Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: might c-KIT represent a therapeutic target?

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8-11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials.

Protection of colostral antibodies against bovine leukemia virus infection in calves on a California dairy.

A three-year prospective study involving 244 calves was undertaken on a California dairy to evaluate the protective role of colostral antibodies against bovine leukemia virus (BLV) infection in calves. Calves were followed from birth to the time they left their individual hutch (TLIH), at about 90 days of age. The probability of being infected at TLIH and the daily risk of infection between birth and TLIH were modelled using the logistic and the Cox models, respectively. Calves with no detectable antibodies during the first week of life were up to 2.00 and 2.75 times more likely to be infected at TLIH compared to calves with low and high concentrations of antibodies during the first week of life, respectively (p = 0.01). When the daily risk was modelled, calves without antibodies at the estimated day of infection were up to 3.4 and 11.6 times more likely to become infected than calves with low and high concentrations of antibodies on that day, respectively (p less than 0.001). Results indicated that calfhood infection may be reduced by about 45% through the feeding of colostrum with BLV antibodies. Further reduction in infection may be possible by feeding calves milk powder, milk replacer, and/or milk from noninfected cows. Results also indicated that quantification of the effect of a time-dependent risk factor, such as colostral antibody concentration, might be affected if treated as a fixed factor.

[Bovine leukemia virus antibody levels in the blood and mammary gland secretion in cows in the perinatal period]. / Ksztaltowanie sie miana przeciwcial dla wirusa enzootycznej bialaczki bydla (ebb) w surowicy krwi i wydzielinie gruczoLu mlekowego krów w okresie okoloporodowym.

The titer of BLV-antibodies was estimated in the mammary gland secretion of 18 cows, naturally infected with BLV. Mammary gland secretion samples were collected every week since the 8th week ante partum and every day during the first week post partum. At the same time, blood samples were collected. The examination showed a marked decrease of antibody titer in the blood serum since the 5th week ante partum to the 2nd day post partum. Negative serological results were noticed temporary in the blood serum. The results indicate that serological examination of mammary gland secretion (dry secretion, colostrum) may be helpful because of high concentration of antibodies in the these secretions.

[New trends in the control of enzootic bovine leukemia in Bulgaria]. / Novi nasoki v borbata s enzootichnata levkoza po govedata u nas.

Experimental and literature data as well as personal observations with herds infected with enzootic bovine leukosis have revealed some new aspects that could be made use of in the control of the disease. The more important ones are stated: newborn calves of cows with positive leukosis response can be given untreated colostrum which has been shown to neutralize the virus; in individual regions and Agro-Industrial Complexes where sporadic cases of leukosis have been recorded isolation premises for positively reacting animals could be designed; cows in the last two months of pregnancy do not always give reliable results with the immunodiffusion test, so such animals should not be investigated at that time with this method.

[Importance of saliva and colostrum in leukemia transmission in cattle]. / Znachenieto na sliunkata i kolastrata pri predavane na levkozata u govedata.

Studied was the occasional role played by the saliva of leukotic cows in the transmission of the infection to test lambs along with the role of colostrum as a factor neutralizing the leukosis virus in experimentally infected lambs. It was found that bovine leukosis could be transmitted by the saliva of affected animals. Colostrum that contained hightiter specific antibodies was found to be effective in neutralizing the leukosis virus in newborn calves provided no subsidiary infection took place.

Transmission of bovine leukaemia virus in milk.

Nineteen calves born to dams free of bovine leukaemia virus (BLV) did not possess maternally derived precipitating antibody to BLV in their sera after the ingestion of colostrum. Eight of these calves remained serologically negative after being fed milk from BLV-free cows while three (27.3%) of 11 similar calves that had been fed milk from BLV-infected cows developed antibody. Forty-four of 47 calves born to BLV-infected dams acquired maternal antibody to BLV after ingesting colostrum. Two (8.7%) of the 23 calves fed milk from BLV-free cows developed antibody to BLV probably as a result of transplacental or colostrum infection whereas four (16.7%) of the 24 calves fed milk from BLV-infected cows developed antibody. It is concluded that milk transmission of BLV is responsible in part for the high rates of infection encountered in our dairy herds and that calves lacking specific maternal antibody are more susceptible to BLV infection through the ingestion of milk than are calves with maternal antibody.

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.

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.

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.

Polypeptides of feline leukaemia virus: identification of p15(E) and p12(E).

Antiserum to the p15(E) polypeptide of Rauscher murine leukaemia virus (R-MuLV) precipitated two proteins from purified virions of feline leukaemia virus (FeLV) with apparent mol. wt. of 18500 and 155000 on SDS-polyacrylamide gels. These proteins have been designated p15(E) and p12(E), in line with the nomenclature for MuLV proteins. Like the analogous protein of MuLV, FeLV p15(E) was found to be disulphide-linked to the virion glycoprotein, gp70. FeLV p15(E) was sensitive to digestion of intact virus particles with the proteolytic enzyme, bromelain, indicating that this protein is on the outer surface of the virion. An analysis of cat sera for precipitating activity for FeLV p12(E) showed this only in sera from cats which had recovered from FeLV infection and had virus-neutralizing activity.

Infectivity tests of secretions and excretions from cattle infected with bovine leukemia virus.

Secretions and excretions from cattle with persistent bovine leukemia virus (BLV) infections were tested for BLV by inoculation into sheep. Development of antibody to the BLV glycoprotein antigen and reisolation of virus from peripheral blood leukocytes were the two criteria used to confirm infection in test sheep. The virus was detected in milk from 4 of 6 cows examined and in colostrum from 1 of 4 cows examined. The virus was not detected in semen from 8 bulls or in nasal secretions, saliva, and urine from 2 cows.

Results of an epidemiological survey of enzootic bovine leukosis in the northern part of Lower Saxony and a preliminary communication of an examination into relationship between BLV-antibody development and calving.

The Agar-Gel-Immunodiffusion-Test as proposed by EEC has been carried out during the last 2 1/2 years within a defined test area of Lower Saxony. About 500 leukosis-infected herds with a total of about 20.000 head of cattle have been tested five times now within three to six months sequences. The AGIDT has proved to be an easy and very practical test for routine diagnosis in large scale surveys. Our results described again show the superiority of the glykoprotein-antigen over the ether-treated p24-antigen in the AGIDT. Slaughter of AGIDT-reactors within this population led to a fast decrease of leukosis-infection as detected by the test. Epidemiological herd-data show, that spreading of infection within a herd depends largely on direct contact between infected and noninfected animals. Moreover our data give rise to the suspicion that the manipulations during blood-sampling seem to be implicated in the spread of the disease within a herd. Possible reasons for inconsistencies of antibody-titer in infected animals are discussed.