Effects of species and sex on plasma hormone and metabolite concentrations in crossbred Brahman cattle and crossbred water buffalo

Buffalo meat is tasty and indistinguishable from beef, and has been described as being lean and low in cholesterol in comparison with beef. However, little is known about the plasma hormone and metabolic profiles related to fat metabolism in buffalo, and how their levels are affected by species and sex. In this study levels of plasma hormones and metabolites were compared between species and sexes. Ten growing crossbred Brahman cattle and ten growing crossbred buffaloes (five males and five females in each), with an average age of 22 months were used. The animals were fed on an experimental diet composed of corn silage, brewer's spent grain and a concentrate mixture at a 50:30:20 ratio on a dry matter basis during a fattening period of 6 months. Blood samples were collected every month during the experiment, and were analysed for plasma leptin, insulin, glucose, triglyceride (TG) and total cholesterol (TC) concentrations. Plasma leptin, insulin and TC concentrations increased during fattening and were significantly (P < 0.05) higher in cattle than in buffaloes. These concentrations in cattle showed a big difference between sexes in comparison with buffaloes, and were higher in females than in males. The plasma glucose and TG concentrations did not differ (P > 0.05) between cattle and buffaloes.

The crude fat content of longissimus muscle was significantly higher in cattle than in buffalo. Differences in body fat deposition seem to have an influence on plasma leptin, insulin and TC concentrations differently between cattle and buffaloes.     

Genetic diversity in Trypanosoma theileri from Sri Lankan cattle and water buffaloes

Trypanosoma theileri is a hemoprotozoan parasite that infects various ruminant species. We investigated the epidemiology of this parasite among cattle and water buffalo populations bred in Sri Lanka, using a diagnostic PCR assay based on the cathepsin L-like protein (CATL) gene. Blood DNA samples sourced from cattle (n = 316) and water buffaloes (n = 320) bred in different geographical areas of Sri Lanka were PCR screened for T. theileri. Parasite DNA was detected in cattle and water buffaloes alike in all the sampling locations. The overall T. theileri-positive rate was higher in water buffaloes (15.9%) than in cattle (7.6%). Subsequently, PCR amplicons were sequenced and the partial CATL sequences were phylogenetically analyzed. The identity values for the CATL gene were 89.6–99.7% among the cattle-derived sequences, compared with values of 90.7–100% for the buffalo-derived sequences. However, the cattle-derived sequences shared 88.2–100% identity values with those from buffaloes. In the phylogenetic tree, the Sri Lankan CATL gene sequences fell into two major clades (TthI and TthII), both of which contain CATL sequences from several other countries. Although most of the CATL sequences from Sri Lankan cattle and buffaloes clustered independently, two buffalo-derived sequences were observed to be closely related to those of the Sri Lankan cattle. Furthermore, a Sri Lankan buffalo sequence clustered with CATL gene sequences from Brazilian buffalo and Thai cattle. In addition to reporting the first PCR-based survey of T. theileri among Sri Lankan-bred cattle and water buffaloes, the present study found that some of the CATL gene fragments sourced from water buffaloes shared similarity with those determined from cattle in this country.     

Ruminal fermentation and microbial ecology of buffaloes and cattle fed the same diet

Although buffaloes and cattle are ruminants, their digestive capabilities and rumen microbial compositions are considered to be different. The purpose of this study was to compare the rumen microbial ecology of crossbred water buffaloes and cattle that were fed the same diet. Cattle exhibited a higher fermentation rate than buffaloes. Methane production and methanogen density were lower in buffaloes. Phylogenetic analysis of Fibrobacter succinogenes‐specific 16S ribosomal RNA gene clone library showed that the diversity of groups within a species was significantly different (P < 0.05) between buffalo and cattle and most of the clones were affiliated with group 2 of the species. Population densities of F. succinogenes, Ruminococcus albus and R. flavefaciens were higher until 6 h post‐feeding in cattle; however, buffaloes exhibited different traits. The population of anaerobic fungi decreased at 3 h in cattle compared to buffaloes and was similar at 0 h and 6 h. The diversity profiles of bacteria and fungi were similar in the two species. The present study showed that the profiles of the fermentation process, microbial population and diversity were similar in crossbred water buffaloes and crossbred cattle.     

Lumpy skin disease outbreaks investigation in Egyptian cattle and buffaloes: Serological evidence and molecular characterization of genome termini

Lumpy skin disease (LSD) is an endemic highly infectious viral disease affecting cattle in Egypt. This study aimed to identify and characterize the LSD virus (LSDV) outbreaks in Egypt between 2016 and 2018 and to determine the role of Egyptian buffaloes in the epidemiology of LSD. A total of 44 skin biopsies (41 from cattle and 3 from buffaloes) and 31 blood samples from asymptomatic buffaloes in contact with clinically infected cattle were collected from 7 Egyptian governorates and tested by real-time (rt)-PCR. The positive samples were further isolated, and the isolates were analyzed by conventional PCR to amplify the LSDV001 and LSDV002 genes; three isolates were sequenced, and the phylogenetic tree was constructed. In addition, 198 serum samples (102 from cattle and 96 from contact buffaloes) were examined using ELISA. Out of 44 skin nodules analyzed by rt-PCR, 31 (70.45 %) were positive while, non of the buffalo samples were positive. Out of 31 positive rt-PCR samples, LSDV was isolated on CAM (n=19; 61.29%) and MDBK cell culture. The virus isolates were confirmed by conventional PCR where 1237 bp product size was successfully amplified. The phylogenetic analysis of LSDV002 gene revealed that three sequenced LSDV isolates were identical to each other and to LSDV isolates from different countries in Africa, Asia, and Europe with 99–100 % identity. ELISA analyses showed seroreactivity of LSDV in Egyptian cattle and buffaloes. In conclusion, the Egyptian water buffalo serves as an accidental non-adapted host for the disease and this point requires more deep investigation. In addition, the current vaccine strategy should be re-evaluated for more coverage and effectiveness.     

Infection of bovine immunodeficiency virus and bovine leukemia virus in water buffalo and cattle populations in Pakistan

A survey of antibodies to bovine immunodeficiency virus (BIV) known as bovine lentivirus and bovine leukemia virus (BLV) was conducted with samples from water buffalo and cattle populations in Pakistan. A total of 370 water buffaloes and 76 cattle were tested, and 10.3% and 15.8%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting, while 0.8% of water buffaloes and no cattle were positive for anti-BLV antibodies determined by immunodiffusion test. BIV-seropositive water buffaloes and cattle were found to have BIV proviral DNA in the peripheral blood mononuclear cells determined by nested polymerase chain reaction. This is the first report of BIV infections in water buffaloes.     

Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

ABSTRACT: The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.     

Comparison of fattening performance in Brahman grade cattle (Bos indicus) and crossbred water buffalo (Bubalus bubalis) fed on high roughage diet

The objective of this study was to compare the fattening performance in Brahman grade cattle (crossbred cattle) and crossbred water buffalo at the same young age and fed with high roughage based fattening rations in the Philippines. Ten crossbred cattle and 10 crossbred water buffalo, aged between 18 and 24 months old were used in this experiment. The animals were fed diets consisting of 85% Napier or Para grass and 15% concentrate mixture (CM) on a dry matter (DM) basis. The grass, total DM intake and bodyweight gain were significantly (P < 0.01) higher for the crossbred water buffalo than for the cattle. There was no species significant difference in the digestion coefficient and feed conversion rate between the crossbred cattle and water buffalo. The return over feed cost for fattening was significantly (P < 0.05) higher in the crossbred water buffalo than in the cattle. These results clearly indicate that under high roughage‐based fattening rations, young crossbred water buffalo are better able to utilize the roughage and they perform better in terms of feed intake and live weight gains than the crossbred cattle in the Philippines.     

Comparison of voluntary feed intake, rumen passage and degradation kinetics between crossbred Brahmam cattle (Bos indicus) and swamp buffaloes (Bubalus bubalis) fed a fattening diet based on corn silage

Three crossbred tropical cattle and three crossbred buffaloes, fitted with rumen cannulas, were used to investigate the differences in feed intake, digestibility and parameters with rumen degradation kinetics between cattle and buffalo fed a fattening diet in the Philippines. The animals were fed a diet consisting of 50% corn silage (CS), 30% brewer's grain (BG) and 20% concentrate mixture (CM) on a dry matter (DM) basis, at a level of 3% bodyweight (BW) as DM. Total DM intake (DMI) and rumen fill were greater (P < 0.05) for buffaloes than for cattle, but significant differences were not detected between the animal species when those were expressed as percentage of BW. The DMI per metabolic body size for BG and CM were greater for buffaloes than for cattle (P < 0.05), whereas that of CS did not differ between the species (P > 0.05). The weight proportion of consumed feedstuffs differed between cattle and buffaloes (P < 0.05), although nutrient intake per total DMI did not differ significantly (P > 0.05) between the animal species. Nutrients digestibility was higher (P < 0.05) for buffaloes than for cattle. Ruminal passage rate constant of feed particles and rumen fluid did not differ (P > 0.05) between the animal species. Higher DM degradation rate constant and effective degradability with CS were prominent for buffaloes than for cattle (P < 0.05). Degradation parameters did not differ (P > 0.05) between cattle and buffaloes, neither with BG nor CM. The results indicate that the greater digestibility with DM and energy for buffaloes are ascribable to greater effective degradability of CS fed ad libitum level.     

Nucleotide and octapeptide-repeat variations of the prion protein coding gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Anatolian,Murrah, and crossbred water buffaloes

Resistance to bovine spongiform encephalopathy (BSE) that is significantly associated with insertion/deletion (indel) polymorphisms at two loci (putative promoter and intron 1) on the prion protein gene (PRNP) in cattle has been well documented. Studies suggest that the insertion alleles are related to BSE resistance. Until recently, BSE has never been reported in water buffaloes (unlike cattle). Previous studies have demonstrated that the PRNP gene in water buffalo consists mostly of insertion alleles at both loci; nevertheless, whether or not water buffaloes are genetically resistant to BSE and the role of indel polymorphisms in their resistance status is not clear. We examined the coding region of PRNP to determine the nucleotide and octapeptide-repeat (octarepeats) variations of Anatolian, Murrah and Murrah × Anatolian (M?×?A) water buffaloes. Three synonymous single nucleotide polymorphisms (SNP) at positions 126, 234, and 285, and a non-synonymous SNP at position 322 (G108S) were detected. Triplet G/A/T base substitutions were observed at position 126 and two additional genotypes, T/A and T/G, at this position were determined. We also found six octarepeats that indicated the presence of the wild-type PRNP6 allele in the coding region. To the best of our knowledge, this is the first report of the T/A and T/G genotypes in water buffaloes.     

Comparison of carcass and meat characteristics of Brahman grade cattle (Bos indicus) and crossbred water buffalo (Bubalus bubalis) fed on high roughage diet

The objective of this study was to compare the carcass and meat quality in crossbred cattle and crossbred water buffalo at the same younger age and fed with high roughage in the Philippines. Ten crossbred cattle and 10 crossbred water buffalo, with an average age of 22 months (18–24 months) were used in this experiment. The animals were fed a similar diet, which consisted of 85% Napier or Para grass and 15% concentrate mixture on a dry matter basis, for 180 days before slaughter. Slaughter weight of the crossbred water buffalo was higher (P < 0.05) than that of the cattle, although the dressing percentage was significantly (P < 0.01) lower for the former than for the latter. The estimated lean yield was higher (P < 0.05) in crossbred cattle than crossbred water buffalo. The muscle fiber diameter of loin in buffalo meat was smaller (P < 0.05) than that in cattle. In regard to carcass and meat quality, the crossbred water buffalo was comparable or slightly superior to the crossbred cattle in tenderness, sensory score of color (P < 0.01) and flavor (P < 0.05). These results clearly indicate that at a younger age and even under high roughage‐based fattening rations, crossbred water buffalo are able to produce good quality meat, which will be by no means inferior to that of crossbred cattle in the Philippines.     

广西的尼里—拉菲水牛和摩拉水牛mtDNA D-loop区遗传多样性研究

[目的]探究引入广西南宁的尼里-拉菲水牛和摩拉水牛的mtDNA D-loop区遗传多样性与母系起源。[方法]采用PCR扩增、测序及生物信息学方法。[结果]对从广西南宁采集的52个江河型水牛（尼里-拉菲水牛25个,摩拉水牛27个）mtDNA D-loop序列与GenBank下载的20条尼里-拉菲水牛和23条摩拉水牛序列进行联合分析,共检测到112个变异位点,定义42个单倍型,发现摩拉水牛（Hd: 0.934±0.027）与尼里-拉菲水牛的遗传多样性（Hd: 0.929±0.017）都很丰富。NJ系统发育树显示尼里-拉菲水牛和摩拉水牛含有江河型和沼泽型水牛mtDNA支系,表明尼里-拉菲水牛和摩拉水牛引入中国后均与沼泽型水牛进行了杂交,在外貌上很难区分。[结论]引入广西南宁的尼里-拉菲水牛和摩拉水牛与沼泽型水牛存在广泛的血缘混杂现象。     

Divergent development of testosterone secretion in male zebu (Bos indicus) and crossbred cattle (Bos indicus x Bos taurus) and buffaloes (Bubalus bubalis) during growth

Delayed pubertal development and low fertility of Bos indicus x Bos taurus crossbred male cattle and domestic buffaloes is hardly understood hence, a sensitive enzymeimmunoassay (EIA) was developed using the second antibody-coating technique and testosterone-3-O-carboxymethyloxime-horseradish peroxidase conjugate as a label for determination of testosterone in blood plasma. The EIA was validated by standard criteria. Blood samples were collected by venipuncture from growing male cattle (Karan Fries and Sahiwal) and buffalo (Murrah) and testosterone was estimated using the EIA procedure. Plasma testosterone concentrations increased significantly (P < 0.05) with advancing age. Testosterone concentrations were significantly (P < 0.01) higher in Sahiwal males in comparison to Karan Fries males. The low testosterone levels in crossbred than Sahiwal could imply that crossbred males have either not stabilized genetically or not adapted well in Indian climatic conditions resulting in poor libido and poor semen quality. The low testosterone levels in Murrah buffalo males may be the possible reason for delayed maturity in this species. The direct, sensitive EIA validated for estimating the plasma testosterone concentration was reliable for studying the testosterone profile in blood plasma of males. The results suggest that there could be a requirement for higher testosterone secretion by males during early stages of growth for attaining early sexual maturity.     

Comparison of feed intake, digestibility and fattening performance of Brahman grade cattle (Bos indicus) and crossbred water buffalo (Bubalus bubalis)

Twenty growing crossbred cattle and crossbred water buffalo (carabao) with an average age of 22 (18–24 months) months were equally distributed into two treatment groups according to species. The animals were fed with the same ration made up of corn silage (50%) + wet brewer's spent grain (30%) + concentrate mixture (20%), and their fattening performance was monitored. The digestibilities of the different nutrients were likewise determined. The economics of raising the animals under intensive production system was calculated. Species differences did not influence total dry matter intake of the animals, when expressed as percentage of the bodyweight and per metabolic body size. There were no significant differences in digestion coefficients of the different nutrients, except for crude protein in crossbred water buffalo and crossbred cattle, although the digestibility of dry matter, organic matter and nitrogen free extract tended to be high in the former than in the latter. Likewise, average daily gain (ADG) was similar, although crossbred water buffalo had numerically higher ADG (828.6 vs 785.5 g) than crossbred cattle during the 6 months feeding. During the first 3 months of feeding (1–90 days), the ADG of crossbred water buffalo was 1066.1 g compared to 940.1 g for crossbred cattle. From 91 to 180 days, the crossbred cattle had slightly higher ADG (630.1 vs 591.1 g) but also the difference was not significant. The return above feed cost was comparable for crossbred cattle and crossbred water buffalo during the first 90 days of feeding. However, extending the feeding period from 91 to 180 days , income over feed cost was higher (P < 0.05) for crossbred cattle by PhP 5.3/kg gain than crossbred water buffalo. Results showed that crossbred water buffalo could attain similar growth rate with that of crossbred cattle under intensive system, when fed with high quality feed materials.     

Transmission and virological studies of a malignant catarrhal fever syndrome in the Indonesian swamp buffalo (Bubalus bubalis)

A malignant catarrhal fever-like syndrome in indonesian swamp buffalo was experimentally transmitted to one of 2 Bos indicus and 3 of 3 Bos javanicus cattle by intravenous inoculation of 250 ml of citrated, whole blood from affected buffaloes. The 4 cattle developed clinical signs of disease on average 32.5 days after receiving the inoculation of blood. The 4 cattle died after a variable period of illness. None of a further 3 B. javanicus cattle inoculated intravenously with a spleen homogenate prepared from another affected buffalo developed the disease. The experimental disease was clinically and pathologically similar to the natural disease in buffaloes although differences were noted. Attempts to adapt the agent to mice, guinea pigs and rabbits failed. A cytopathic agent (Japanese encephalitis virus) was isolated from the spleen of one buffalo with clinical signs but was not considered significant. Sixty-three B. indicus, 7 B. javanicus (and 6 of their crosses), 3 B. taurus and 4 Bubalus bubalis (Murrah buffalo) were kept in the same quarters where 50 of 177 swamp buffaloes died between September 1979 and May 1982. Four of the 7 B. javanicus cattle developed the clinical signs of disease and died. All the other cattle in contact remained healthy.     

Serological survey of Neospora caninum and Toxoplasma gondii in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in ten provinces of Brazil

The objective of this study was to determine the prevalence of antibodies to Neospora caninum and Toxoplasma gondii among 500 cattle (Bos indicus) and 500 buffaloes (Bubalus bubalis) using the indirect fluorescent antibody test (IFAT) technique. Blood samples from were collected from water buffalo and cattle in 10 municipalities in the northern region of Brazil. The frequency of cattle and water buffaloes seropositive for Neospora caninum in Pará state, Brazil, was 55% and 44%, respectively, and the frequency of cattle and water buffaloes seropositive for Toxoplasma gondii was 52% and 39%, respectively. Seropositivity for both N. caninum and T. gondii was detected in 10.6% of the cattle samples and 14.8% of the buffalo samples. The frequency of cattle positive for N. caninum and T. gondii was significantly (p < 0.05) higher than that of buffalo in two and three provinces, respectively. Buffaloes had a lower seroprevalence for N. caninum or T. gondii in all of the provinces studied. These results suggest that both species, when exposed to the same risks for N. caninum and T. gondii infection, have a high serological prevalence. Cattle showed a higher probability of being seropositive when exposed to the same risks for N. caninum and T. gondii. Our study, which included an extensive number of blood samples, provides important epidemiological information pertinent to buffalo production in tropical countries that can be used as a basis for disease-management practices in Latin America.     

Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer)

Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.     

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.     

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4⁺CD25⁺Foxp3⁺ T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3⁺CD4⁺ cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4⁺ cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4⁺ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4⁺ and CD25⁺ T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4⁺ cells in the CD4⁺ T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4⁺CTLA-4⁺ T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.