Abstract Viggo Bitsch: Characteristics of the reactions in tests for antibodies to viruses and their significance for standard assays and adequate routine tests. Interactions between antibodies and viruses are diverse and are used in different ways in tests for the demonstration of either virus or antibody. Three tests, each based on one of three different neutralization reactions by antibodies, and another two ELISA modifications of which one demonstrates a binding reaction and the other one a blocking reaction by antibodies, represent the five basic types of antibody tests. These five tests are 1) the first-order virus neutralization test (f-ord-VNT), 2) the virus aggregation neutralization test (aggr-VNT), 3) the complement-enriched virus neutralization test (C-enr-VNT), 4) the conventional antibody ELISA (conv-ab-ELISA), and 5) the blocking antibody ELISA (bl-ab-ELISA). Basic versions of these five tests are evaluated. The reaction in each test is described. The reacting antibodies in the highest concentration determining the antibody titer and the test sensitivity are defined as either neutralizing or non-neutralizing, and modifications with a sensitivity found optimal are evaluated for routine practical use and for the potential use as a reference or even gold standard assay (cf. Definitions). In the f-ord-VNT with appropriately extended reaction, the reacting antibodies are exclusively neutralizing. The reaction is of first order, implying that the sensitivity is depending on the temperature and is proportional to the reaction time. A 37oC/24h configuration (reaction at 37 oC for 24 hours), being approx. 16 times more sensitive than a 37oC/1h version, is judged to be the ideal reference and gold standard assay for measuring truly neutralizing antibodies to viruses. In the aggr-VNT, the inactivation by aggregation is explosive and short-lasting. All antibodies to the various antigenic determinants react synergistically. The reaction is highly dependent on the antibody concentration and can readily be diluted away, but the sensitivity is low and not variable, and the test is of no significance for the demonstration of antibodies to viruses. The reaction in the C-enr-VNT is explosive immediately after the addition of complement, but otherwise of first order with appropriately extended reaction times. The sensitivity of a 37oC/24h modification is equal to that of a conv-ab-ELISA with identical reaction conditions, but the test is more laborious and therefore not relevant for practical use. The conv-ab-ELISA is a first-order assay, and the reacting antibodies in the highest concentration determining the test sensitivity are non-neutralizing. Aggregation of virions is impossible, and the sensitivity is directly proportional to the length of the reaction time. The 37oC/24h conv-ab-ELISA is an ideal reference and gold standard antibody assay for comparative measurements of the test sensitivity. Because of its high sensitivity, it is excellent for controlling questionable results by other tests, and it is well-suited for routine large-scale examinations. The sensitivity of the bl-ab-ELISA, is dependent on both the reaction temperature and time, but the reaction is not immediately of first order. An increase of the reaction time from 1 to 24 hours at 37 oC will raise the sensitivity by approx. a factor of 4, but still the sensitivity is relatively high. The reactants can easily be varied in different ways to serve different objectives. In its basic configuration, it is the test best suited for large-scale antibody examinations in connection with the diagnosis and control of viral infections. The sensitivity and specificity of 37oC/24h modifications of the three antibody tests found of special value are regularly over 99 percent when undiluted samples are examined. SARS-CoV-2 antibody tests are referred to in relevant sections.

In the period December 1967 through April 1969 eight spontaneous cases of pseudorabies in Danish red foxes were demonstrated by virus isolation. One fox had been kept in captivity, the remaining seven were free-living. Five of the foxes were found dead, while three, presenting abnormal behaviour when encountered, had been killed.

Serologic diagnosis of bovine paratuberculosis (Johne's disease) with currently available tests may give false-positive results due to cross-reactions with avian and bovine tuberculosis viruses and other infectious agents. Indirect enzyme-linked immunosorbent assays (ELISA) for detection of antibodies against paratuberculosis based on antigens from Mycobacterium avium subsp. avium (A-ELISA) and M. avium subsp. paratuberculosis (P-ELISA) were compared. Despite an expected higher specificity for M. a. paratuberculosis in the P-ELISA, the 2 antigens were equally suitable for demonstration of antibody to M. a. paratuberculosis in cattle. Receiver operating characteristic (ROC) curves was used to demonstrate the possible antigenic relationship. The area under the curve (AUC) was calculated for each of the 2 ROC curves. The AUC for the P-ELISA ROC curve was 0.9197, and the AUC for the A-ELISA ROC curve was 0.9149, demonstrating a negligible difference in efficiency of the 2 tests ( z = 0.182).

To study how routinely recorded data (also called "register data") might be used in disease monitoring on a regional or national level, a database for bovine virus-diarrhoea virus (BVDV) was made from existing databases, covering the period January 1995-November 1999. This paper includes a general description of the database, including basic statistics for selected variables. Information was largely complete for cattle herds in the milk-recording scheme (MRS), but only partly available for other herds. A methodology was developed to identify when and how a herd initially was infected. For most herds, it was possible to determine when and how BVDV first was introduced. Among the infected herds, most were already infected by the start of the study. BVDV had been present in 40% of the MRS herds and in 9% of the non-MRS herds. In the MRS herds, most new infections were associated with a dam that had been present in the same herd during gestation. Among the non-MRS herds, most new infections were associated with movement of a persistently infected animal. The monthly number of newly infected herds is presented; it is seen that the incidence declined substantially during the study period.

The usefulness of enzyme-linked immunosorbent assay (ELISA) was investigated as a simple method to screen for Salmonella Dublin infection in dairy herds, examining bulk tank milk samples for lipopolysaccharide (O:1,9,12) antibodies. The cut-off value for the ELISA on bulk tank milk was established based on individual milk samples (n = 2887) and bulk tank milk from 52 herds. Bulk tank milk samples (n = 5108) were collected from 1464 dairy herds located in 19 different areas. About 10% of the dairy herds in Denmark participated in the study. The percentage of herds changing from test-negative to test-positive in each area was correlated with the incidence of S. Dublin outbreaks in the corresponding county (r = 0.48, n = 19; P < 0.025). The mean level of the OD values obtained in the first and third test rounds was not constant (Pr /t/ = 0.0001). The study demonstrated that the probability of being test-negative in the third test round was 0.926 for a herd with 2 previous test-negative results. It was concluded that the investigated ELISA method was in general accordance with the cases of clinical S. Dublin infection recorded, and that the method has a potential for national screening purposes.

Free-living deer are suggested as a possible source of infection of cattle with bovine virus diarrhea (BVD) virus. To examine this hypothesis blood samples from 476 free-living deer were collected during two different periods and tested for BVD virus and antibody in Denmark. In 1995-96, 207 animals were tested. These included 149 roe deer (Capreolus capreolus), 29 fallow deer (Dama dama), 20 red deer (Cervus elaphus) and one sika deer (Cervus sika). For the remaining eight animals no species information was available. In 1998-99, 269 animals were tested including 212 roe deer and 57 red deer. The animals were selected from areas with a relatively high prevalence of cattle herds with a BVD persistent infection status in 1997 and 1998. All 207 samples from 1995-96 were found antibody-negative except two samples from red deer. Only 158 of the 207 samples were tested for virus and were all found negative. Of the 269 samples from 1998-99 all but one were antibody negative. The positive sample was from a red deer. All samples were virus-negative. It appears that BVD infection does not occur in roe deer in Denmark. The presence of antibody in a few red deer from various districts in Jutland probably results from cattle to deer transmission, rather than spread among deer. Hence, the possibility of free-living deer as a source of infection for cattle in Denmark seems to be remote.

The main experiences from the Danish bovine virus diarrhoea (BVD) eradication programme over 5 years from 1994 to 1999 are presented. The last 3 years of the programme has been strongly supported by legislation. The most important regulations have been blood testing of live animals before movement to other herds, common pastures or exhibitions, and monitoring of all herds at regular intervals for the presence of the infection. Nevertheless, free herds have experienced infection, e.g., 204 dairy herds in 1998. Of herds found to be infected in the period from July 1997 through June 1998 after previously having been registered to be BVD-free, 67 herds were thoroughly investigated. Nineteen herds (28%) were found infected because of purchase of pregnant cows or heifers which delivered persistently infected (PI) calves, and 24 (36%) and two (3%) because of PI animals on neighbouring pastures or in neighbouring farm houses, respectively. In five herds (7%) pregnant heifers had become infected on one and the same common pasture, while in 17 herds (25%) no immediate cause of infection could be demonstrated. Yet, airborne spread from PI herds as a source of infection was suspected in some of these cases. It was furthermore concluded from investigations presented, that antibody-positive AI bulls were a remote but unlikely possibility. Free-living deer in Denmark had to be considered uninfected. Presence of PI-animals in sheep on infected farms has been seen and is paid attention to in individual cases. The results underline the need for legislation to be used in eradication programmes in areas with a high prevalence of infection and to be introduced right from the beginning in order to minimise the risk of infection for free herds.

Paratuberculosis (Johne's disease) has been widespread in Danish dairy herds for a long time but the herd-level prevalence has never been determined precisely. To evaluate the prevalence of paratuberculosis in Danish dairy herds in various regions, an ELISA based on a commercially available antigen was adapted for testing bulk-tank milk for the presence of antibodies to Mycobacterium avium subsp. paratuberculosis. Bulk-tank milk samples were collected from six milk-collecting centres from six different areas of the country. Samples from 900 herds (about 7.5% of all Danish dairy herds) were examined, and 70% were positive at the statistically optimal cut-off (sensitivity 97.1%; specificity 83.3%). The technical performance of the ELISA was not sufficient to provide a tool for surveillance because even slight changes in optical density for the samples would change the classification of some samples. The infection is more widespread than previous investigations have shown.

Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from all stalled animals in 10 herds with recent (< 6 months) outbreaks of salmonellosis located in Jutland, where salmonella infection is enzootic. Using herd history of salmonellosis, herd location and clinical status of the herds as criteria, the optimal cutoff in the milk ELISA was determined as being at least 5% of the samples having optical density > 0.5, resulting in herd sensitivity of 1.0 and herd specificity of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, rs = 0.69, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Successful control and eradication of BVDV infection presuppose sufficient knowledge of its epidemiology, particularly sources of infection and ways of transmission. Furthermore, it is crucial to have tests that can be trusted to give the true infection of individual animals and indicate the infection status of herds. PI animals are considered to be the main source of infection. In preliminary experiments in Denmark, it was found that eradication in herds could be based on the identification and removal of PI animals. Actual and possible means of herd-to-herd transmission of importance for infection control are reviewed. Special attention is paid to the possibility of airborne transmission, which must be anticipated in areas with high BVDV prevalence and a high cattle population density. BVDV control programs have been initiated only in the Scandinavian countries including Finland, where the occurrence of BVDV varies from a very low prevalence in Finland to a very high prevalence in Denmark. The BVDV programs in Finland, Norway, and Sweden are basically the same. The primary aim of each is the identification of the herds free from infection and prevention of introduction of the infection to these herds. A secondary aim is to reduce gradually the number of infected herds. In Denmark, which has a high prevalence of BVDV, the program is a combined control and eradication program, and different tests are used. The control programs in Scandinavian countries and the eradication program in Denmark are described together with the tests involved. With respect to control, special emphasis is given to herd tests applied to bulk tank milk or to specially selected blood samples to indicate the infection status of individual herds. The initial bulk tank milk testings were the main basis for the conclusions that in Finland, Norway, and Denmark approximately 1%, 9%, and 39% of the dairy herds, respectively, seemed to have PI animals. With respect to eradication, an ELISA developed in Denmark for demonstration of virus in blood has proved to be extremely reliable for identification of PI animals. The BVDV programs are generally voluntary, although in Norway, where BVD is a notifiable disease, restrictions have been placed on infected herds to prevent a further spread of the infection. The annual losses in Denmark from BVDV have been calculated to be approximately 100 million DKr (17 million dollars), whereas the total costs of the control and eradication program for a 3-year period including testing of trade animals are estimated to be approximately 160 million DKr (27 million dollars).

A total of 2585 serum samples from 62 dairy herds located in four different regions of Denmark were tested in an O-antigen (0:1,4,5,12)-based ELISA for the detection of antibodies against Salmonella typhimurium. Ten closed herds from an island with no reported occurrence of salmonellosis for several years, and 12 herds from a salmonella enzootic area which had had clinical outbreaks of S typhimurium were used to define a herd ELISA cut-off value. When herds with at least 5 per cent of the serum samples having an optical density of > 0.5 were considered ELISA-positive, all 10 herds from the salmonellosis-free island were ELISA-negative, and all but one of the 12 S typhimurium-infected herds were ELISA-positive, which resulted in a herd test sensitivity of 0.92 and herd test specificity of 1.0. Eleven of the 12 S typhimurium-infected herds were negative in a blocking ELISA based on a monoclonal antibody to the 0:9 antigen of the serogroup D salmonellas, indicating the possibility of rapid serogroup-specific screening of herds by means of these two tests. Ten other randomly selected herds with clinical outbreaks of S dublin were all, to a large extent, positive in the 0:1,4,5,12-ELISA, whereas a S dublin (0:1,9,12)-ELISA described previously appeared to be more serogroup D-specific. Thus, the 0:1,4,5,12-ELISA appears to be useful for detecting herd infections with S typhimurium, and positive reactions may be further discriminated by the serogroup D-specific ELISA.

Usefulness of two enzyme-linked immunosorbent assays (ELISA) for screening of dairy herds for antibodies to lipopolysaccharide (LPS) of Salmonella dublin (O:1,9,12) was investigated. Sera (3097) were collected from 40 dairy herds located in three areas of Denmark with different prevalence of salmonellosis: ten salmonellosis-free herds from the island of Samsø where there is no history of salmonellosis, ten salmonellosis-free herds from the island of Sealand where outbreaks are infrequent, and 20 salmonella infected herds from Jutland where salmonellosis is enzootic. The samples were analyzed for antibodies to S. dublin LPS using an indirect (O:9,12) and a blocking (O:9) ELISA. Using herd history of salmonellosis, herd location and clinical state of the herds as reference, the herd sensitivity and herd specificity of the tests were 100% and 100% in the indirect ELISA and 95% and 100% in the blocking ELISA, respectively. A significant correlation was found between the two tests (rs = 0.46, p < 0.001). However, the indirect ELISA detected more seropositive animals than the blocking ELISA (17% vs. 7%, respectively). In calves from Sealand, level of background reaction was significantly lower (p < 0.001) compared to the heifers and the cows. The percentages of seropositive calves in both tests were higher (p < 0.01) in comparison to cows (19 vs. 8 in indirect ELISA, and 14 vs. 6 in blocking ELISA, respectively). Results of the study indicated that it is possible to apply LPS ELISA in serological screening for salmonellosis.