Restriction fragment length polymorphism (RFLP) analysis was used to assist epidemiological investigations following the recent introduction of infectious laryngotracheitis virus (ILTV) to commercial poultry flocks in Northern Ireland (NI). A 4.9 kbp PCR product of the ILTV ICP4 gene was generated from each of 16 field isolates of ILTV originating from England, Scotland, NI and the Republic of Ireland (RoI) and of the single vaccine strain currently licenced for use within the United Kingdom. With the exception of isolate PV6/94 from RoI, all field isolates generated RFLP patterns, following digestion with HaeIII, similar to that obtained using the vaccinal strain. Following MspI digestion, NI isolates were indistinguishable from the vaccinal strain and recent English isolates. However, one English and one Scottish isolate, both made prior to the introduction of vaccination, and two isolates from RoI generated a second pattern following digestion with MspI.

Abstract.A single-dilution quantitative enzyme-linked immunosorbent assay (ELISA) system, based on commercial ELISA kits, for the simultaneous detection of seroconversion to bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI3V), and infectious bovine rhinotracheitis virus (IBRV) was evaluated by testing acute and convalescent serum pairs from 564 cattle in 145 outbreaks of respiratory disease. Seroconversion to BVDV, BRSV, PI3V and IBRV was detected in 8.0%, 19.0%, 13.7%, and 7.4%, respectively, of serum pairs tested. Seroconversion was detected in 60.7% of herds and 34.6% of animals tested. Infection with 2 or more viruses was found in 46.6% of these herds and in 27.2% of these animals. The majority of BVDV infections (62%) were associated with other virus infections, suggesting that BVDV may potentiate infection with other agents rather than being a primary pathogen of the respiratory tract. The results were compared with those obtained by virus neutralization and hemagglutination inhibition testing, and the sensitivity, specificity, and overall correlation were calculated. Sensitivities of 92%, 95%, 100%, and 100% were obtained for BVDV, BRSV, PI3V, and IBRV, respectively. The corresponding specificity values were 89%, 92%, 86%, and 91%. The overall correlation for each virus was 90%, 93%, 90%, and 93%, respectively. These results demonstrate that this ELISA system may be used successfully to detect seroconversion in serum pairs, highlight the frequency of multiple viral infections in outbreaks of respiratory disease, and provide further evidence of an immunosuppressive role for BVDV infections.A single-dilution enzyme-linked immunosorbent assay (ELISA) system for quantifying antibody levels to bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), parainfluenza-3 virus (PI3V), and infectious bovine rhinotracheitis virus (IBRV) has previously been described. 9 This system is based on the standardization of commercially available kits and offers the potential for simultaneously testing acute and convalescent serum pairs for evidence of seroconversion to these 4 viruses. Such a system offers considerable savings over more labor-intensive tests, such as virus neutralization (VN), hemagglutination inhibition (HI), complement fixation, immunofluorescent antibody testing (IFAT), and ELISA end-point titration, that are conventionally used for testing serum pairs and that require testing of serial dilutions of each sample. 1,23 The purpose of this work was to evaluate this ELI-SA system for the detection of seroconversion to BVDV, BRSV, PI3V, and IBRV by using it to test serum pairs submitted from 145 outbreaks of respiratory disease in cattle. To determine the sensitivity and specificity of the system, the results obtained were compared with the results of testing by VN and HI. Material and methodsBovine sera. Private veterinary practitioners were asked to submit paired acute and convalescent bovine sera from samples obtained 2-4 wk apart ...

Salmonella typhimurium DT204C infection is the commonest cause of salmonellosis in calves. On five calf rearing farms a distinct strain, as indicated by plasmid profile analysis, was found to have persisted on the premises for periods ranging from four months to two years, the average being 14 months. The persistence of salmonellae in the environment appears to be an important factor in the epidemiology of calf salmonellosis and clearly indicates the inadequacy of many cleaning and disinfection routines.

Eighty-four pairs of acute and convalescent serum samples collected in 1998 and 1999 from 17 outbreaks of respiratory disease, milk drop syndrome or diarrhoea in cattle were tested by haemagglutination inhibition against human influenza viruses A/Eng/333/80 (HIN1) and A/Eng/427/88 (H3N2). Antibodies to these viruses were present in the convalescent sera of 56.5 per cent and 58.8 per cent cattle tested, respectively, with 56 per cent of the animals seroconverting to one or both viruses. Titres were typically higher to A/Eng/427/88 (H3N2). Further testing of a subset of 21 of these serum pairs against the predominant H1N1 and H3N2 human and porcine strains circulating when the samples were collected revealed that the highest reactivity, in terms of both the magnitude of the recorded titres and the number of positive sera, was to human H3N2 strains. The titres to human H1N1 strains and to both porcine subtypes were low or absent. Attempts to isolate influenza A virus from nasal mucus or swab samples from 142 cattle from 46 cases of respiratory disease and/or milk drop syndrome by passage in embryonated specific pathogen-free eggs were unsuccessful.

The fetal fluid samples were also tested for BVDV-specific antibodies using a blocking ELISA kit (BVD/BD p80 Blocking One-Step; LSI). IFAT was performed using a commercial poly clonal BVDV antiserum (VMRD). The spleen, ileum and lung samples from each fetus were pooled, and a 10 per cent w/v suspension was prepared and processed for virus isolation according to standard procedures (Graham and others 1998). Each pool received two passages on fetal bovine lung cells followed by IFAT for BVDV. Viral RNA was extracted from a 200 µl volume of each fetal fluid using the QIAamp Virus BioRobot MDX kit on a BioRobot Universal system (Qiagen). Real-time RT-PCR was subsequently carried out using the Quantitect SYBR Green RT-PCR kit (Qiagen) with the primer pair F2/PESTR (Letellier and Kerkhofs 2003). Briefly, 25 µl of the reaction mixture, containing 2·5 µl of extracted RNA and 300 pmol of each primer, was subjected to the following thermal conditions: 50°C for 30 minutes, 95°C for 15 minutes, then 40 cycles of 95°C for 15 seconds and 55°C for 30 seconds, followed by 72°C for 30 seconds. Thereafter, melt analysis was performed, with samples that gave both a typical amplifi cation curve and a melt temperature between 83°C and 86°C being considered positive. All kits were used according to the manufacturers' instructions. For a minority of fetuses, not all tests were performed, due primarily to insufficient sample being available.Based on the calculation reported by Rexroad and others (1974), the fetuses examined ranged from 122 to 302 days of gestation (median 210 days). One or more tests were positive from a total of 25 (17·9 per cent) fetuses (Table 1). Virus isolation gave positive results in three of 134 (2·2 per cent) fetuses, and IFAT gave a positive result in one of 134 (0·7 per cent) fetuses. Specific fluorescence was detected in all three tissues (spleen, ileum and lung) of the IFAT-positive fetus, which was also culture positive. In contrast, 20 of 140 (14·3 per cent) fetal fluids and 16 of 129 (12·4 per cent) ear skin samples were positive by antigen ELISA. The overall agreement between antigen ELISA results for fetal fluids and ear skin was 95·3 per cent. The real-time RT-PCR was positive in 18 of 138 (13 per cent) fetal fluids, with positive cycle threshold values ranging from 17·9 to 26·9 (mean 23·5). The agreement between RT-PCR results and antigen ELISA results on fetal fluids was 97·1 per cent. Three fetal fluid samples positive by ELISA were negative by RT-PCR. This could have been due to inhibitors in the samples, although this cannot be stated conclusively. Alternatively, it could reflect a greater resistance of viral proteins relative to RNA to degradation in the fetal environment. All three of the culture-positive fetuses were also positive by antigen ELISA on fetal fluid and by realtime RT-PCR, with the ear skin sample from two of these also positive by ELISA. Antibodies were detected in two of 140 (1·4 per cent) samples. No other tests for BVDV on these two fetuses were positive. A possible alternativ...