Study of the proviral load levels and mRNA expression of cytokines in peripheral blood mononuclear cells and somatic milk cells in cattle with different BLV infection profiles.

The retrovirus bovine leukemia virus (BLV) might produce abnormal immune function, associated with susceptibility to developing other infectious diseases, including mastitis. This study aimed to determine the proviral load and cytokines gene expression in peripheral blood mononuclear cells (PMBC) and milk somatic cells (SC) in BLV-infected and non-infected cattle. Of 27 BLV-infected cows in PBMC, 17 (62.96%) had a high proviral load (HPL), and 10 (37.04%) had a low proviral load (LPL). All SC samples had low proviral load (LPL-SC). Higher IFN-γ and IL-10 expression, and lower IL-12 and IL-6 expression, were found in PBMC from BLV-infected compared to BLV non-infected cattle. Moreover, higher IFN-γ, IL-12, and IL-6 expression, and lower IL-10 expression were observed in cattle with LPL-PBMC compared to HPL-PBMC. In milk samples, lower IFN-γ and higher IL-12 mRNA expression were observed in LPL-SC compared to BLV non-infected cattle in SC. IL-10 and IL-6 expression mRNA was significantly lower in LPL-SC than in SC from BLV non-infected cattle. This study shows that milk SC maintains lower proviral load levels than PBMC. This first report on Th1 and Th2 cytokines expression levels in SC may be relevant to future control strategies for BLV infection, mastitis, and udder health management.

Effect of heat stress on TNF-α, TNFRI and TNFRII expression in BLV infected dairy cattle.

High temperatures for extended periods, which do not allow animals to recover from heat stress, affect in particular those BLV-infected animals that carry a high proviral load. For this study, animals were discriminated between BLV (+) and BLV (-), and those belonging to the first group, were classified based on their proviral load. The expression of the inflammatory cytokine TNF-α and its receptors, which play an important role in disease progression, were quantified by qPCR in two different seasons. During the summer, average temperature was 19.8 °C, maximums higher than 30 °C were frequent. Instead, during the autumn, the average temperature was 12.63 °C, and temperatures never exceeded 27 °C. During this season, almost no periods of temperatures exceeded the comfort limit. Our results revealed that the expression levels of TNF-α and its receptors were downregulated in animals with high proviral load. This fact could affect their antiviral response and predispose to viral dissemination; over time, animals with a poorer immune system are prone to acquiring opportunistic diseases. Conversely, animals with LPL maintained their expression profile, with behavior comparable to non-infected animals. These findings should be considered by producers and researchers, given the problems that global warming is causing lately to the planet.

Cytokine TNF-α and its receptors TNFRI and TNFRII play a key role in the in vitro proliferative response of BLV infected animals.

Bovine leukemia virus (BLV) main host cells are B lymphocytes. Infected animals can be classified into high or low proviral load (HPL or LPL respectively), regarding the number of proviral copies infected lymphocytes they carry. After infection, there is an overexpression of several cytokines, particularly TNF-α, which has a delicate regulation mediated by receptors TNFRI and TNFRII; the first one involved with apoptosis, while the other stimulates cell proliferation. The study aimed to quantify TNF-α and its receptors mRNA expression, and in which extent in vitro proliferation was affected, in peripheral blood mononuclear cells (PBMC) from BLV-infected animals with different proviral loads, after the addition or not of synthetic TNF-α (rTNF-α) for 48 h. PBMC from BLV-infected animals showed spontaneous proliferation after 48 h in culture but did not show changes in proliferation rates after 48 h incubation in the presence of the rTNF-α. TNF-α mRNA expression after 48 h culture without exogenous stimulation was significantly lower, regardless of the proviral load of the donor, compared to non-infected animals. In the LPL animals, the expression of TNF-α mRNA was significantly lower with respect to the control group while the expression of TNFRI mRNA was significantly increased. The HPL animals showed a significant decrease in the expression of TNF-α and TNFRII mRNA respect to the control group. After 48 h incubation with rTNF-α, PBMC from infected animals had different responses: TNF-α and TNFRI mRNA expression was reduced in PBMC from the LPL group compared to the BLV negative group, but no differences were observed in PBMC from the HPL group. TNFRII mRNA expression showed no differences between HPL, LPL, and BLV negative groups, though HPL animals expressed 10.35 times more TNFRI mRNA than LPL. These results support the hypothesis that LPL animals, when faced with viral reactivation, present a pro-apoptotic and anti-proliferative state. However, complementary studies are needed to explain the influence of TNFRII on the development of the HLP profile. On the other hand, exogenous stimulation studies reinforce the hypothesis that BLV infection compromises the immune response of the animals.

Alterations in TNF-α and its receptors expression in cows undergoing heat stress.

Heat stress is one of the environmental factors that most severely affects milk industry, as it has impact on production, immune responses and reproductive performance. The present study was conducted with high-performance Holando-Argentino cows. Our objective was to study TNF-α and its receptors pattern expression in cows from a region characterized by extreme climatic seasonality. Animals were evaluated in three periods: spring (n = 15), summer (n = 14) and autumn (n = 11). Meteorological records from a local station were used to estimate the temperature and humidity index (THI) by means of an equation previously defined. A THI higher than 68 is indicative of stressing conditions. During the summer period, the animals were exposed to 8.5 ±â€¯1.09 h of heat stress, or THI > 68. In spring, stress hours were reduced to 1.4 ±â€¯0.5 every day, while during the autumn, there were no recorded heat stress events. Expression of TNF-α, and its receptors was determined by qPCR. During the summer, TNF-α and its receptors expression diminished drastically compared to the rest of the year, when stressful conditions were infrequent. We conclude that animals that are not physiologically prepared to resist high temperatures might have a less efficient immune response, reinforcing the need to develop new strategies to improve animal welfare.

Effect of bovine leukemia virus (BLV) infection on bovine mammary epithelial cells RNA-seq transcriptome profile.

Bovine leukemia virus (BLV) is a δ-retrovirus responsible for Enzootic Bovine Leukosis (EBL), a lymphoproliferative disease that affects cattle. The virus causes immune system deregulation, favoring the development of secondary infections. In that context, mastitis incidence is believed to be increased in BLV infected cattle. The aim of this study was to analyze the transcriptome profile of a BLV infected mammary epithelial cell line (MAC-T). Our results show that BLV infected MAC-T cells have an altered expression of IFN I signal pathway and genes involved in defense response to virus, as well as a collagen catabolic process and some protooncogenes and tumor suppressor genes. Our results provide evidence to better understand the effect of BLV on bovine mammary epithelial cell's immune response.

Lymphocyte proliferation and apoptosis of lymphocyte subpopulations in bovine leukemia virus-infected dairy cows with high and low proviral load.

Bovine leukemia virus (BLV) is one of the most important virus in dairy cattle. The infection behavior follows what we call the iceberg phenomenon: 60% of infected animals do not show clinical signs; 30% develop persistent lymphocytosis (PL); and the remaining 10%, die due to lymphosarcoma. BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms by which cattle governs the control of viral dissemination will be desirable for designing effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. It is known that BLV affects cellular homeostasis: proliferation and apoptosis. It is also known that the BLV tropism is directed towards B lymphocytes, and that lymphocytotic animals have elevated amounts of these cells. Usually, when an animal is infected by BLV, the B markers that increase are CD21, CD5 and CD11b. This increase could be related to the modulation of apoptosis in these cells. This is the first work in which animals infected with BLV are classified according to their proviral load and the subpopulations of B and T lymphocytes are evaluated in terms of their percentage in peripheral blood and its stage of apoptosis and viability. PBMCs from HPL animals proliferated more than LPL and non-infected animals. CD11b+/CD5+ lymphocytes in LPL animals presented greater early and late apoptosis than HPL animals and cells of HPL animals had increased viability than LPL animals. Our results confirm that BLV alters the mechanism of apoptosis and proliferation of infected cells.

Stable infection of a bovine mammary epithelial cell line (MAC-T) with bovine leukemia virus (BLV).

Bovine leukemia virus (BLV) is a retrovirus that affects cattle causing a lymphoproliferative disease. BLV infection has been associated with misbalance of the immune response causing a higher incidence of other infections. Mastitis is one of the most important conditions that affect milk production in cattle. The aim of this study was to stably infect a bovine mammary epithelial cell line (MAC-T). MAC-T cell line was successfully infected with BLV and the infection was confirmed by nested PCR, qPCR, immunocytochemistry, western blot and transmission electron microscopy. This is the first report of a bovine mammary epithelial cell line stably infected with BLV. This new cell line could be used as an in vitro model to study the effect of BLV on the immune response in the mammary gland and the relationship with other agents causing mastitis.

Can Bovine Leukemia Virus Be Related to Human Breast Cancer? A Review of the Evidence.

The incidence of breast cancer is continuously increasing worldwide, as influenced by many factors that act synergistically. In the last decade there was an increasing interest in the possible viral etiology of human breast cancer. Since then, many viruses have been associated with this disease (murine mammary tumor virus, MMTV; Epstein-Barr virus, EBV; and human papillomavirus, HPV). Recently, BLV has been identified in human breast cancers giving rise to the hypothesis that it could be one of the causative agents of this condition. BLV is a retrovirus distributed worldwide that affects cattle, causing lymphosarcoma in a small proportion of infected animals. Because of its similarity with human retroviruses like HTLV and HIV, BLV was assumed to also be involved in tumor emergence. Based on this assumption, studies were focused on the possible role of BLV in human breast cancer development. We present a compilation of the current knowledge on the subject and some prospective analysis that is required to fully end this controversy.

Toll-like receptors, IFN-γ and IL-12 expression in bovine leukemia virus-infected animals with low or high proviral load.

Bovine leukemia virus (BLV) infection is widespread mainly in dairy cattle and 5-10% of infected animals will die due to lymphosarcoma; most cattle remain asymptomatic but 30% develop persistent lymphocytosis (PL). BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms which govern the control of viral dissemination will be desirable for the design of effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. We aimed to characterize cytokines and toll-like receptors (TLR) expression related to the proviral load profiles. IFN-γ and IL-12 mRNA expression level was significantly higher in PBMC from infected cattle (LPL n=6 and HPL n=7) compared to uninfected animals (n=5). While no significant differences were observed in IL-12 expression between LPL and HPL group, IFN-γ expression was significantly higher in LPL animals. Infected cattle exhibited higher expression levels of TLR3, 7-9. Animals with HPL had significantly higher expression of TLR7/8 than uninfected cattle. TLR8 and TLR9 were up-regulated in HPL group, and TLR3 was up-regulated in LPL group. This is the first report related to TLR gene expression in BLV infected cattle and represents evidence of the involvement of these receptors in BLV recognition. Further studies on different subpopulations of immune cells may help clarify their role in response to BLV and its consequences on viral dissemination.

Association of TNF-α gene promoter region polymorphisms in bovine leukemia virus (BLV)-infected cattle with different proviral loads.

Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine involved in the immune response against viral and other infections. Its expression levels are affected by a polymorphism in the promoter region of the gene. Bovine leukemia virus is a retrovirus that infects cattle and develops two different infection profiles in the host. One profile is characterized by a high number of proviral copies integrated into the host genome and a strong immune response against the virus, while the most relevant property of the other profile is that the number of copies integrated into the host genome is almost undetectable and the immune response is very weak. We selected a population of cattle sufficiently large for statistical analysis and classified them according to whether they had a high or low proviral load (HPL or LPL). Polymorphisms in the promoter region were identified by PCR-RFLP. The results indicated that, in the HPL group, the three possible genotypes were normally distributed and that, in the LPL group, there was a significant association between the proviral load and a low frequency of the G/G genotype at position -824.

Trypanosoma cruzi (Chagas' disease agent) reduces HIV-1 replication in human placenta.

BACKGROUND: Several factors determine the risk of HIV mother-to-child transmission (MTCT), such as coinfections in placentas from HIV-1 positive mothers with other pathogens. Chagas' disease is one of the most endemic zoonoses in Latin America, caused by the protozoan Trypanosoma cruzi. The purpose of the study was to determine whether T. cruzi modifies HIV infection of the placenta at the tissue or cellular level. RESULTS: Simple and double infections were carried out on a placental histoculture system (chorionic villi isolated from term placentas from HIV and Chagas negative mothers) and on the choriocarcinoma BeWo cell line. Trypomastigotes of T. cruzi (VD lethal strain), either purified from mouse blood or from Vero cell cultures, 24 h-supernatants of blood and cellular trypomastigotes, and the VSV-G pseudotyped HIV-1 reporter virus were used for the coinfections. Viral transduction was evaluated by quantification of luciferase activity. Coinfection with whole trypomastigotes, either from mouse blood or from cell cultures, decreased viral pseudotype luciferase activity in placental histocultures. Similar results were obtained from BeWo cells. Supernatants of stimulated histocultures were used for the simultaneous determination of 29 cytokines and chemokines with the Luminex technology. In histocultures infected with trypomastigotes, as well as in coinfected tissues, IL-6, IL-8, IP-10 and MCP-1 production was significantly lower than in controls or HIV-1 transducted tissue. A similar decrease was observed in histocultures treated with 24 h-supernatants of blood trypomastigotes, but not in coinfected tissues. CONCLUSION: Our results demonstrated that the presence of an intracellular pathogen, such as T. cruzi, is able to impair HIV-1 transduction in an in vitro system of human placental histoculture. Direct effects of the parasite on cellular structures as well as on cellular/viral proteins essential for HIV-1 replication might influence viral transduction in this model. Nonetheless, additional mechanisms including modulation of cytokines/chemokines at placental level could not be excluded in the inhibition observed. Further experiments need to be conducted in order to elucidate the mechanism(s) involved in this phenomenon. Therefore, coinfection with T. cruzi may have a deleterious effect on HIV-1 transduction and thus could play an important role in viral outcome at the placental level.