Eradication of bovine leukemia virus infection from a high-prevalence herd, using radioimmunoassay for identification of infected animals

Radioimmunoassay (RIA), using the virion glycoprotein antigen, was applied in an attempt to eradicate bovine leukemia virus (BLV) infection from a herd in which virtually all the adult cattle are infected. Considering that most calves born to BLV-infected cows are negative for BLV at birth and remain negative for the first several months of life, the eradication program was based on the identification and isolation of the BLV-free calves born to infected cows. Twenty-five calves raised on colostrum and milk from their infected dams were classified as BLV-free on the basis of negative results in the RIA at 6 to 8 and 9 to 11 months of age. These animals were maintained in either complete (10 calves) or partial (15 calves) isolation from infected cattle and were examined at regular intervals for BLV and BLV antibodies. With the exception of 1 calf in the group raised in partial isolation, the animals have remained free of BLV up to the time of the last evaluation, when they were 32 to 35 months old. At these ages, more than 90% of the nonisolated cattle in the herd are BLV-positive. The data also show that this eradication trial would have failed if, in the initial procedure used to classify the calves as BLV-free, the agar gel immunodiffusion test instead of the RIA had been used. Inasmuch as the 25 calves in this study were fed colostrum and milk from their dams, the fact that only 1 of the calves became infected during the 26 to 29 months of observation provides further evidence that milk-borne transmission of BLV is infrequent and perhaps inconsequential.     

Evaluation of the stable fly (Stomoxys calcitrans) as a vector of enzootic bovine leukosis

Experiments reported here were directed at 2 questions: (1) Can the stable fly (Stomoxys calcitrans) transmit enzootic bovine leukosis? (2) Could early viremia augment the probability of transmission by this insect? In one vector experiment, calves and bovine leukemia virus (BLV)-infected cows were housed with and without stable flies. The calves were monitored serologically during a 3-month postexposure period, using the agar gel immunodiffusion test. All fly-infested and fly-free calves remained BLV-seronegative. For a second vector experiment, donor calves, newly injected with blood from BLV-infected cows with high virus expression, were tethered alternately between uninoculated, weaned BLV-seronegative calves. These groups were housed with or without flies in 2 replicate trials. The inoculated calves from the first replicate seroconverted at 16 and 23 days after inoculation; the inoculated calves from the second replicate seroconverted at 11, 16, 16, and 37 days after inoculation. All uninoculated calves remained BLV-seronegative. In a manual transmission experiment, 50 unfed stable flies were allowed to complete a meal on each of 3 BLV-seronegative calves after feeding on a BLV-seropositive cow with high (42%) virus expression. One control calf was injected with blood from the cow. Seroconversion occurred in the control calf and 1 calf on which flies were given access. A scanning electron microscopic study was made of the everted and closed mouth parts of the stable fly. Given the lymphocyte count in blood from the cow used in the manual vector transmission experiment, it was calculated that 3,950 mouth part volumes would be necessary to transmit BLV.(ABSTRACT TRUNCATED AT 250 WORDS)     

An attempt to protect calves against experimental bovine leukosis using allografts

Six calves sensitised by implanting skin from a calf were later inoculated with lymphocytes from the same calf after the calf had been infected with bovine leukosis virus (BLV). Two out of 6 calves challenged did not develop BLV antibodies and BLV was not isolated from these animals, whereas all of the 5 control calves became infected with BLV.     

Effect of colostral antibody on bovine leukemia virus infection of neonatal calves

Pairs of newborn calves were exposed to bovine leukemia virus (BLV) when they were given their 1st colostrum feeding. Calves that were given 10(6) BLV-infected lymphocytes in colostrum free of BLV-specific antibody became infected. Calves that were fed 10(7), 10(8), or 10(9) infected lymphocytes in colostrum that contained BLV-specific antibody did not become infected. One of 2 calves inoculated intradermally with 250,000 infected lymphocytes was protected by colostral antibody, but the other was not. Colostral antibody titers in the unprotected calf decreased normally until the calf was 4 months old and then increased markedly; this pattern indicates that the presence of colostral antibody may have prolonged the latent period of the BLV infection.     

In utero transmission of bovine leukemia virus

In an initial study, 18 calves born to cows persistently infected with bovine leukemia virus (BLV) were tested for infective virus and antibodies at birth, and no infected or seropositive animals were found. Four of these calves were maintained in quarters where infected animals were housed, and 3 of the 4 subsequently became infected. These were probably contact infections acquired during, or at some time after, birth. The remaining 14 calves were kept in isolation pens in a building housing no infected cattle. None of this group was found to be BLV infected during 1 year of observation. In further studies, 15 pregnant cows inoculated with BLV became infected. One abortion, considered to be unrelated to the BLV inoculation, occurred 38 days later. The remaining 14 cows gave birth to 1 dead and 14 live calves. The dead calf and its live twin were seropositive for BLV at birth, indicating that they had been infected in utero. The remaining 13 calves were negative for BLV antibodies at birth and remained so during 1 year of observation.     

Timing of seroconversion and acquisition of positive polymerase chain reaction assay results in calves experimentally infected with bovine leukemia virus

OBJECTIVE: To determine the interval to provirus and serum antibody detection (via PCR assay and ELISA, respectively) in calves after experimental inoculation with bovine leukemia virus (BLV). ANIMALS: 8 colostrum-deprived, BLV-negative Holstein bull calves (> or = 6 weeks old). PROCEDURES: Via IM injection, each calf received a fresh whole-blood inoculum (day 0) calculated to contain 2 x 10(6) lymphocytes. Blood samples for the ELISA and PCR assay were collected from calves immediately prior to inoculation and weekly thereafter for 7 weeks. Mean and median number of weeks to PCR-detected conversion of BLV status and seroconversion were calculated. Point sensitivity and cumulative sensitivity of the 2 assays were calculated at each sample collection. At each sampling time, the proportion of calves identified as infected by the cumulative weekly ELISA and PCR assay results was compared by use of a Fisher exact test. RESULTS: In 5 calves, conversion of BLV status was detected via PCR assay before seroconversion was identified. However, seroconversion preceded PCR-detected conversion in 2 calves. In 1 calf, both assays yielded positive results at the same test date. These differences were not significant. CONCLUSIONS AND CLINICAL RELEVANCE: In experimentally inoculated BLV-negative calves, conversion of BLV status was detected via PCR assay more quickly than via ELISA; this difference was not significant and probably not clinically important. The PCR assay may be useful as a confirmatory test in animals of exceptional value; tests based on viral identification may become critically important if vaccines against BLV infection are developed and marketed.     

Decreased periparturient transmission of bovine leukosis virus in colostrum-fed calves

BACKGROUND: The relation between calf bovine leukosis virus (BLV) infection status and colostrum ingestion is unclear. Two conclusions have been drawn from previous studies. One suggests that colostrum ingestion transmits BLV to neonatal calves. The second suggests that colostral antibodies are protective. HYPOTHESIS: Colostrum from BLV-positive cattle is protective in naturally exposed calves. ANIMALS: Twelve colostrum-deprived Holstein calves and 20 colostrum-fed Holstein calves born to BLV-infected cows. METHODS: Prospective study. Colostrum-deprived calves were tested weekly by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) tests for BLV antibody and provirus for 12 weeks or until the animal became positive for BLV infection. Colostrum-fed calves were fed colostrum derived from BLV-positive cows. Thereafter, ELISA and PCR tests for BLV antibody and provirus were performed every other week until 2 consecutive negative ELISA tests or 1 positive PCR test was achieved. The proportion of calves that converted to BLV-positive status was calculated for each group and compared between groups by using the Fisher exact test. RESULTS: Four of 12 colostrum-deprived calves (33%) became BLV positive, whereas 0 of 20 colostrum-fed calves (0%) became BLV positive. The proportion of calves that became infected was significantly higher in the colostrum-deprived group (P = .014). CONCLUSIONS AND CLINICAL RELEVANCE: Calves born to BLV-positive cows are exposed during parturition, and a proportion of these calves will become infected with BLV. Administration of colostrum from BLV-positive cows greatly decreases the risk of infection.     

Vertical transmission of bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds.

Vertical transmission of bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) was investigated in five dairy cattle herds in Hokkaido, where 36.1 and 17.0% of cattle were BLV and BIV seropositive, respectively, and 9.9% of dams were co-infected with both BIV and BLV. Twenty six cases of offspring born from dams infected with only BLV (17 cases) or with both BIV and BLV (9 cases) were examined for the presence of BLV and BIV before and after colostrum feeding by polymerase chain reaction (PCR) and syncytium assay. After birth, all calves were separated immediately from their dams. The offspring born from BLV-positive dams were BLV-negative before colostrum feeding, suggesting that no transplacental transmission had occurred. Thereafter, these offspring were fed colostrum or milk from their dams, but still remained BLV-negative. The other offspring born from BLV-positive dams were fed with BLV-negative colostrum, or with pasteurized BLV-positive colostrum. All these calves remained negative for BLV infection, suggesting that in utero transmission of BLV is negligible. In the case of offspring born from dams co-infected with BLV and BIV, calves were BIV-positive before colostrum feeding at 1 day after the birth, indicating in utero transmission of BIV. After colostrum feeding from their dams, newborn calves became BLV-positive. In addition, one calf was BLV-positive even before colostrum feeding. These results suggest that BIV can be transmitted to offspring in utero, and that BLV can be transmitted through colostrum or milk if dams are infected with both BIV and BLV.     

Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage

A non-surgical embryo collection was completed on a day 7 superovulated Chianina donor cow. Because all but two of the ova from the collection were unfertilised and a surplus of potential recipients was available, one embryo (an excellent quality late morula) was dissected into four equal portioned 'quarter' embryos using a simplified micromanipulation procedure. Each quarter embryo was then placed in a 0.25 ml French straw and non-surgically transplanted to four different crossbred beef recipient females. The remaining embryo was similarly transplanted to a herd mate recipient as an intact embryo. One recipient returned to oestrus, one recipient had an extended post transfer cycle and the two remaining recipients produced a live quarter embryo transplant calf each within 24 hours of the other. The intact embryo placed in a herd mate recipient did not produce a transplant calf. To the authors' knowledge, these transplant offspring are the first live births reported from a non-surgically collected later-stage bovine morula (day 7), which had been dissected into quarters and then individually transplanted non-surgically to recipient females. The procedure was relatively simple to perform and was completed in less than one hour.     

Bovine leukemia virus: Duration of BLV colostral antibodies in calves from commercial dairy herds

A study was conducted to determine the duration of colostral antibodies to bovine leukemia virus (BLV) in diary calves from commercial dairy farms. Sera of pregnant dams from four different dairy farms and sera of the calves of these dams were analyzed for BLV antibodies using the agar gel immunodiffusion (AGID) test. Precolostral serum samples were collected from the female calves of known BLV serologically positive dams. Postcolostral serum samples of the same calves were collected on Day 2 and biweekly until 2 consecutively negative AGID tests performed 4 weeks apart were obtained. Subsequently, the biweekly serum samples from each calf were analyzed quantitatively for BLV antibodies with the AGID test. End-point titers were determined using phosphate buffered saline to make two-fold dilutions. A logarithmic transformation of the inverse of the end-point titer was used to determine the regression line of antibody decay for each calf. Estimated weighted regression analysis was used to determine the least-squares regression line for 27 of the 38 calves sampled. The duration of colostral antibodies was calculated as 71 days using the prediction equation. The minium and maximum durations of colostral antibodies were 14 days and 147 days, respectively. The half-life of the antibodies was 36.05 days. Factors affecting the duration of BLV colostral antibodies and the practical applicability of this study were discussed.     

Embryo transfer and transmission of bovine leukosis virus in a dairy herd

Transmission of bovine leukosis virus (BLV) by embryo transfer was investigated in a large commercial Holstein herd. One hundred and sixteen calves, transplanted as embryos from BLV-positive cows into BLV-negative heifers, were serologically negative, as were recipients, whereas 5 of 29 (17%) calves transplanted as embryos into BLV-positive recipients were infected with BLV, as evidenced by antibodies in the agar gel immunodiffusion test.     

Reproductive performance of apparently healthy cattle persistently infected with bovine viral diarrhea virus.

A Holstein-Friesian bull and three Holstein-Friesian cows were seronegative for bovine viral diarrhea (BVD) virus but were persistently infected with the virus. Virus was isolated from buffy coat cells and nasal and lacrimal secretions during their lifetime, and they remained free of clinical signs of BVD. The three cows were pregnant when purchased, and they gave birth to full-term calves. One calf lived only a few hours, one calf became ill and died within a few days, and one calf became ill and was euthanatized within a few weeks. One cow was then bred and became pregnant but aborted a 7-month fetus. A second cow was bred approximately 5 months after parturition but did not conceive. The third cow was necropsied 6 weeks after calving, because of loss of weight. Although the bull's semen contained BVD virus when seropositive cows were bred, normal calves were born. When seronegative heifers were bred, they became seropositive to BVD virus within two weeks, with higher titers in six weeks. On heifer conceived after one service but aborted a 6-month fetus. Three others continued to have estrous cycles until their titers rose to 1:128, then they conceived and gave birth to normal calves. Another heifer conceived on the first service, had a titer of 1:128 two weeks after breeding, and gave birth to a normal calf.     

Decay of colostral antibodies to bovine leukemia virus with application to detection of calfhood infection

An estimated weighted-regression method was used to describe the decay of colostral bovine leukemia virus (BLV) antibodies in the calf, as measured by agar-gel immunodiffusion with glycoprotein antigen. The prediction equation, based on 473 observations from 130 animals, was log10 inverse titer = 1.29 -0.012 age (days). The half-life of BLV antibodies was estimated to be 25.8 days. Ages at which colostral antibodies were last detected were between 51 and 187 days. Normal limits of antibody decay were estimated and used to identify virus-induced active antibodies in calves during the colostral antibody period. Calves known to be infected were identified between 2 and 180 days of age, using 95% limits. Application of this procedure for the early serologic detection of BLV-infected calves in eradication or control programs is discussed.     

Direct detection of bovine leukemia virus infection: practical applicability of a double polymerase chain reaction.

A double polymerase chain reaction (PCR) assay has been devised for the direct detection of bovine leukemia virus (BLV). The assay was directly performed on blood leukocytes, avoiding the DNA-purification procedures. The PCR products were identified by gel-electrophoresis and the specificity of the test was confirmed by hybridization with a biotinylated oligonucleotide probe. When testing the sensitivity of PCR, less than eight genome copies of the provirus were detected in the background of two million negative lymphocytes. In a BLV infected herd 22 animals of various age groups were examined by the indirect (serological) diagnostic tests of agar-gel immunodiffusion and indirect ELISA as well as by the direct detection method of PCR. The tests were repeated at monthly intervals on five occasions. When examining the specimens from cows and heifers, a close agreement was found between the results of the various methods. The newborn calves, which were the offspring of BLV infected mothers, were consequently negative in PCR throughout the experimental period. However, in the indirect tests the calves were positive during the first samplings and became negative only around four months of age. Since the indirect tests can not discriminate infection from colostral immunity, PCR proved to be a useful complementary assay for the safe diagnosis of BLV infection in young calves.     

Investigation of the possible role of the tuberculin intradermal test in the spread of enzootic bovine leukosis

The single intradermal comparative test was used with both avian and bovine tuberculin. Three cattle infected with bovine leukosis virus (BLV) were used as a source of infection. BLV-positive and susceptible animals were tuberculin tested alternately. Fifteen susceptible calves and 15 susceptible sheep were tested. A further three calves and three sheep were used as controls; the needles of the tuberculin syringes were deliberately contaminated with blood from the BLV-infected cattle, before being used in the test. Whereas all three calves and the three sheep inoculated intradermally with contaminated needles developed BLV infections, all of the other 30 animals have remained serologically negative to BLV for 10 months. Transmission of BLV with needles contaminated with BLV-infected blood was prevented by wiping the needles with absorbent cotton wool.     

Transmission of bovine leukemia virus by Tabanus fuscicostatus

Bovine leukemia virus (BLV) was transmitted by horse flies, Tabanus fuscicostatus, from a cow with a lymphocyte count of 31,500/mm3 to goats and dairy calves. As few as 10 and 20 flies transmitted BLV to goats and calves respectively, but the minimal number of flies required to transmit the infection was not established. Groups of 150 and 100 T fuscicostatus transmitted BLV to beef calves from a cow with a lymphocyte count of 14,600/mm3. These results support a role for horse flies in the horizontal transmission of BLV.     

Prenatal and postnatal maternal contributions to reproductive, maternal, and size-related traits of beef cattle

Brown Swiss-Hereford (BS-H) reciprocal cross embryos were transferred to BS and H recipient cows and Red Poll-Angus (RP-A) reciprocal cross embryos were transferred to RP and A recipient cows to estimate the relative contributions of ovum cytoplasm and uterine influences to prenatal maternal effects. Calves resulting from embryo transfers (ET) were weaned early (3 to 5 d). Reciprocal cross mating also were made by natural service (NS) between BS and H and between RP and A breeds; part of the offspring were weaned at 3 to 5 d, and the remainder nursed their dams to an age of 150 to 180 d. This was done to estimate breed differences in prenatal and postnatal effects combined and to separate the effects of prenatal maternal influences from postnatal maternal influences of these breeds. Females produced in both ET and NS parts of the experiment were retained to produce three calf crops to an age of about 4.5 yr. The following traits were analyzed: percentage of conception rate; percentage of calf survival; percentage of calves produced per cow exposed; birth and weaning weights of calves produced; and periodic weights, heights, and condition scores of females to an age of 4.5 yr. Neither breed of donor (cytoplasmic influence) nor breed of recipient (uterine influence) had consistently important effects on the traits evaluated. In NS matings, differences between reciprocal crosses were small for most of the traits evaluated. Method of rearing (nursed vs weaned at 3 to 5 d) had no effect on reproductive and maternal traits for RP-A reciprocal cross females, but females that nursed generally were heavier, were taller, and had higher condition scores at most ages than early-weaned females. For the BS-H reciprocal cross, early-weaned females were favored over females reared by their dams in percentage of calves produced per cow exposed, but the method of rearing did not affect other reproductive or maternal traits. BS-H reciprocal cross females that nursed their dams were heavier at 550 d and were heavier and had higher condition scores at an age of 34 mo than early-weaned females.     

Role of insects in the transmission of bovine leukosis virus: potential for transmission by stable flies, horn flies, and tabanids

The ability of stable flies (Stomoxys calcitrans), horn flies (Haematobia irritans), and tabanids (Diptera: Tabanidae) to transmit bovine leukosis virus (BLV) was investigated. Stable flies and horn flies were fed on blood collected from an infected cow, and the flies' mouthparts were immediately removed, placed in RPMI-1640 medium, ground, and inoculated into sheep and calves. Infection of sheep occurred with mouthparts from as few as 25 stable flies or 25 horn flies. However, sheep were not infected when removal of stable fly mouthparts was delayed greater than or equal to 1 hour after blood feeding. Infection of calves occurred after inoculation of mouthparts removed immediately after feeding from as few as 50 stable flies or 100 horn flies. Infected blood, applied by capillary action to the mouthparts (labella) of 15 deer flies (Chrysops sp) and a single horse fly (Tabanus atratus) caused infection in each of 2 sheep. Infection did not occur in 2 calves inoculated daily for 5 days with mouthparts from 50 horn flies collected after feeding on a BLV-infected steer. Four calves receiving bites from 75 stable flies interrupted from feeding on a BLV-positive cow also were not infected. Seronegative cattle held for 1 to 4 months in a screened enclosure with positive cattle in the presence of biting flies were not infected with BLV. The feeding behavior of each insect is discussed to assess their potential as vectors of BLV.     

Development and serial passage of persistent lymphocytosis associated with bovine leukemia virus infection in cattle

Two calves each were inoculated with 1.5 x 10(8) or 5 x 10(9) lymphocytes collected from each one cow which had persistent lymphocytosis (PL) and antibodies to bovine leukemia virus (BLV). A sudden increase in the number of peripheral blood lymphocytes (PBL) was observed 14 and 23 days, respectively, after inoculation and the maximum number reached 29,000 and 52,000/microliters 72 and 57 days after inoculation. Although the degree of PL decreased gradually in these cattle, it continued until 14 and 44 months after inoculation when one animal was sacrificed and the other died of lymphosarcoma. The PL was passaged in cattle by inoculation of a large number of PBL obtained from cattle at the stage of PL (PLL). The degree of PL was severer in cattle inoculated with a larger number of PLL. PL was not caused by inoculation of PBL obtained from either BLV-infected non-PL cattle or cattle free of BLV. The PL was also caused by inoculation of PLL into BLV-infected non-PL cattle. On the other hand, it was not observed after inoculation of a large amount of cell-free virus obtained from short-term cultures of PLL. Antibodies to BLV developed earlier and to higher levels in cattle inoculated with PLL than in those inoculated with cell-free virus. These facts show that infection with BLV was established more effectively by PLL than by cell-free virus, the infection may occur by lymphocyte to lymphocyte interaction and the actual number of infected BLV may have an important role in development of PL.     

Role of the spleen in the pathogenesis of experimentally induced bovine leukemia virus infection

In an extension of a previous pathogenesis study, bone marrow and other tissues from four experimentally inoculated cattle were tested for virus between the 13th and 20th days after experimental inoculation with bovine leukemia virus. BLV was detected in the blood of three, spleen of two, lymph node of two and bone marrow of only one of the inoculated cattle. In additional studies, four splenectomized and two intact control calves were also examined. Two of these calves were splenectomized before BLV inoculation and two after a persistent virus infection had been established. Results indicated that the removal of the spleen affected neither the establishment and persistence of virus infection nor the development and maintenance of serological responses to viral antigens.