Characterization of monoclonal antibodies to avian leukosis viruses

Hybridoma cell lines secreting monoclonal antibody (MCA) to avian leukosis virus (ALV) structural proteins p27 and p19 have been established. In an indirect enzyme-linked immunosorbent assay (ELISA), MCA 6AL20 (IgG1 isotype) reacted with RPL-40 (ALV subgroup A), avian myeloblastosis virus (AMV) (a mixture of subgroups A and B), Rous-associated virus (RAV)-2 (subgroup B), and Carr-Zilber strain of Rous sarcoma virus (CZ-RSV) (subgroup D) but not with Prague strain of RSV (PrC-RSV) (subgroup C) or the endogenous virus RAV-0 (subgroup E). MCA 6AL22 reacted as above and also reacted marginally with PrC-RSV. Both MCAs immunoprecipitated p19 from 35S-methionine-labeled chicken embryo fibroblasts (CEFs) infected with RPL-40 or RAV-1, but not from CEFs infected with RAV-0, thus identifying the viral structural protein p19 as a polypeptide with subgroup-specific epitopes. Both MCAs can be used to differentiate RPL-40 from RAV-0 infection either in an indirect antibody ELISA or by immunoprecipitation. A third MCA, 6AL42 (IgG2a isotype), reacted with the above viruses of subgroups A, B, C, and D at an antibody titer up to 1000-fold higher than with subgroup E RAV-0 virus in indirect ELISAs. MCA 6AL42 immunoprecipitated p27 from cells infected with RPL-40, RAV-1, or RAV-0. These MCAs are potentially useful in developing immunological tests for differentiation of ALV strains.     

Prevalence of antibodies to four bovine rotavirus strains in different age groups of cattle

Neutralizing antibody titers to four bovine rotavirus strains, representing three serotypes, were measured in 160 sera from cattle of different age groups. Age-specific seroprevalence analysis revealed serotype 6, represented by bovine rotavirus (BRV) NCDV, as the predominant rotavirus serotype infecting German cattle and serotype 10, represented by BRV V1005, as the least prominent. Infections with serotype 8, represented by BRV 678, occurred with intermediate frequency. Antibodies of young calves distinguished between NCDV and UK virus, two serotype 6 BRV strains differing in VP4 antigen.     

Actinobacillus pleuropneumoniae in Thai pig herds. Prevalence of serum antibodies and relation to performance

The aim of the study was to investigate the prevalence of Actinobacillus pleuropneumoniae infections in market weight pigs in Thailand. ELISA systems employing purified lipopolysaccharide antigens were used to detect antibodies in 549 serum samples collected from pigs of 22 herds. Relevant cut-off values were established from three herds defined seronegative. Serum antibodies were detected to all serotypes except serotype 10. Almost 60% of the samples were seropositive to at least one serotype and 45% of the pigs were seropositive to more than one serotype. Antibodies to the cross-reacting serotypes 1, 9 or 11 were found in 29% of the pigs. Other common serotypes included the cross-reacting serotypes 3, 6 or 8 (26% seropositive pigs) and serotype 5a (also 26%). Antibodies to serotypes 2, 5b and 12 were low in prevalence (<10%). Three herds were regarded to be seronegative and six to have a low pathogen load with respect to the prevalence of seropositive pigs. The remaining 13 herds had a high incidence of pigs with antibodies to A. pleuropneumoniae, dominated by serotypes 1-9-11 and 5a (n = 6), serotypes 3-6-8, and 5a (n = 4) or 1-9-11, 3-6-8, 5a and 4-7 (n = 3). A low pathogen load with respect to A. pleuropneumoniae, as well as small herd size and age-segregated rearing, tended to improve the performance of growers.     

Enzyme-linked immunosorbent assay to detect subgroup-specific antibodies to avian leukosis viruses

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against avian leukosis viruses (ALV), using antigens extracted from Rous-associated virus-inoculated chicken embryo fibroblast (CEF) cells by Nonidet P-40 treatments. The antigens reacted strongly to sera of chickens immunized with antigenically homologous viruses, but weakly to those of chickens immunized with heterologous viruses. Antigens extracted from noninoculated CEF cells by the same procedures did not react to either of the immune sera. Normal control sera did not react to any of the antigens. Reactivities of immune sera were decreased markedly by the sera adsorbing with homologous Rous-associated virus-inoculated CEF cells, but not with heterologous CEF cells. The ELISA-specific optimal doses (the differences between the optimal doses with antigens from ALV-inoculated and noninoculated CEF cells) were correlated strongly with the virus-neutralization titers (r = 0.876, P less than 0.01). Examination of the antibody response from ALV-inoculated chickens revealed that ELISA detected antibodies at the same time or several weeks earlier than did the virus-neutralization test.     

Protein retention in relation to age

Male Norway rats of the Wistar strain, aged 30, 75, 90, 150, 180, and 360 days, were given ad libitum diets with optimum nutrient contents at every age. Determining the net protein utilization, the values of protein retention were calculated from protein intake. The energy demand per unit of protein retention was determined from the saccharide and fat intake. The energy ratios of proteins, fat, and saccharide were calculated from the optimum nutrition values; this ratio was 13:(57-29):(30-58) in the period of rapid growth (30-90 days old animals). 12:28:60 in the period of maturing (slower growth, 150 days old animals), and 10.5:28:61.5 at maturity (finished growth, 180-360 days old animals). The daily weight gains of the animals given optimum diets (5.52, 5.22, 5.09-3.73-2.11, 1.85 g daily) and the values of protein retention also corresponded to the division of the animals according to their growth (after administration of the standard Larsen diet). In the period of rapid growth, protein retention was increasing up to the age of 90 days--this suggests that the proteins are utilized for building the body and for its development. A decrease in protein retention in 150 days old animals means that the animals have entered the stage of slow growth. Finally, the low retention values at adult age suggest that the proteins are utilized only for the regeneration of worn tissues and for maintenance. It is indicated by the higher values of energy demand per unit of body protein retention at adult age that more energy (mainly the energy of saccharides--starch) is needed to secure the processes of tissue maintenance and regeneration.     

Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus

Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A.     

Prevalence of antibodies to pestiviruses in goats in Austria

Serological investigations were carried out to determine the prevalence of pestiviral infections in goats in Austria, and to investigate the possible relations to herd management practices. The prevalence of antibodies to pestiviruses was investigated in 549 goats in 80 flocks from four regions of Austria. The examination for antibodies was performed using an indirect enzyme-linked immunosorbent assay detecting antibodies to the border disease virus (BDV) and bovine viral diarrhoea virus (BVDV). The observed individual prevalence was 11.5% and the flock prevalence was 31.3%. Comparative neutralization studies on the 63 seropositive samples with BVDV-1, BVDV-2 and the BDV yielded in 32 samples higher titres (> or =4-fold) to BVDV-1 and in two samples to BDV. The remaining samples did not show distinct differences in antibody titres against the pestivirus strains tested because of the cross-reactions. There was a significant (P < 0.05) association between the prevalence of antibodies to pestiviruses and the presence of cattle on the farm. Significant (P < 0.05) geographical variations in individual prevalence were obtained, ranging from 3.5% in lower Austria to 20.2% in Vorarlberg.     

Chemical contamination of the cervical mucus in cows in relation to pregnancy and age factors]

Applying the method of absorption atom spectrometry (AAS), the contamination of cervical mucus by chemical elements (Cd, Pb, Hg, Cr, Cu, Zn) was demonstrated in the oestrus period of cows in different ecological agricultural regions of the North Moravian region. The results of observation revealed only a statistically significant difference (P less than 0.05) in Cd contents in the test groups; test group mean = 0.015 micrograms.g-1 Cd, control group mean = 0.006 micrograms.g-1. Cr findings in cervical mucus are of priority importance, the same applies to Cu findings. Zinc was found to be an element influencing negatively the conception of cows. The Zn values in cervical mucus of nonpregnant cows were demonstrated to be significantly higher (P less than 0.01) (conception--group = 0.841 micrograms.g-1, conception + group = 0.219 micrograms.g-1 Zn. So called sum of chemical elements: Cd + Pb + Hg + Cr + Cu + Zn was proposed and evaluated as a picture of the total contamination of cervical mucus. This characteristic was also influenced by the Zn findings in the group of pregnant and nonpregnant cows. Zn: Cd antagonism in cervical secretions of barren and pregnant cows was statistically highly significant (P less than 0.01). Chemical elements in cervical mucus were determined for the first time in a digested sample of cervical secretion. The correlation coefficients used for the cows (n = 99) from a contaminated region showed that the content of chemical elements in cervical secretion was not influenced by the age of cows. The content of chemical elements in cervical secretion was not increasing with the age.     

Prevalence of antibodies to Neospora caninum in dogs.

Serum samples from 1077 dogs suspected of having Neospora caninum infections from 35 states in the United States and 3 provinces in Canada were tested for N. caninum IgG antibodies by the indirect fluorescent antibody test. Antibodies to N. caninum were found in 75 of 1077 (7%) of the samples. Twenty of the positive dogs were females, 17 were males and the sex was not recorded on 38 dogs. Chi square analysis indicated no differences (P > 0.05) based on sex were present. Dogs from the Southeast and Midwest regions of the United States were more likely to be N. caninum antibody positive than were dogs from the Northeast or West regions.     

Prevalence of antibodies to equine viruses in the Netherlands

Summary

The prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the Netherlands between 1963 and 1966 and from 1972 onwards. Neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested.

The observed incidence of serum samples positive to equine adenovirus in the complement fixation test was 39%. Precipitating antibodies to equine infectious anaemia virus were detected only in serum samples from two horses imported from abroad. Haemagglutination inhibiting antibodies to Myxovirus influenzae A / equi‐1, M. Influenzae A / equi‐2, and Reovirus types 1, 2, and 3 were present in respectively 82%, 50%, 10%, 33% and 3.6% of the serum samples tested.

The most frequently observed incidence of antibodies to the various equine respiratory viruses occurred in the groups of horses having repeatedly contact with other horses.     

Prevalence of antibodies to equine viruses in the Netherlands

Summary The prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the Netherlands between 1963 and 1966 and from 1972 onwards. Neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested. The observed incidence of serum samples positive to equine adenovirus in the complement fixation test was 39%. Precipitating antibodies to equine infectious anaemia virus were detected only in serum samples from two horses imported from abroad. Haemagglutination inhibiting antibodies to Myxovirus influenzae A / equi-1, M. Influenzae A / equi-2, and Reovirus types 1, 2, and 3 were present in respectively 82%, 50%, 10%, 33% and 3.6% of the serum samples tested. The most frequently observed incidence of antibodies to the various equine respiratory viruses occurred in the groups of horses having repeatedly contact with other horses.     

Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus.

In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far.