Replication of the bovine immunodeficiency-like virus in diploid and aneuploid cells: permanent, latent and virus-productive infections in vitro.

Bovine immunodeficiency-like virus (BIV) is an infectious and leukotropic retrovirus, the sole lentivirus candidate which has been isolated from cattle. Although BIV has recently been shown to be related to the human immunodeficiency virus, there is very limited information on the replication and the pathogenesis of BIV. It is reported here that BIV can permanently infect diploid and aneuploid cells from four different species: bovine, canine, ferret and ovine. With the exceptions of a bovine diploid and a canine aneuploid cell line, all lines were virus non-productive. However, BIV was rescued by co-cultivation of virus non-productive cells with homologous BIV-free or canine cells (Cf2Th). A permanent and BIV-productive infection was established for 90-serial subcultures in a canine cell line. A BIV titre of 1 x 10(6)/0.1 ml was observed in stationary culture and 1 x 10(10)/0.1 ml in suspension culture. The canine cell line above was used for production of BIV antigens, whereas BIV-free canine cells were routinely used to isolate BIV from BIV non-productive cells infected in vitro and from blood from experimentally BIV-infected cattle. The different steps of virus maturation were similar by electron microscopy to those of lentiviruses. BIV results are compared to those of lentiviruses.

Characterization of an atypical biotype of Brucella abortus.

Brucella abortus strains were isolated from bovine tissue and milk samples from seven Ontario herds. The isolates were characterized by colonial morphology, requirement of CO2 for growth, lysis by Tbilisi phage, biochemical tests and agglutination in monospecific sera. They resembled B. abortus biotype 2 (on the basis of sensitivity to thionin and basic fuchsin) and biotype 4 (on the basis of agglutination with anti-Brucella "M" but not anti-Brucella "A" absorbed sera). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these isolates and B. abortus biotypes 1, 2 and 4 showed similar profiles. Immunoblots with anti-A and anti-M absorbed sera showed different antigenic regions reacting with the specific sera and also confirmed that the atypical B. abortus isolates were serologically similar to biotype 4.

A comparison of the bovine leukemia and bovine syncytial virus status in utero-tubal cells recovered from fluids used to flush the uterus and oviducts of BLV-infected, superovulated cattle.

Twenty-four cell lines were established from uterine-oviductal flush fluid (UOFF) cells from 20 bovine leukosis virus (BLV)-infected embryo-donor cows and 4 BLV-free control cows harvested by the Ficoll-gradient technique. Similar epithelial-like and fibroblast-like cells were observed in the primary cultures of UOFF from both groups. BLV-antigens were not detected with direct immunofluorescence test in any of the cell-lines from the 20 positive BLV-cows but a positive reaction was observed with the competitive radioimmunoassay in one cell line only. Bovine syncytial virus (BSV) was detected (multinucleated cells) in five of the 20 cows, bovine virus diarrhea (BVD) in six and infectious bovine rhino-tracheitis (IBR) in one (by D-IF). Some of the cell lines had antigens to one (3/20) two (2/20) or three (1/20) different viruses as demonstrated by D-IF. There were no antigens to BLV, BSV or IBRV demonstrated in the BLV-free cows by both immunofluorescence test and radioimmunoassay. Permissiveness to the growth of BLV in the cell lines of bovine utero-tubal (BUT) origin was demonstrated by inoculating three of the 20 cell lines from BLV-infected cows and three cell lines from the 4 BLV-free cow by BLV suspensions. All six cell lines permitted the growth of BLV as shown by syncytia, and positive reactions with the immunofluorescence test but only three out of six lines were BLV-positive by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of five serological tests for bovine brucellosis.

Five serological assays: the buffered plate antigen test, the standard tube agglutination test, the complement fixation test, the hemolysis-in-gel test and the indirect enzyme immunoassay were diagnostically evaluated. Test data consisted of results from 1208 cattle in brucellosis-free herds, 1578 cattle in reactor herds of unknown infection status and 174 cattle from which Brucella abortus had been cultured. The complement fixation test had the highest specificity in both nonvaccinated and vaccinated cattle. The indirect enzyme immunoassay, if interpreted at a high threshold, also exhibited a high specificity in both groups of cattle. The hemolysis-in-gel test had a very high specificity when used in nonvaccinated cattle but quite a low specificity among vaccinates. With the exception of the complement fixation test, all tests had high sensitivities if interpreted at the minimum threshold. However, the sensitivities of the standard tube agglutination test and indirect enzyme immunoassay, when interpreted at high thresholds were comparable to that of the complement fixation test. A kappa statistic was used to measure the agreement between the various tests. In general the kappa statistics were quite low, suggesting that the various tests may detect different antibody isotypes. There was however, good agreement between the buffered plate antigen test and standard tube agglutination test (the two agglutination tests evaluated) and between the complement fixation test and the indirect enzyme immunoassay when interpreted at a high threshold. With the exception of the buffered plate antigen test, all tests were evaluated as confirmatory tests by estimating their specificity and sensitivity on screening-test positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)

The persistent infection of a canine thymus cell line by equine infectious anaemia virus and preliminary data on the production of viral antigens.

Equine infectious anaemia virus (EIAV) was adapted to the Cf2Th cell line, a heterologous malignant line from canine thymus. A persistent infection was monitored for 100 serial passages by demonstrating the presence of virus and viral antigens at each 10th passage by electron-microscopy, immunodiffusion and immunofluorescence. Chromosome analysis of EIAV-infected cells indicated they had a karyotype resembling the control cells of similar passage history. Virus-infected cells, grown in roller cultures for 65 days without subculturing, continuously produced viral antigens into supernatant fluids which were harvested every 3-4 days. Antigen peaks were observed at approximately 12-day intervals. Immunoprecipitin lines of identity were demonstrated between ether-extracted antigens from virus-infected canine cell line and known positive EIAV antigen extracted from infected equine spleen and a commercial source. Replication of a non-oncogenic retrovirus (EIAV) resulted in the continuous release of viral antigens from a persistently infected and infinite cell line.

Embryo transfer in relation to bovine leukemia virus control and eradication.

Two hundred and seven, zona pellucida-intact bovine embryos were collected from bovine leukemia virus-infected donors, washed, and transferred to uninfected recipients: 111 of these embryos were sired by bovine leukemia virus-infected bulls. Fifty live calves were obtained from the 57 pregnancies resulting from the transfers. None of the recipients or calves developed antibodies to bovine leukemia virus. Nine zona-intact ova, 12 zona-intact morulae and 15 hatched blastocysts, exposed "in vitro" to bovine leukemia virus, washed and then tested for bovine leukemia virus were negative. Twenty-seven, zona-intact embryos and 14 hatched embryos were similarly exposed and washed prior to being transferred in groups to two uninfected recipients: no pregnancies resulted, nor did the recipients develop antibodies to bovine leukemia virus up to 120 days posttransfer. The conclusion from these and other bovine leukemia virus studies is that zona-intact embryos can be transferred from bovine leukemia virus-infected donors, including those bred by bovine leukemia virus-infected bulls, without risk of transmitting bovine leukemia virus, providing that they are properly washed prior to transfer.

Campylobacter fetus in artificial insemination unit and slaughterhouse bulls in Ontario.

Preputial fluid samples were collected from 90 bulls in two Ontario artificial insemination units using a penial glove swab technique previously developed by one of us for use in donor bulls. No Campylobacter fetus organisms were identified from the prepuce or from samples of semen collected at the same time from these bulls. The distal genitalia of 200 bulls were collected at a slaughter house. One isolation of a Campylobacter fetus subspecies venerealis was obtained on a culture from the fornix area of the prepuce of one of these bulls.

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle.

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.

Quantitative evaluation of a transport-enrichment medium for Campylobacter fetus.

The enrichment feature of a selective serum-based transport medium for Campylobacter fetus was quantitatively examined. Preputial samples from artificial insemination bulls were spiked with known numbers of C fetus strains and inoculated into transport-enrichment medium (TEM). The survival and multiplication of these strains in TEM under different incubation periods and temperatures were assessed by plate counts. Mean enrichment values of 3.72 log and 4.42 log were observed after incubation at 37 degrees C for two and four days, respectively. There was no significant difference in the enrichment values between the C fetus subspecies venerealis strains and a C fetus subspecies fetus strain. Incubation of inoculated TEM vials at room temperature for up to two days neither improved the growth of C fetus nor affected its subsequent enrichment when the vials were reincubated at 37 degrees C. Comparison of the survival of C fetus with and without the use of TEM under simulated transport conditions demonstrated the superiority of TEM.

Isolation of bovine syncytial virus from lymphocytes recovered from fluids used to flush uterus and oviducts of superovulated cattle.

A Ficoll-gradient method was applied to isolate lymphocytes from fluids used to flush the uterus and oviducts of superovulated cows. Bovine syncytial virus antigens were demonstrated in 15 of 19 cows by cocultivation of lymphocytes with fetal lamb spleen cells and examining them with direct immunofluorescence. Viral serum antibodies were found in the same 15 of 19 cows as above by the modified direct complement-fixation test. The virus was also detected in one of four uterotubal cell cultures.

An hemolysis-in-gel test for bovine brucellosis.

An hemolysis-in-gel test (HIGT) for bovine antibody against Brucella abortus was developed and evaluated. Sera to be tested were placed in wells in an agarose gel containing guinea pig complement and J-negative bovine erythrocytes coated with lipopolysaccharide prepared from B. abortus biotype 1. After incubation, zones of hemolysis were produced by positive sera. The activity of some positive sera was heat labile, but was restored to heated sera by addition of a crude preparation of the first component of bovine complement. The HIGT was compared with the complement fixation test (CFT), the buffered antigen plate test (BPAT), the standard tube agglutination test (STAT) and the standard plate agglutination test (SPAT), using sera from 1041 brucellosis-free cattle, from 51 cattle infected with B. abortus biotype 1 or biotype 4, and from six heifers vaccinated with strain 19. Three of 1041 sera (0.29%) from brucellosis-free cattle were HIGT-positive, ten (0.96%) were BPAT-positive, and none were positive in the CFT, STAT, or SPAT. The HIGT was more sensitive, and detected infection earlier than the other tests in the case of B. abortus biotype 1 infection, but was less sensitive for biotype 4 infection. In vaccinated heifers, HIGT reactions appeared later than reactions to the other tests, and persisted as long as 565 days. Further studies are needed to standardize the HIGT test conditions and to improve its sensitivity for B. abortus biotype 4 infections. Attempts to determine the effects of LPS antigen prepared from different biotypes of B. abortus are in progress.

Detection of Campylobacter fetus in artificial insemination bulls with a transport enrichment medium.

One hundred and five bulls from an artificial insemination unit were tested for the presence of Campylobacter fetus subspecies venerealis. The method involved the inoculation of preputial samples into a new transport enrichment medium prior to culture and immunofluorescence tests. Seventeen bulls (16%) were found to be either positive or suspected carriers of C. fetus at one or more sampling times. The average age of these 17 bulls was about two years greater than the average age of all the bulls in the unit. A combined treatment of vaccination and dihydrostreptomycin sulfate injection suppressed or eliminated the organism from carrier bulls. The use of transport enrichment medium has increased our capability and effectiveness to monitor the presence of C. fetus in artificial insemination bulls.

Attempts to isolate bovine leukemia and bovine syncytial viruses from blood, uterine flush fluid, unfertilized ova and embryos from infected donor cattle.

Blood leucocytes, sediments of uterine flush fluid (UFF), eggs and embryos from 25 BLV-positive donor cows were tested for bovine leukemia (BLV) and bovine syncytial (BSV) viruses by cocultivation with fetal lamb spleen cells and by applying syncytium induction and immunofluorescence tests. BLV was diagnosed in 11/15 (73.3%) leucocyte and 4/25 (16.0%) UFF-sediment specimens as compared to BSV in 14/15 (93.3%) and 21/25 (84.0%) of the similar specimens and neither BLV or BSV were found in 26 eggs and 60 embryos collected from 20 of the 25 cows. Detection of BLV antigens by immunofluorescence was hampered by the competitive replication of both BLV and BSV and competitive growth in indicator cells and uterine cells. As BLV has not been observed in cells of UFF sediments, it was probably isolated from leucocytes present in the lumen of uterus or from blood seeping out from inapparent vessel damage during flushing. Isolation of BLV in UFF sediments gives additional evidence to the concept of a transplacental transmission by a not yet elucidated mechanism. The high rate of BSV recovery from cells of UFF sediments indicated that this virus is more wide-spread than previously shown and that it may play a role in causing disorders of the reproductive tract.

Demonstration of parvovirus in Canadian swine and antigenic relationships with isolates from other countries.

A Canadian isolate of porcine parvovirus, isolated from cultured pig thyroid cells, was shown to be antigenically indistinguishable from a British (59e/63) and a German (G10/1) strain when treated by the modified direct complement-fixation, the hemagglutination-inhibition and the fluorescent antibody tests. These tests also revealed that antibodies to parvoviruses were detectable in a large proportion of the conventionally raised pigs in the provinces of Quebec and Ontario. Cell cultures, prepared from tissues collected in a slaughterhouse, were often found to be infected with parvovirus. In cell cultures the infection was demonstrated more effectively by immunofluorescence than by the hemagglutination test.

Transmissible gastroenteritis: demonstration of the virus from field specimens by means of cell culture and pig inoculation.

Isolation of transmissible gastroenteritis virus was attempted from segments of jejunum collected from piglets submitted for diagnosis of transmissible gastroenteritis. The virus was isolated more frequently in susceptible piglets than in pig kidney or pig thyroid cells. Practically, both cell systems were equally capable of demonstrating the virus when the tissue suspensions were sonicated. The pig thyroid cells prepared with glands collected from minimal disease pigs were preferred to the pig kidney cells for initial virus isolation because of their ability to respond to transmissible gastroenteritis virus with a progressive cytopathic effect. However, the pig thyroid cells, prepared from pool of glands collected in abattoirs, were often contaminated with parvoviruses and could not be used for diagnostic work. Controlled ultrasound treatments of the inoculum increased the frequency of virus isolation in both cell systems.

Vibriosis: demonstration of Vibrio fetus and Vibrio bubulus organisms in preputial fluid by immunofluorescence and cultural techniques.

Fluorescent conjugates were prepared from the sera of calves immunized with four Vibrio fetus strains and one Vibrio bubulus strain. The fluorescent antibody technique (FAT) was then used to detect vibrio organisms in preputial fluid collected from 67 bulls belonging to a Canadian artificial insemination (AI) unit. The V. fetus conjugates reacted with both V. fetus var venerealis and V. fetus var intestinalis. V. fetus was found in 20 animals (29.9%), 13 of which also harboured V. bubulus. In two cases, the FAT failed to detect V. fetus which was isolated by concurrent bacteriological examinations. It was concluded that the FAT can be a rapid method of detecting some carrier bulls but more reliable results are obtained when a combination of FAT and bacteriological methods is employed. It was found that a single sample giving negative results is inconclusive and additional tests are required before making a final diagnosis. The FAT can also be used to differentiate V. fetus isolates from V. bubulus.

A study of complement-fixation and serum agglutination tests on vibrio fetus infected and immunized cattle.

Complement-fixation (CF) and tube agglutination (TA) tests for demonstration of Vibrio fetus antibodies were conducted on the sera of three groups of ten heifers. One group was vaccinated subcutaneously with a commercial V. fetus var venerealis bacterin and challenged intra-utero, at the external os cervicus one month later; the second was infected only and the third vaccinated only. The vaccinated cattle developed high CF serum titers, but no such increase was observed in animals infected only. A moderate increase in serum antibody titers was demonstrated by the TA test following either infection or vaccination; although titers observed were not higher than those observed in the sera of some apparently normal uninfected animals. The group receiving both vaccine and challenge was the only one in which significant serum antibody titers were demonstrable by the TA test. The sera of these animals also had significant titers in the CF tests. The CF and TA tests detected serum antibodies produced by the parenteral inoculation of V. fetus antigen. These two tests were of limited value in detecting serum antibodies from animals with genital V. fetus var venerealis infection, although the formation of local antibodies was demonstrable by the vaginal mucus agglutination test.

Studies on bovine virus diarrhea: serum neutralization, complement-fixation and immunofluorescence.

The complement-fixation, the serum neutralization tests and the fluorescent-antibody technique were the serological methods applied in this laboratory for the detection of antigens for bovine virus diarrhea (BVD). As observed previously, the modified direct complement-fixation (MDCF) test was required to demonstrate antibodies against virus infections of cattle. At a certain stage of infection, the MDCF test was found to be as accurate and less time-consuming than the serum neutralization test for the detection of antibodies in bovine sera. The modified direct complement-fixing antibodies were detectable in the serum from approximately three weeks up to a few months after infection as compared to several years for the serum neutralization test. Thus, as in most other viral diseases, the MDCF test was of value for detecting recent infections while the serum neutralization test detects both recent and long-standing infections. The fluorescent antibody technique was of value to detect viral antigens of both cytopathogenic and noncytopathogenic strains of BVD in primary fetal kidney cell cultures inoculated with field specimens. In addition, the virus was detected in six of 220 fetuses collected at a local slaughter house for the preparation of primary cell cultures. The length of time required for the detection and identification of specific viral antigens by immunofluorescence was considerably reduced over that of the serum neutralization and virus interference tests.

Anaplasmosis: control of the first outbreak in Canada by serological identification and slaughter.

On August 13, 1968 Canada experienced its first outbreak of anaplasmosis. The initial diagnosis based on hematological and clinical evidence was made by the Provincial Veterinary Laboratory, Winnipeg, Manitoba, and later confirmed in our laboratory by use of the complement-fixation test, hematology, and animal transmission studies. Sixteen herds (1,717 cattle) were examined but the outbreak was found to be localized mainly in one herd of 830 cattle. A low degree of infection was also found in four other herds. None of the remaining 11 herds in the area were infected.The infection was controlled by serological testing, and a slaughter policy. In the four herds with low grade infection, no clinical signs were evident, and serological tests made five and six months after the discovery of the outbreak were negative. In the main herd, the tests were negative at six and nine months. Even though no clinical manifestations of anaplasmosis were detected, surveillance of the animals in the area was continued. Sera from all the cattle were tested 16 months after the initial test. Four reactors were detected in the herd in which the main infection had previously been located. In addition, single borderline reactions were observed in a herd which previously had only one questionable reactor, and in another herd which had heretofore been negative. All of these reactive animals were slaughtered including the two with low grade reactions of doubtful significance. Following the removal of the reactive animals, tests were performed until negative results were obtained twice at six week intervals. The last test was conducted at the end of January 1970, 18 months after the original test.