza A primers designed in the M-gene segment [9].
Both RT-PCR tests were performed in a two-step protocol, using Superscript III (Invitrogen) for reverse transcription, and Hotstar Taq DNA polymerase (Qiagen) for PCR. The reverse transcription was performed at 50°C for 45 min for the avian paramyxovirus detection, and at 55°C for 30 min for the avian influenza detection. Both reactions were followed by inactivation of the reverse transcriptase at 70°C for 15 min prior to PCR. The amplification programs consisted of an initial polymerase activation step for 15 min at 95°C, followed by 40 cycles with the following conditions for the avian paramyxovirus PCR protocol: 94°C for 40 s, 55°C for 20 s and 72°C for 60 s, and by 45 cycles with the following conditions for the avian influenza protocol: 94°C for 45 s, 58°C for 45 s and 72°C for 30 s. A final elongation step at 72°C for 5–10 min was performed, followed by chilling to 4°C. The concentration of Mg2+ in the reactions was 1.5 mM for both protocols, and Qiagen's Q solution was added in the PCR for avian paramyxovirus detection. Amplified products were separated by gel electrophoresis and visualised by UV illumination of the gel stained with ethidium bromide.
Cultivation of bacterial pathogens
Examination for the presence of Campylobacter spp. was carried out on all the fresh samples (those that had not been frozen) from the pigeons from Stavanger and Oslo, the mallards, and the geese from Smøla, but not the geese faecal samples from Rogaland. The fresh samples were cultivated directly on CAT-agar: Campylobacter blood free selective agar (Oxoid CM 739, Oxoid, UK) [4] supplemented with cefoperazone, amphotericin B and teicoplanin (Oxoid SR 174), and incubated in a microaerophilic atmosphere at 37°C for 2–3 days. Presumptive Campylobacter spp. colonies were tested for typical appearance and motility by phase contrast microscopy and subcultured on 5% bovine blood agar under the same conditions as described above. The different species were identified by phenotypic assays, including growth pattern at 42°C, catalase production and hippurate hydrolysis.
The same samples were tested for the presence of Salmonella spp. by use of the Nordic Committee on Food Analyses (NMKL) method no. 71, which has been validated and is considered equivalent to ISO no. 6579:1993, and revised editions, according to the EU Commission Decision 97/278/EC. The principle for the method is nonselective pre-enrichment in phosphate buffered peptone water, selective enrichment in Rappaport-Vassiliadis soya peptone broth, and plating out on red violet bile agar plates. Colonies of presumptive Salmonella spp. were subcultured on lactose sucrose bromthymol blue agar plates and tested for hydrogen sulfide production on triple sugar/iron (TSI) agar and urease production on urea agar. Hydrogen sulfide positive and urease negative isolates were further tested by API20E (bioMérieux, Marcy l'Etoile, France).
Sequence analysis
The PCR products were sequenced using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 (Applied Biosystems, California, USA) according to manufacturer's instructions, and analysed on an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems).
The software used for sequence analysis was Sequencher™ version 4.1.4 (Gene Codes Corporation, http://www.genecodes.com), Clustal W Multiple Sequence Alignment Program version 1.83 http://www.ebi.ac.uk/clustalw/, BioEdit Sequence Alignment Editor version 5.0.9 (Tom Hall, Department of Microbiology, North Carolina State University, North Carolina, USA, http://www.mbio.ncsu.edu/Bio%20Edit/bioedit.html), the Influenza Sequence Database at LANL, http://www.flu.lanl.gov/[18], PHYLIP Package version 3.6 (Joe Felsenstein, Department of Genome Sciences, University of Washington, Seattle, Washington, USA, http://evolution.gs.washington.edu/phylip.html), and TreeView (Win32) version 1.6.6 (Roderick D. M. Page, Division of Environmental and Evolutionary Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK, http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).