Original Article Bovine immunodeficiency virus and bovine leukemia virus and their mixed infection in Iranian Holstein cattle Gholamreza Nikbakht Brujeni1, Taghi Taghi Poorbazargani2, Susan Nadin-Davis3, Mohammad Tolooie4 and Neda Barjesteh1 1Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 3Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, ON, Canada 4Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran Abstract Introduction: Bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) have worldwide distributions, but their prevalences in Iran are unknown. We investigated the presence of infections in Iranian Holstein cattle and determined changes in hematological values for infected animals. Methodology: Nested PCR was used on blood samples from 143 animals Holstein cattle to detect proviral BIV and BLV gag sequences. Flow cytometric analysis was performed using monoclonal antibodies (mAbs) against CD4, CD8, and CD21 bovine T lymphocyte subsets. Results: Proviral BIV and BLV gag sequences were detected in 20.3% and 17% of the animals, respectively. BIV-BLV confection was also detected in 4.2% of the study population but this was not statistically significant. Flow cytometric analysis showed that both BIV-infected cows and non-infected ones had CD4/CD8 ratios of 2.45 and 1.43, respectively, and this difference was significant. BLV infected and non- infected animals had no significant differences in their CD4/CD8 ratio. In comparison to non-infected cattle, those with both BIV and BLV had a significant decrease in their CD4/CD8 ratios (1.5 % vs. 2.3; P = 0.01). Conclusion: This is the first report of BIV and BLV infections in Iran. We found no evidence that infection with one agent predisposed an animal to infection with the other. BIV infection may have a role in decreasing T CD8 counts, but this may depend on the genetics of the cattle and virus strains involved. Key words: bovine immunodeficiency virus; bovine leukemia virus/ nested PCR; flow cytometry; Iran J Infect Dev Ctries 2010; 4(9):576-579. (Received 29 November 2009 – Accepted 03 June 2010) Copyright © 2010 Brujeni et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Bovine immunodeficiency or immunodeficiency- The clinical significance of BIV infection may like virus (BIV) is a lentivirus of the family depend upon the strain of BIV, the breed of cattle, Retroviridae. It occurs widely with infections being and environmental stressors [2,6]. lifelong and generally subclinical [1,2]. In some Bovine leukaemia virus (BLV) is a lymphotropic instances, however, infections may be of economic retrovirus structurally related to human T-cell importance with seropositive cows having decreased leukaemia virus type 1 (HTLV-1) [14]. Following milk production [3-5]. Also, there is evidence that infection, the BLV can remain clinically dormant, BIV can cause immunosuppression with increased cause a persistent lymphocytosis (PL) with increased incidences of secondary bacterial infections and B lymphocytes, or cause B-cell lymphomas in lymph encephalitis found in herds with high seroprevalences nodes and other tissues. Only a few (< 5%) BLV- [4-9]. Following experimental infections, cattle may infected animals develop tumors [14]. have transient increases in lymphocytes, lymphoid As there is no data on BIV and BLV in Iran, we hyperplasia, atypical lymphosarcoma, and secondary conducted a survey of dairy cattle to determine the bacterial infections [6,10,11]. It has also been prevalence of single and concurrent infections. We suspected that the stress of parturition in BIV also determined the hematological effects of infected cows is associated with the progression of infections. bovine leukemia virus (BLV) infections [8,12,13]. Brujeni et al. - BIV and BLV in Iranian Holstein cattle J Infect Dev Ctries 2010; 4(9):576-579. Methodology PCR was also used to detect BLV proviral DNA as Whole blood and blood in EDTA were collected described previously by Wang et al. [18]. DNA from 143 apparently healthy Holstein cows in extracted from BLV-infected FLK cells (kindly Maysam slaughterhouse (Tehran, Iran). Complete provided by Razi Institute, Iran) was used as a blood counts were established by routine procedures positive control and the DNA sample prepared from a in the Tehran University laboratory. BLV-seronegative cow was used as negative control Single-colour staining for flow cytometric for PCR amplification. analysis was performed using the following PCR products generated from three BIV positive commercially available mAbs against bovine samples were cloned using a QIAGEN PCR cloning lymphocyte subsets (Serotec, Kidlington, England): kit (QIAGEN, Hilden, Germany) and sequenced CD4- fluorescein isothiocyanate (FITC), CD8-FITC commercially (Macrogen Inc., Seoul, Korea). and CD21- FITC. Mouse IgG1-FITC and IgG2a- Sequences were aligned and phylogenetic analyses were used as negative controls. performed with the Clustal programme available in Peripheral blood cells were lysed the software package of Bioedit version 7.0.5.3 (DakoCytomation Uti-Lyse Erythrocyte Lysing (http://jwbrown.mbio.ncsu.edu/Bioedit/bioedit.html). Reagent, Dako, Glostrup, Denmark) and lymphocytes Sequences obtained were deposited in GenBank were labeled using the mAbs according to the (EF661978, EF661979 and EF661980). manufacturer’s instructions. Flow cytometric analysis Data obtained from hematological and flow was performed by counting 10,000 cells from each cytometric experiments were analyzed by the SPSS sample on a FACS flow cytometer (Becton– 15.0 program using chi-square and Student’s t test. Dickinson and Co. Immunocytometry Systems, Franklin Lakes NJ, USA) and analyzed with Results CELLQuest software. Nested PCR identified 29 BIV-positive samples For the preparation of DNA, aliquots of whole giving an overall positivity rate of 20.3% (29/143). blood (500 μl) were added to 1 ml red blood cell lysis Similarly, 16.8% (24/143) were positive with the buffer (10 mM Tris-HCl pH 7.6; 5 mM MgCl2, 100 BLV-gag nested PCR. Six animals were positive for mM NaCl, 0.75 %Triton X-100) mixed and incubated both viruses (6/143; 4.2 %). at room temperature. After 2 minutes the tube was There was 98-100% identity when sequences of centrifuged at 12000 g for 20 seconds and the Iranian BIV amplicons (EF661978, EF661979 supernatant was discarded. The pellet was and EF661980) were compared with those of isolate resuspended in 600 μl of cell lysis buffer (10 mM R-29, confirming that BIV was detected in the Tris-HCl, pH 8; 0.1 M NaCl, 25 mM EDTA, pH 8; Iranian cattle we studied using our BIV specific 0.5% sodium dodecyl sulphate) and homogenized. primers. The sequences had no similarity with the Then 300 μl denaturing solution containing 4M bovine genome and other known related organisms guanidinium isothiocyanate, 25 mM sodium citrate (JDVCG). pH 7; 0.1 m 2ME and 0.5 % Sarkosyl was added and The only significant differences found, when incubated at room temperature. After 10 minutes, 300 comparing hematological and flow cytometery μl potassium acetate (3M potassium and 5M acetate) results, were the proportions of CD8 cells in BIV were added and vortexed for 30 seconds. After infected and non infected animals (27% and 21%; P ≤ centrifugation for 3 minutes at 12000g, 900 μl 0.05); the number of BIV and BLV infected animals supernatant were recovered and DNA precipitated (26% and 17%; P ≤ 0.5); and the proportions of CD4 with 600 μl isopropanol. The pellet was washed with cells in BLV infected and non infected animals (36% 70% ethanol and resuspended in 50 μl of TE (pH, and 30%; P ≤ 0.05). When CD4/CD8 ratios were 7.6). All samples were stored at -20°C until used. calculated, significant differences were also observed The DNA extracted from each blood sample was between BIV infected and non infected animals (1.4 used as a template to detect BIV proviral DNA by and 2.4; P ≤ 0.05) and BIV-BLV coinfected animals nested PCR as described previously by Nadin-Davis (1.5 and 2.3; P ≤ 0.5). et al. [17]. Plasmid DNA containing the complete BIV gag coding region (pGEM7-gag kindly provided Discussion by S. Nadin-Davis) served as a positive control. DNA Although BIV infected cattle have been from the animals that was consistently assayed as identified in some countries neighboring Iran [4,19], BIV negative was used as negative control. Nested to the best of our knowledge this is the first report of 577 Brujeni et al. - BIV and BLV in Iranian Holstein cattle J Infect Dev Ctries 2010; 4(9):576-579. BIV infection in Iranian Holstein cattle. Although we buffalo and cattle populations in Pakistan. J Vet Med Sci 62: studied only a small number of animals, the 329-331. 5. Snider TG, III, Hoyt PG, Coats KS, Graves KF, Cooper CR, prevalence we found of 20.3% is similar to that Storts RW, Luther DG, Jenny BF (2003) Natural bovine reported from other Asian countries (12% in Turkey lentiviral type 1 infection in Holstein dairy cattle. I. Clinical, [19] to 35% in Korea [20]), and neighboring serological, and pathological observations. Comp Immunol countries such as Pakistan (16 %) [4,13]. The Microbiol Infect Dis 26: 89-101. 6. Belloc C, Polack B, Schwartz-Cornil I, Brownlie J, Levy D prevalence of BLV infection (17%) in the current (1996) Bovine immunodeficiency virus: facts and questions. study was also similar to that of other Asian and non Vet Res 27: 395-402. Asian countries (9 to 33 %) [21-24]. 7. Isaacson JA, Flaming KP, Roth JA (1998) Effects of long- There was a significant increase in the CD4/CD8 term infection with bovine immunodeficiency virus and/or ratio in BIV-infected cows due to a significant bovine leukemia virus on antibody and lymphocyte proliferative responses in cattle.