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.

Isolation and molecular characterization of a new Neospora caninum isolate from cattle in Argentina.

Neospora caninum is one of the most important causes of bovine abortion, but isolation of live parasites from infected tissue is difficult. The aims of the present study were to obtain new isolates of N. caninum from congenitally infected asymptomatic newborn cattle in Argentina and to perform characterization by multilocus-microsatellite analysis. Five clinically normal born calves, with demonstrable N. caninum antibodies in precolostrum serum by indirect fluorescent antibody test, were euthanized and their brain samples were processed for histopathological, immunohistochemical, polymerase chain reaction (PCR) analysis, and for bioassay in γ-interferon knockout (GKO) mice. Although N. caninum DNA was detected in brain from all the calves by PCR, viable N. caninum was isolated in GKO mice from only one calf. Neospora caninum tachyzoites of this Argentinean isolate, designated NC-Argentina LP1, were propagated in VERO cell cultures seeded with tachyzoites from the infected GKO mice tissues. Multilocus-microsatellite typing on DNA derived from cell cultured tachyzoites revealed a unique genetic pattern, different from reported isolates. This is the first bovine isolation and genetic characterization of N. caninum in Argentina.

Risk factors associated with Neospora caninum infections in cattle in Argentina.

From 2003 through 2007, serum samples from 5594 dairy and beef heifers and cows in Argentina were assessed to quantify the association between presence of Neospora caninum antibodies and history of abortion, type of exploitation, and age category of animals. Animals with a history of abortion were 85% more likely (P<0.01) to be positive to N. caninum than animals without a record of abortion. For a given category (age) of animals, being in a dairy operation increased the odds of being N. caninum-positive. Replacement dairy heifers were 76% more likely (P<0.01) to be N. caninum-positive than beef cows. These results suggest that postnatal exposure may be more frequent in dairy operations than in beef herds and provide insight into the epidemiology of the disease in one of the most important livestock production regions of the world.

In vitro amplification of BVDV field strains isolated in Argentina: effect of cell line and culture conditions.

The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD-420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.

The role of Neospora caninum and Toxoplasma gondii in spontaneous bovine abortion in Argentina.

The aim of the present work was to evaluate the role of Neospora caninum and Toxoplasma gondii infections in spontaneous bovine abortions in Argentina. Based on histopathological results, 70 presumptive cases of apicomplexan protozoal abortion from a total of 666 cases of spontaneous bovine abortion submitted to the National Institute of Agrarian Technology, Balcarce, from 1999 to 2007 were included in this study. N. caninum infection was diagnosed by an indirect fluorescent antibody test (IFAT), by immunohistochemistry (IHC) and by nested-PCR. T. gondii infection also was diagnosed by nested-PCR. DNA from fetuses was extracted primarily from CNS tissues. Heart, liver, muscle and/or placenta were processed when nervous tissue was not available. Sixty-six (9.9%) fetuses were positive by at least one technique (IFAT, IHC or nested-PCR) for N. caninum infection. Overall, there was poor agreement among results obtained by these diagnostic techniques. In contrast, no Toxoplasma-infection was detected in any aborted bovine fetus.

Retrospective study of bovine neonatal mortality: cases reported from INTA Balcarce, Argentina.

A retrospective study was performed on 169 beef and dairy calves aged from 1 to 7 days old submitted to the Diagnostic Laboratories at INTA Balcarce, Argentina. Bacterial culture was performed for aerobic and microaerophilic organisms. Samples from spleen and lymph nodes, and peripheral blood mononuclear cells were also cultured for viral isolation on cell culture. Bovine rotavirus was detected by direct-ELISA. Multiple tissue samples were fixed in 10% formalin, routinely processed and Stained with hematoxylin and eosin for microscopic examination. Etiological diagnosis was made in 70 of the 169 calves. Infectious agents were identified in 49 cases, the most common being Escherichia coli. When the histopathological examination was performed in cases with undetermined diagnosis, it was noted that 44 specimens had histological lesions, which suggested the presence of an infectious agent. In order to characterize the causes of bovine neonatal mortality, the protocols and methodology should be improved in further works.

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle.

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 +/- 0.27 on the day of challenge and reached a top value of 3.36 +/- 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

Humoral immune response in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival route.

The aim of this study was to compare systemic humoral immune responses in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival and intravenous routes. Twenty nine heifers separated in three experimental groups were studied: Group 1 (n=10 animals) and Group 2 (n=9 animals) were inoculated with 10(8) of N. caninum tachyzoites by conjunctival and intravenous routes at 5th month of gestation, respectively; Group 3 (n=10 animals) were non-inoculated control animals. An indirect fluorescent antibody test (IFAT) and western immunoblotting (IB) were used to analyze the humoral immune response. All animals from Group 1 developed N. caninum specific antibody responses after conjunctival inoculation recording the highest antibody titer (mean+/-SE: 160+/-49.9) at 6th month of gestation. There were statistical differences between humoral immune responses found in Group 1 and 2 being higher in the second one at 6.5th, 8.5th and 9th months of gestation (P<0.05). Interestingly, all heifers from Group 1 reverted to seronegative status at the end of gestation. No increase in antibody was detected in the uninfected control group. Same pattern of N. caninum antigens was recognized by sera from heifers inoculated by conjunctival route and heifers inoculated by intravenous route. Recognized antigens were 116, 92, 84, 77, 45, 40, 25-26 and 17-18 kDa. The conjunctival instillation of N. caninum tachyzoites in pregnant heifers induces specific systemic antibodies. Further work is needed in order to clarify the consequences of this novel experimental route of infection not only on the fetus but also on the dam.

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.

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.

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation.

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-gamma production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450+/-252), peaked at week 23 (mean 16,000+/-6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467+/-2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 (P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-gamma in similar concentrations to those of infected animals (P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers > or = 200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.

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.

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.

Immune response and protection provided by live tachyzoites and native antigens from the NC-6 Argentina strain of Neospora caninum in pregnant heifers.

The aim of the present study was to compare the immunogenicity and protective efficacy of live tachyzoites and native antigen extract obtained from the NC-6 Argentina strain against vertical transmission of Neospora caninum, following experimental challenge in pregnant heifers with the NC-1 strain. Sixteen pregnant heifers were divided in 4 groups of 4 animals, each receiving different inoculation before mating: group A animals were intravenously (iv) inoculated with 6.25×10(7) live tachyzoites of the NC-6 strain, group B heifers were inoculated twice subcutaneously (sc) with N. caninum native antigen extract formulated with ISCOMs, group C heifers were sc injected with sterile phosphate-buffered saline (PBS) and group D heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv challenged with the NC-1 strain at 70 days of gestation. Serum and heparinized blood samples were collected eight times on weeks 0, 2, 3, 5, 9, 13, 16 and 17 post-inoculation. Dams were slaughtered at the 17th week of experiment (104 days of pregnancy) and placental and fetal tissue samples were collected. Specific antibody responses in heifers were tested by indirect enzyme linked immunosorbent assay (iELISA). The cellular immune response in dams was assessed by quantifying IFN-γ production and the percentages of T-cells (CD4(+), CD8(+) and γδ(+)) and monocytes in peripheral blood mononuclear cells (PBMC). Fetal fluids and tissue samples were tested using the indirect fluorescence antibody test, western blot, histopathology, immunohistochemistry and nested-PCR. A significant increase in N. caninum antibody response was detected in heifers of groups A and B from week 3 after inoculation (P<0.001). IFN-γ production was similar in groups A and B at week 13 (P>0.05). All fetuses were viable at necropsy. Specific IgG against N. caninum was detected in 1/4 fetal fluids recovered from groups A, C and D heifers and 3/4 fetal fluids from group B. Transplacental transmission could be determined in one fetus from group A and three fetuses from group B by nPCR. All fetuses from groups C and D were positive by nPCR. It is noteworthy that dams with higher CD4(+)/CD8(+) ratios in PBMC, regardless of the experimental group, had lower pathology scores. The results of this study confirm that inoculation with live parasites pre-mating may provide at least partial protection against vertical transmission of N. caninum following challenge in heifers at early gestation.

In vitro replication of bovine herpesvirus types 1 and 5.

The aim of this work was to study the in vitro replication of bovine herpesvirus types 1 and 5 (BoHV-1 and 5) at the beginning and end of the logarithmic growth phase of Madin-Darby Bovine Kidney (MDBK) cells. The replication kinetics and size of lysis and infection plaques of the field isolates 09/210 (BoHV-1) and 97/613 (BoHV-5) and the reference strains BoHV-1.1 Los Angeles 38 (LA38), BoHV-1.1 Cooper, BoHV-5a N569 and BoHV-5b A663 were evaluated. The highest mean virus titre was recorded for N569, followed by LA38 and 97/613. For most of the viruses, the virus titre values increased from 24 h post-infection (hpi) up to 48 hpi and then, they remained unchanged up to 72 hpi. However, the virus titre for 09/210 was significantly lower and a slight, steady increase was observed from 24 to 72 hpi. Furthermore, the largest lysis and infection plaques were recorded for 97/613 and LA38, respectively. According to this work, it is evident that there is a relationship between the replication of BoHV and the multiplication stage of MDBK cells. The results of this study contribute to the understanding of the replication behaviour in cell cultures of several strains of BoHV, which is critical for the rational design of in vitro experiments and vaccine production.

Genomic analysis of bovine herpesvirus type 4 (BoHV-4) from Argentina: high genetic variability and novel phylogenetic groups.

Bovine herpesvirus 4 (BoHV-4) is a γ-herpesvirus that has been isolated both from apparently healthy animals and from cattle with a variety of clinical signs, including post-partum endometritis and abortion. BoHV-4 causes either a persistent or a latent infection in cells of the monocyte/macrophage lineage. Two groups of BoVH-4 strains have been defined based on their restriction patterns: the Movar-like strains (European prototype) and the DN 599-like strains (American prototype). The purpose of the present study was to genetically characterize wild type BoHV-4 strains isolated from vaginal discharges of aborted cows in Argentina. The virus was identified by isolation and nested PCR in all vaginal discharge samples from aborted cows, either as a sole agent or in association with other pathogens. Restriction enzyme profiling and phylogenetic analysis demonstrated that there is a high genetic variability among the studied field isolates. The existence of three groups of strains, which were designated as genotypes 1, 2 and 3, is described. Genotypes 1 and 2 possibly correspond to the Movar-like and DN 599-like groups, respectively, whereas Genotype 3 corresponds to a novel group. Two viral strains did not cluster into any of these three groups, indicating that other genotypes could be circulating in Argentina. These results suggest a complex epidemiological background for the Argentinean BoHV-4 strains, probably influenced by independent events of genetic drift. This hypothesis cannot be rejected based on the available data. However, there is no direct evidence supporting this possibility. Thus, it seems speculative to suggest that interspecific jumps are responsible for the observed phylogenetic grouping. On the other hand, our analyses suggest a geographical structure for the observed viral genotypes, since genotypes 1 and 2 included the European (Movar-like) and American (DN599-like) reference strains, respectively. Geographic dispersion is known to be a driver of herpes viruses diversification, and independent evolution in geographical isolated places ensures the emergence of particular mutations in each location due to genetic drift (Compans, 2007; Zong et al., 1999). Therefore, at this point, the genetic drift hypothesis is the one that requires less ad-hoc considerations and thus, to our understanding, is the one that fits to the findings from this study. The involvement of this genetic variability in the detection and pathogenesis of BoHV-4 remains to be investigated.

Clinical and reproductive consequences of using BVDV-contaminated semen in artificial insemination in a beef herd in Argentina.

The current report was prompted by an atypical outbreak of mucosal disease that occurred in a beef herd in the southwestern part of Buenos Aires Province, Argentina, where a total of 9/41 (21.9%) yearling bulls died. Blood samples from 73 bulls and 189 heifers were tested for evidence of persistent BVDV infection with Bovine Viral Diarrhea Virus (BVDV). Non-cytopathic BVDV was isolated from 7 (9.6%) 24- to 36-month-old bulls, and 3 (1.6%) 36-month-old heifers. Non-cytopathic BVDV was also detected in the seminal plasma of three of six persistently infected (PI) bulls. Furthermore, a 171 bp genomic fragment of BVDV was consistently detected by nested RT-PCR in one of the two samples of the commercial semen used for artificial insemination, indicating that this semen could be a possible source of infection for the whole herd. To evaluate the possible reproductive consequences of PI heifers and bulls, ovaries and semen were obtained from PI cattle for in vitro assays. The in vitro fertilization of oocytes with semen from PI bulls was associated with decreased cleavage and embryo development rates. Additionally, non-cytopathic BVDV was isolated from the follicular fluid of PI heifers. Genetic typing revealed that all isolates BVDV from the present study had a high percentage of homology and that all of the fragments from the RT-PCR clearly fit with the BVDV 1b cluster. These findings confirm the negative impact that BVDV can have on the reproductive performance of cattle and the importance of applying the proper sanitary controls to minimize the risk of BVDV infection.