Molecular Epidemiological Investigation Of Bovine Leukemia Virus And Research On Its Pathogenicity

Bovine leukemia virus(BLV),which belongs to the family of Retroviridae and genus Deltaretrovirus,is genetically closely related to human T-cell leukemia virus-1(HTLV-1).The BLV genome consists of 8,714 nucleotides including gag,pro,pol,env,G3,G4,TAX,REX genes which are flanked by two identical long terminal repeats(LTRs).The virus is the causative agent of enzootic bovine leukosis(EBL),and approximately 30%infected cattle develop persistent lymphocytosis(PL)while a small percentage of the infected cattle will die from malignant lymphoma.In addition to cattle,the virus can infect also zebu,buffalo,sheep and capybara.Surprisingly,the provirus DNA has been identified in breast tissues of the patients with breast cancer by nested PCRs.Some studies have demonstrated a negative effect of BLV infection on milk production at the herd level However,no association has been found between BLV infection and milk production in several studies in individual animals.This study attempts to establish a highly sensitive and specific molecular detection method for BLV detection.The established method was then used to investigate the prevalence of BLV in four islands of the Caribbean and in 19 provinces of China.Most importantly,this study investigate the pathogenicity of BLV and its effect on the production of dairies based on a retrospective cohort study and a model of BLV-infected dairy cattle.1.Establishment of BLV FRET-qPCRA variety of existing techniques are available for diagnosis of EBL worldwide.Histopathological test can be used to identify suspected cattle by observing the pathological changes in lymph nodes and other diseased regions.The hematological examination can also screen the infection of BLV by peripheral lymphocytes and it is possible to suffer from the disease if the number and percentage of lymphocytes increases significantly.Serological methods include indirect ELISA and blocking ELISA,which are based on the anti gp51 and p24 antibodies in the serum of the infected animals.Molecular assay are popular in the recent years as its high sensitivity and specificity,and can detect BLV provirus immediately after infection.In this study,we designed pol gene-based primers and probes while aligning all available BLV complete sequences in GenBank before May 2015,and the established system enables us reliably to detect single copy of BLV DNA per reaction.The BLV FRET-qPCR was negative for bovine herpesvirus 1,bovine viral diarrhea virus,bovine respiratory syncytial virus,Mycobacterium bovis,Brucella abortus,Babesia spp.,Theileria spp.and Anaplasma spp..The FRET-qPCR was performed with blood,virginal secretions,milk and feces of 105 cows,and the highest positive rate and BLV copy number was found in the whole blood.The BLV FRET-qPCR kit developed in this study has been granted as a China Invention Patent(ZL201410597176.X).2.Molecular epidemiology and characterization of BLV in the CaribbeanThe Caribbean,located between North America and South America,is a region that consists of the Caribbean Sea,its islands and the surrounding coasts.To date,few researcher has identified the presence of BLV in this region by molecular tests.To investigate the BLV prevalence in this region,DNA from whole blood of cattle(n=325)from three Caribbean island countries were collected and screened using HMBS-based endogenous internal control system.BLV FRET-qPCR gave positive result on Dominica(5.2%;4/77)and St.Kitts(19.2%;37/193),and negative result on Montserrat(n=12)and Nevis(n=43).In order to find out the genotypes of BLV in the Caribbean,two pairs of primers were designed targeting the env gene.Phylogenetic tree(neighbor-joining)based on full-length of env(1,548 bp)and env gp51(807 bp)showed that the BLV strains in the Caribbean were belonged to genotype 1(99.4%similarity),which is the most widely spread genotype in the world.This is the first molecular characterization of BLV infections in the Caribbean.3.Molecular epidemiology and characterization of BLV in ChinaIn China,the department of agriculture and holders pays little attention to EBL as infected animals always appear asymptomatic.However,some developed countries in Western Europe,Australia and New Zealand,have nearly succeeded in eradicating this disease.For the decades,the United States Department of Agriculture(USDA)carried on national wide epidemiology of EBL for several times and found it is widely spread in American dairy and beef cattle.Accordingly,it is highly necessary for us to investigate the BLV prevalence in China.Whole blood samples of 1,963 dairies and 1,390 beefs from 19 provinces of China were tested with the FRET-qPCR and commercial blocking ELISA kit.Overall,significantly more dairy cattle(49.1%,964/1,963)were positive for BLV proviral DNA as compared with beef cattle(1.6%,22/1,390;P<0.0001).The copy number of BLV were 103.57±1.55 per milliliter in cows,which were significantly higher than 101.42±1.32per milliliters in beefs.Serological test showed 86.3%identity with the molecular test.In order to find out the genotypes of BLV in China,we performed regular PCR targeting the env gp51 gene and sequencing.The phylogenetic tree(neighbor-joining)gave the result that the BLV strains in China were closest to genotype 6(97.9%similarity).To compare the env gp51 nucleotide and deduced amino acid sequences of Chinese BLV strains,we found the strains from Yancheng,Shanghai and Bengbu had identical substitutions and the strains from Yangzhou and Tianjin had identical substitutions.We reported the prevalence of BLV in China in an international journal at the first time.4.The pathogenicity of BLV:Based on a retrospective cohort studyBLV is widely spread in dairy cattle and the copy number of BLV in dairies is significantly higher.To investigate the association between BLV infection and milk yield and milk quality among cows,a retrospective cohort study was performed in a BLV-positive dairy farm in Shanghai,China.Whole-blood samples were collected at the beginning of this study and a year later for PCR detection of BLV,but only those cows positive or negative for BLV at both time points were included in the data analysis.Totally 655 Holstein cows,which were free with theileriosis,anaplasmosis and ehrlichiosis,had been selected in this study.Among the 655 cows,81.7%were BLV-positive and 18.3%were BLV-negative.At the outset,44.3(290/655),38.8(254/655),and 16.9%(111/655)of the cows were in the early,middle,or late lactation stages,respectively.Most were in their second pregnancy(parity 1 = 36.5%,239/655)with fewer in their subsequent pregnancies(third = 25.3%,166/655;fourth,14.4%,94/655;fifth or more = 23.8%,156/655).Most animals(14%,92/655)were in their early lactation following their second pregnancy(early lactation,parity 1),with the least in their late lactation and their fourth or fifth pregnancy(lactation≥3,parity 3 or ≥4).The result demonstrated that BLV infection was associated with a significantly reduced milk yield and increased SCS when cows with a parity of>4 were in their early(26.8 vs.30.9 kg;13.2%reduction compared with non-infected;P=0.0002)and middle(22.2 vs.26.1 kg;14.9%reduction compared with non-infected;P=0.0002)stages of lactation(Figure 2).Similarly,these cows with parity ≥4 had increased SCS compared with uninfected cows in their early(5.2 vs.4.3;P<0.0001)and middle(4.9 vs.3.9;P=0.0002)stages of lactation.The milk production and SCS did not differ significantly between the BLV-infected and-uninfected cows when the cows were in the late stage of lactation or when they had parity ≤3.5.The pathogenicity of BLV:Based on dairy cattle infection modelWe established a BLV cow infection model to study the complete blood count(CBC),immunity related cytokines,milk yield quality,the transcriptome of peripheral white blood cells and virus distribution in cattle under the infection BLV.Thirty cows(all in their second pregnancy)of the similar genetic background and performance were selected and randomly divided into two groups.Before BLV infection,we detected or obtained the CBC,cytokines(GM-CSF,IFN-γ,IL-lb,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12p70,IL-13),milk production and DHI records(fat%,lactose%,protein%,and somatic cell count)data of all 30 cows,and no significant difference was shown between the two groups with all the parameters.Cows in group-1 were challenged with the whole blood from a BLV positive cow while group-2 cows were challenged with 1 × phosphate buffered solution(1 × PBS).All the management procedures were the same except the blocks where they were housed.For the molecular detection,the DNA of BLV previous was successfully identified in the peripheral blood from 6 cows at 2 hours post-injection.Five days past infection,6 cows were detected to be BLV positive,among which five of them were consistent with previous detection.From fifteen days post-injection,all the cows were detected to be positive.For the antibody detection,no anti-BLV gp51 protein could be detected until 45 days post-injection.Based on the BLV infection model in this study,during the 8 months post-injection,BLV infection was associated with a significantly increased milk yield(33.00±8.42 vs.32.45±8.18,kg,1.67%increment compared with noninfected,P=0.03)and milk fat(4.45±1.40 vs.3.88± 1.04,%;P=0.02),and no association was found between BLV infection and other parameters.The results of CBC and cytokines indicated that cows had an obvious inflammatory reaction after BLV injection,and the inflammatory reaction was mainly concentrated on the days 15-45 after injection.CBC parameters(WBC,lymphocyte,monocyte,granulocyte,lymphocyte%,monocyte%,granulocyte%,hemoglobin,hematocrit,MCHC,RDW,platelet and RDW)showed significant differences(P<0.05)between injected and control groups after injection.Interestingly,the WBC,lymphocyte and monocyte decreased significantly at 5 days post-injection and increased significantly at 15 days and 45 days post-injection and stay smooth after then.The expression level of several cytokines increased significantly at 15 days post injection,including IFN-γ(75,794.73±42,529.12 vs.41,065.30±25,216.67,pg/mL,P=0.01),IL-10(108,555.82±50,554.92 vs.69,994.84±39,582.16,pg/mL,P=0.03)and IL-12p70(4,304.16±2,483.99 vs.2,306.02±779.66,pg/mL,P=0.008).Furthermore,the expression of IL-12p70 increased significantly at 165 days(3,791.58±1,200.86 vs.2,926.13±711.69,pg/mL,P=0.05)post injection.The transcriptome analysis of peripheral blood leucocytes gave us 12 differentially expressed genes(DEGs),and seven of which were closely related to cancer occurrence.These DEGs are RECQL4,KIFC1,CDC20,DTYMK,NCAPH,UBE2C and MKI67.To study the distribution of BLV in cows,totally 16 kinds of viscera were sampled from three injected cows.FRET-qPCR gave positive result on blood,muscle,synovial fluid,heart,liver,spleen,kidney,rumen,breast,gills lymph nodes,submaxillary lymph nodes,superficial inguinal lymph nodes,common iliac lymph nodes and negative results on jejunum and ovary.The copy number of BLV in blood and spleen was significantly higher than that in other viscera.In conclusion,this study established the BLV FRET-qPCR and performed the molecular epidemiology and characterization of BLV infection in the Caribbean and China.The association between BLV infection and dairy production,CBC,cytokines and transcriptome was investigated based on retrospective cohort study and dairy cattle infection model.This study lays the foundation for further discover the diagnostic,source-tracking,pathogenicity and elimination of BLV.