Effect of bovine viral diarrhea virus on subsequent infectivity of bovine gammaherpesvirus 4 in endometrial cells in primary culture: An in vitro model of viral co-infection.

Bovine viral diarrhea virus (BVDV) and bovine gammaherpesvirus 4 (BoHV-4) infect the uterus of cattle, being responsible for huge economic losses. Most of the pathogenesis of BoHV-4 in the bovine reproductive tract has been elucidated by conducting tests on primary cultures. Thus, it is important to have optimal in vitro conditions, avoiding the presence of other pathogens that can alter the results. BVDV is one of the most frequent viral contaminants of cell cultures. Considering that non-cytopathic (NCP) BVDV biotype can generate persistently infected (PI) cattle, which are the major source for virus transmission in susceptible herds, it is important to check products derived from cattle that are intended to be used in research laboratories. The aim of this work was to evaluate how the natural infection of bovine endometrial cells (BEC) with a NCP BVDV strain (BEC + BVDV) affects BoHV-4 replication. We have demonstrated a delay in BoHV-4 gene expression and a decrease in viral load in the extracellular environment in BEC + BDVD cells compared to BEC (BVDV-free) cells. These results confirm that replication of BoHV-4 in BEC primary cultures is affected by previous infection with BVDV. This finding highlights the importance of ruling out BVDV infection in bovine primary cell cultures to avoid biological interference or misinterpretation of results at the time of performing in vitro studies with BoHV-4.

Gene expression and in vitro replication of bovine gammaherpesvirus type 4.

In vitro cell cultures are widely used models for dissecting cellular and molecular mechanisms that lead to certain physiological conditions and diseases. The pathogenesis of BoHV-4 in the bovine reproductive tract has been studied by conducting tests on primary cultures. However, many questions remain to be answered about the role of BoHV-4 in endometrial cells. The aim of this study was to compare the replication and gene expression of BoHV-4 in cell lines and bovine reproductive tract primary cells as an in vitro model for the study of this virus. We demonstrated that BoHV-4 strains differ in their in vitro growth kinetics and gene expression but have the same cell type preference. Our results demonstrate that BoHV-4 replicates preferentially in bovine endometrial cells (BEC). However, its replication capacity extends to various cell types, since all cells that were tested were permissive to BoHV-4 infection. The highest virus titers were obtained in BEC cells. Nevertheless, virus replication efficiency could not be fully predicted from the mRNA expression profiles. This implies that there are multiple cell-type-dependent factors and strain properties that determine the level of BoHV-4 replication. The results of this study provide relevant information about the in vitro behavior of two field isolates of BoHV-4 in different cell cultures. These findings may be useful for the design of future in vitro experiments to obtain reliable results not only about the pathogenic role of BoHV-4 in the bovine female reproductive tract but also in the development of efficient antiviral strategies.

Cathelicidin bovine myeloid antimicrobial peptide (BMAP) 28 is involved in the inflammatory response against alpha-herpesviruses in the bovine nervous system.

The role of the local innate immune response in the neuropathogenesis of bovine herpesvirus (BoHV) type 1 and 5 remains largely unknown. This study determined the gene transcriptional expression of relevant bovine cathelicidins, TNFα and IFNß in the nervous system of experimentally-infected cattle during the different stages of BoHV-1 and BoHV-5 infectious cycle. We studied the modulation of bovine myeloid antimicrobial peptide (BMAP) 27 and 28 by alpha-herpesviruses during acute infection of the central nervous system (CNS). However, BMAP28 was the main cathelicidin modulated. BoHV-5 supressed BMAP28 expression mainly in frontal cortex and cervical medulla whereas BoHV-1 slightly induced the expression of cathelicidins in the olfactory and posterior cortex. The differences in the regulation of the innate response are likely related to distinct replication rates of both alpha-herpesviruses in the CNS. During latency and reactivation, BoHV-1 and -5 decreased BMAP28 and BMAP27 expression, accompanied by high levels of TNFα and IFNß transcripts in the posterior brain region and medulla during BoHV reactivation. In terms of cytokines, a remarkably overexpression of IFNß was induced by BoHV-5 (133.8-fold). In trigeminal ganglion (TG) both alpha-herpesviruses induced cathelidicins gene expression at all stages of the infection cycle, while only acute BoHV-5 infection increased TNFα (129-fold) mRNA levels. This study suggests that the pronounced downregulation of BMAP28 in BoHV-5-acutely-infected CNS is due to a decreased immune stimulation during viral infection, favouring its establishment in the CNS with a low replication rate until latency. Thus, cathelicidins, together with IFNß and TNFα, are differentially regulated by BoHV-5 and BoHV-1 infections and this regulation is dependent on the stage of virus infection in the bovine nervous system.

Spongy Myelinopathy in Newborn Beef Calves Associated with Consumption of Corn Infected with Stenocarpella maydis.

Stillbirth and perinatal mortality with neurological signs and lesions were diagnosed in two calves following ingestion by their dams of corn infected with Stenocarpella maydis during the third trimester of gestation. Grossly, the brain and spinal cord were unremarkable. Microscopically, diffuse severe status spongiosis of the white matter was detected in the cerebral hemispheres, brainstem, spinal cord and cerebellum. To the best of our knowledge this is the first pathological description of congenital disease in calves associated with the consumption of S. maydis-infected corn; the findings resemble those reported for the naturally occurring and experimentally induced disease in lambs.

Phenotypic characterization of immune cells in fetal tissues of cattle immunized and challenged with Neospora caninum.

The purpose of this work was to characterize the cellular phenotype in inflammatory infiltrates of fetal tissues from pregnant heifers immunized and experimentally challenged with Neospora caninum. Fetuses from 20 heifers separated into 5 groups were obtained. The experiment was designed as follow: Group A, heifers inoculated intravenously with live tachyzoites of Argentine strain NC-6 (n = 4); Group B heifers inoculated subcutaneously with soluble native antigen from the same strain formulated with immune stimulant complexes (ISCOMs) (n = 4); Group C heifers inoculated with recombinant proteins, rNcSAG1, rNcHSP20, rNcGRA7 formulated with ISCOMs (n = 4), Group D heifers inoculated subcutaneously with sterile phosphate buffered solution (n = 4) and Group E heifers inoculated subcutaneously with antigen-free ISCOMs (n = 4). Experimental challenge was performed at 70 days of gestation and all heifers were euthanized 34 days later. Fetal tissues were taken for histological studies. Inflammatory lesions were observed in brain and lung, and immunhistochemistry was used to identify CD3+, CD20+ and MHC II+ cells. The majority of the cells that infiltrate and circumscribe the lesions in the brain and lung tissue expressed MHC II antigen; varying between 70-90% of the total cellular infiltrate. CD3+ cells were also present within the lesions, contributing to up to 30% of the inflammatory cells. CD20+ cells appeared as a marginal group, in some cases, with a range between 10 and 25%. As expected, the immunolabeling of MHC II + and CD3 + cells in fetal tissues was associated with fetal infection with N. caninum. There were statistically significant differences in the distribution and population of the inflammatory infiltrate in relation to the immunogenic treatment and the type of tissue, with inflammatory cells being markedly less extensive fetuses from group A (dams previously exposed to N. caninum) and in brain tissue. This work showed that Neospora-infection induced MHC II+ and CD3+ cells in bovine fetuses from dams receiving experimental vaccines.

Modulation of cathelicidins, IFNß and TNFα by bovine alpha-herpesviruses is dependent on the stage of the infectious cycle.

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNß, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNß, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNß in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system.

A Descriptive Study of Lectin Histochemistry of the Placenta in Cattle following Inoculation of Neospora caninum.

The aim of this study was to describe the lectin-binding pattern in the placentas of cows infected experimentally with Neospora caninum. Four cows were inoculated intravenously with 1 × 108 tachyzoites of the NC-1 strain of N. caninum at 150 ± 7 days of pregnancy. Two control cows were administered a placebo. An indirect fluorescence antibody test (IFAT) was performed on serum samples obtained before and after the inoculation. The cows were killed at 30 and 37 days post inoculation. Samples of placenta were taken for histopathology and lectin histochemistry. Fetal tissues and fluids were collected for histopathology and IFAT, respectively. All infected cows had high antibody titres. All fetuses had characteristic histopathological lesions, including non-suppurative meningoencephalitis, myocarditis, hepatitis and myositis, suggesting N. caninum infection. Only two infected fetuses developed specific antibodies. Mild non-suppurative inflammatory infiltrates were recorded in the placentae. Differences in the lectin-binding pattern were observed between infected animals and controls in the glycocalyx (CON-A and WGA) and apical cytoplasm (RCA-I and CON-A) of the trophoblastic cells; giant trophoblastic cells (CON-A and DBA); glycocalyx (PNA, WGA) and apical cytoplasm (CON-A, WGA, PNA, DBA and RCA-I) of endometrial cells; trophoblast of the interplacentomal region (WGA); endothelium (CON-A, SBA, RCA-1 and WGA); and finally, mesenchyme (CON-A, RCA-1, SBA, PNA and DBA). These findings indicate that there is a distinctive pattern of lectin binding in the placenta of cattle infected with N. caninum. The direct effect of the presence of the protozoa as well as the altered expression of cytokines could explain these changes in the maternofetal interface.

Immunization with inactivated antigens of Neospora caninum induces toll-like receptors 3, 7, 8 and 9 in maternal-fetal interface of infected pregnant heifers.

Neospora caninum is an obligate parasite and a major cause of abortion in cattle. Pregnancy failures appear to be associated with weak innate defences on the maternal-fetal interface during infection with N. caninum. Herein, we studied the gene expression of Toll-like receptors (TLRs) in pregnant heifers immunized with different vaccine formulations against N. caninum before mating and then challenged the heifers with live N. caninum on day 70 of gestation. TLR7 and TLR8 expression was upregulated in the placental caruncle of infected-pregnant heifers previously exposed to live N. caninum as immunogen. However, TLR7 and 8 expression in both placenta and caruncle as well as, TLR3 and 9 expression in caruncle were upregulated when heifers were previously immunized with inactivated soluble whole antigens and recombinant NcSAG1, NcHSP20 and NcGRA7 proteins. All dams were carrying viable fetuses when they were culled at day 104 of gestation. Upregulation of TLR7 and IFNγ expression was detected in fetal spleen when their mothers where previously vaccinated with soluble antigens and recombinant NcSAG1, NcHSP20 and NcGRA7 proteins. These studies demonstrate that soluble or recombinant NcSAG1, NcHSP20 and NcGRA7 antigens induce key TLRs expression at the maternal-fetal interface, probably triggering damaging inflammatory cellular immune responses associated with abortion. Previous infection with N. caninum seems to attenuate the innate immune response at the maternal-fetal interface, which could favour pregnancy maintenance and perpetuation of the disease. This finding represents novel information on how N. caninum vaccination and infection modulate TLRs expression at the placenta and fetal spleen, the possible role in the pregnancy outcomes and transplacental transmission of the protozoa.

Toll-like receptors 3, 7 and 8 are upregulated in the placental caruncle and fetal spleen of Neospora caninum experimentally infected cattle.

Innate immune responses at the maternal-fetal interface are key in the pathogenesis of Neospora caninum, an obligate parasite that causes abortion in cattle. Herein, we determined the gene expression of endosomal Toll-like receptors (TLRs) in the placenta and fetuses from both non-infected pregnant heifers and pregnant heifers intravenously challenged with live tachyzoites of N. caninum on day 70 of gestation. On day 104 of pregnancy, mRNA expression of TLRs 3 and 8, as well as that of TLRs 7 and 9, was high in the spleen of fetuses from N. caninum-infected heifers. Gene expression levels of endosomal TLRs were also detectable in the placenta and the maternal caruncle from infected heifers, being TLRs 3, 7 and 8 particularly upregulated, mostly in the caruncle. Basal TLR levels were higher in fetal spleen than in placental tissues. This study provides novel information on how innate TLR responses are induced at the maternal-fetal interface of cattle in response to intracellular N. caninum.

Distribution of bovine alpha-herpesviruses and expression of toll-like receptors in the respiratory system of experimentally infected calves.

This study provides an initial analysis of the toll-like receptors (TLRs) that might be implicated in alpha-herpesvirus infection of the bovine respiratory system. A significant variation in the expression of TLR3 and TLRs 7-9 during bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) acute infections and particularly an up-regulation during viral reactivation in respiratory tissues has been demonstrated. Furthermore, viral distribution in the respiratory tract of BoHV-1- and BoHV-5-infected calves at different stages of the infectious cycle was analysed. The wide distribution of BoHV DNA in the respiratory tract during acute infection was restricted during latent infection and the subsequent reactivation of BoHV-1 and BoHV-5. Overall, the findings presented here contribute to the knowledge on the replication and dissemination of bovine alpha-herpesviruses. Furthermore, some of the immune factors triggered in the host that determine the different outcomes of infection by two closely related pathogens of cattle have been elucidated.

Comparative study on the in vitro replication and genomic variability of Argentinean field isolates of bovine herpesvirus type 4 (BoHV-4).

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to the Rhadinovirus genus, which is increasingly associated with various problems of the reproductive tract of cattle. In Argentina, analysis of BoHV-4 strains isolated from cervico-vaginal mucus of aborted cows revealed a high genetic divergence among strains, which could be classified in three different groups: Genotype 1 comprises Movar-like strains (European prototype), Genotype 2 includes DN599-like strains (American prototype) and Genotype 3 corresponds to a novel genotype group. Understanding the replication behavior in cell cultures and the molecular characteristics of this pathogen of cattle is critical for the rational design of in vitro experiments. The aim of this work was to quantitatively evaluate the replication properties of different Argentinean BoHV-4 strains and to characterize their phylogenetic relationships. Significant differences were evident among the virus titers of the different BoHV-4 isolates in vitro. The most conserved gene was the major capsid protein (ORF25). The glycoprotein B (gB), glycoprotein H (gH), and thymidine kinsase (TK) genes displayed both synonymous and non-synonymous substitutions, with the highest diversity observed for gB, which displayed amino acid substitutions in 24 out of the 178 positions examined. Strains 09/759, 12/512, and 07/568 presented a deletion encompassing amino acid position 27 to 35, whereas strains 07/435 and 09/227 had a deletion from position 28 to 35. Two strains, 07/435 and 09/227, also displayed the highest divergence compared to the other strains analyzed. This study provides information about the in vitro replication and behavior of nine field isolates of BoHV-4. These findings are relevant since available information on the in vitro growth characteristics of BoHV-4 strains is scarce. The results from this study may also be useful for establishing comparisons with other related viruses.

Frequency of Neospora caninum infections in beef cow-calf operations under extensive management.

The aim of this study was to evaluate the frequencies of Neospora caninum horizontal and vertical transmissions in beef cow-calf operations under three different extensive management systems: group A: 0.75 head per hectare pasturing on natural grass; group B: 1.1 head per hectare on natural grass and improved cultured pastures; and group C: 2 head per hectare on natural grass, improved cultured pasture and whole corn silage. Serum samples from 72 multiparous cows assigned to each beef cow-calf operations were obtained every 3 months during 2 years. A group of 30 replacement heifers from each group were tested similarly since they were 10-21 months old. Twenty four, 20 and 34 calves from groups A, B and C respectively, were bled before colostrum intake and again 6 months later. The samples were analyzed by indirect fluorescence antibody test (IFAT) for detection of total IgG against N. caninum at a serological titre ≥ 200 for multiparous cows and replacement heifers, and a serological titre ≥ 25 for calves. Serum samples from seropositive cows were assessed by ELISA to evaluate the avidity of their specific antibodies. There were no differences in the proportion of seropositive cows from groups A, B and C at the beginning of the trial (p>0.05). Interestingly, the lowest serological titres in seropositive cows from all groups were observed during the first trimester (p<0.05). Although seropositive cows had medium to high avidity antibodies, suggesting chronic infection; seroconversion associated with low antibody avidity was found in 2, 3 and 3 seropositive cows from groups A, B and C. All replacement heifers remained seronegative. No abortions were recorded but 2, 1, and 2 calves from groups A, B and C were seropositive before colostrum intake, respectively. Seropositive calves born from cows having intermediate or high avidity remained with the same serostatus at 6 months of age. Even under varying extensive management conditions, both N. caninum horizontal and vertical transmission methods do occur in beef cow-calf operations.

Distribution of bovine herpesvirus type 1 in the nervous system of experimentally infected calves.

Bovine herpesvirus type 1 (BoHV-1) is responsible for respiratory and genital disease in cattle. BoHV-1 encephalitis is only occasionally reported. However, several cases of neurological disease have been recently attributed to BoHV-1. In this study, the distribution and pathological alterations caused by two BoHV-1 strains in the nervous system of experimentally infected calves during acute infection and reactivation are described. Calves were inoculated intranasally with BoHV-1 Los Angeles (BoHV-1.LA) or Cooper (BoHV-1.Cooper) strains. Acutely infected calves were euthanased at 6 days (BoHV-1.Cooper, n = 2) and 7 days post-inoculation (BoHV-1.LA, n = 2). Latently infected calves that were given dexamethasone to induce reactivation were euthanased at 2 days (BoHV-1.Cooper, n = 2) or 5 days (BoHV-1.LA, n = 2) after dexamethasone administration. Both BoHV-1 strains were isolated from the brains of acutely infected calves. Distribution of viral DNA in the neural tissues was similar for both strains. During reactivation, neither BoHV-1.LA nor BoHV-1.Cooper was isolated from any brain section or trigeminal ganglia in infected calves. Macroscopic lesions were not evident in any group. In BoHV-1.LA infected calves, microscopic lesions were found in the brain but not in the trigeminal ganglia. Microscopic lesions in the brain of BoHV-1.Cooper infected calves were not as evident as in BoHV-1.LA infected animals. However, mononuclear infiltrates and neuronophagia were present in trigeminal ganglia. The results of this study demonstrated that respiratory BoHV-1 strains are able to replicate and disseminate within the bovine nervous tissue and provide evidence of the neuroinvasiveness of BoHV-1 strains.

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.

Cell mediated immune responses in the placenta following challenge of vaccinated pregnant heifers with Neospora caninum.

The aim of the present study was to investigate and correlate the cell-mediated immune response and pathological changes at the maternal-fetal interface of Neospora-challenged pregnant cattle previously immunized with live and inactivated experimental vaccines. Pregnant heifers naïve to Neospora caninum were divided in 5 groups of 4 animals, each one immunized before mating: Group A heifers were intravenously (iv) immunized with 6.25 × 10(7) live tachyzoites of the NC-6 strain; group B heifers were immunized twice subcutaneously (sc) 3 weeks apart with native antigen extract of the NC-6 strain formulated with ISCOMs; group C heifers were sc immunized twice 3 weeks apart with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) of the NC-1 strain formulated with ISCOMs; group D heifers were sc injected with sterile phosphate-buffered saline (PBS) and group E heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv-challenged with 4.7 × 10(7) NC-1 tachyzoites at 70 days of gestation. Heifers were culled at day 104 of gestation and placentomes were examined to evaluate lesions and local cellular immune responses using histopathology, immunohistochemistry and real time-PCR. Immunohistochemistry was performed using bovine leucocyte specific antibodies. Cytokine expression and levels (IFN-γ, IL-4, IL-10, IL-12 and TNF-α) were measured using real-time reverse transcription-PCR and ELISA, respectively. Minimal inflammation was observed in group A placentomes; while placentomes from group B, C, D and E had moderate to severe infiltration with CD3(+), CD4(+), γδ-T cells, CD8(+) cells and macrophages being more numerous in groups B and E placentomes, when compared with groups C and D (P<0.001). Cytokine levels were significantly increased in the caruncles of animals of groups B and C in comparison with the other animal groups (P < 0.001). The results from this study showed that the strongest cellular immune responses were observed in the placentomes of animals that were immunized with inactivated vaccines (groups B and C) and in the placentomes of animals that were sc-sham-inoculated (groups D and E). On the other hand, animals that were immunized with live tachyzoites showed a milder immune cell infiltration to the placenta possibly due to the existence of a protective systemic maternal immune response that helped to minimize N. caninum infection at the maternal-fetal interface.

Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994-2010.

Bovine coronavirus (BCoV) is an important viral pathogen associated with neonatal calf diarrhea. Our aim was to investigate the incidence of BCoV in diarrhea outbreaks in beef and dairy herds from Argentina during 1994-2010. A total of 5.365 fecal samples from diarrheic calves were screened for BCoV diagnosis by ELISA. The virus was detected in 1.71% (92/5365) of the samples corresponding to 5.95% (63/1058) of the diarrhea cases in 239 beef and 324 dairy farms. The detection rate of BCoV was significantly higher in dairy than in beef herds: 12.13% (29/239) vs. 4.32% (14/324) respectively. Phylogenetic analysis of the hypervariable S1 region of seven representative samples (from different husbandry systems, farm locations and years of sampling) indicated that BCoV strains circulating in Argentinean beef and dairy herds formed a cluster distinct from other geographical regions. Interestingly, Argentinean strains are distantly related (at both the nucleotide and amino acid levels) with the Mebus historic reference BCoV strain included in the vaccines currently available in Argentina. However, Mebus-induced antibodies were capable of neutralizing the BCoV Arg95, a field strain adapted to grow in vitro, and vice versa, indicating that both strains belong to the same CoV serotype reported in cattle. This work represents the first large survey describing BCoV circulation in Argentinean cattle.

Latency of bovine herpesvirus type 5 (BoHV-5) in tonsils and peripheral blood leukocytes.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) can both establish latency in the trigeminal ganglion. Non-neural sites of latency have been described for BoHV-1 but not for BoHV-5. The aim of this study was to determine whether peripheral blood leukocytes and tonsils are targets for BoHV-5 infection and to establish whether all stages of that virus's infectious cycle can occur in those cell types. Comparisons with BoHV-1 infection of these tissues were also made in order to better understand the pathogenesis of both viruses. BoHV-1 and BoHV-5 were isolated from tonsils of acutely-infected calves. BoHV-5 was also isolated from a tonsil homogenate after dexamethasone-induced reactivation. During latency, infectious virus was recovered from a tonsil explant of one BoHV-5-infected calf. The genomes of BoHV-5 and BoHV-1 were detected in tonsils from acutely-infected calves although were not detected in tonsils from latently-infected calves or from calves treated with dexamethasone. Virus DNA was intermittently detected in leukocytes. The study has shown that BoHV-5 can establish latency in bovine tonsils and peripheral white blood cells, and that it can be reactivated from latently-infected tonsils, which might contribute to viral transmission. The titres of BoHV-1 and BoHV-5 in tonsils were similar, suggesting that replication at this site is a common feature for both viruses.

Toll-like receptor expression in the nervous system of bovine alpha-herpesvirus-infected calves.

In this study, the expression levels of viral Toll-like receptors (TLRs) in the nervous system of bovine herpesvirus type 5 (BoHV-5)-infected calves were investigated. A significant increase in the expression of TLRs 3 and 7-9 was found in the anterior cerebral cortex during acute infection and viral reactivation. In the trigeminal ganglia, only TLR9 expression was significantly affected. The magnitude of the increase was lower in BoHV-1-infected calves, suggesting that a restricted immune response might protect against exacerbated inflammatory responses in the brain. This work describes, for the first time, the involvement of TLRs 3 and 7-9 in the recognition of BoHV in the bovine nervous system, indicating that the expression of these receptors might be associated with the development of neurological disease. Modulation of the signalling pathways mediated by TLRs might provide an effective approach to control the neuro-immune response to BoHV-5, which may be responsible for neurological lesions.

Neospora caninum serostatus is affected by age and species variables in cohabiting water buffaloes and beef cattle.

The aim of this study was to investigate how Neospora caninum serostatus may be affected by variables such as host species (water buffaloes or cattle) and age in animals cohabiting in the same ranch. A convenience cross-sectional study was performed on four ranches in the Northeast of Argentina, where water buffalo are cohabitating with beef cattle. Blood samples were collected from 1350 female water buffaloes (Bubalus bubalis) and 880 female beef cattle (Bos taurus and Bos indicus crossbreeds) from four ranches. Calving and weaning percentages at herd level for each ranch were also recorded. N. caninum antibody levels were measured by an indirect fluorescent antibody test (IFAT) (reciprocal antibody titers ≥ 100). Serological results were classified into 2 categories (0: negative; 1: positive). A logistic regression model was used to describe the relationship between N. caninum serostatus and specie (water buffalo or cattle), age or ranch and their interactions. Likelihood ratio tests were used to assess the significance of the model and their terms. Odds ratios were estimated and 95% profile likelihood (LR) and Wald confidence intervals (CI) obtained. Overall, specific antibody titers were found in 43.3% (584/1350) of water buffaloes and 28.6% (252/880) of cattle. Seropositive water buffaloes and cattle were observed on all ranches. Age was statistically significant (p=0.01) with an overall estimate of logit (log odds) of age of 0.03 for both species. This indicates that for every one year increase in age, the expected change in log odds of being seropositive increased by 0.03. On three of four ranches a water buffalo was 4.48, 1.54 and 2.25 times more likely to be seropositive than cattle for animals of the same age. The N. caninum serostatus was affected by age in the first place, but also by species on at least three of the four ranches. Calving and weaning percentages were higher in water buffaloes than in beef cattle (p<0.05). Even though the low pathogenicity that N. caninum seems to have in water buffaloes, this study reinforces the importance of this specie as maintenance of the disease.

Toll-like receptor activation and expression in bovine alpha-herpesvirus infections.

The involvement of Toll-like receptors (TLRs) in bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) infections has not been analyzed. In this study, the role of TLR signaling on virus replication was investigated. Blood leukocytes consistently express TLRs. Thus, our approach was to study in vitro the effects of agonist stimulation of TLRs expressed by peripheral blood leukocytes on BoHV-1 and BoHV-5 replication. Furthermore, the patterns of TLRs 3, 7-9 expression on virus-infected-bovine leukocytes were analyzed. Only Imiquimod (TLR7/8 agonist) showed anti-viral activity on infected MDBK cells. This is the first evidence that the timely activation of TLR7/8 signaling is effective in impairing BoHV-1 and 5 replication, thereby providing an experimental indication that Imiquimod may be a promising immune modulator. This work describes, for the first time, the expression patterns of TLRs in BoHV-1- or BoHV-5-infected-bovine leukocytes, suggesting the involvement of TLR7 and TLR9 in the recognition of these viruses.