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.

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.

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.

Effect of Bovine Viral Diarrhea Virus on the ovarian functionality and in vitro reproductive performance of persistently infected heifers.

The aim of this study was to study the effect of Bovine Viral Diarrhea Virus on the reproductive female tract by means of analyzing the ovarian follicular population of persistently infected (PI) heifers, and evaluating the performance of oocytes procured form those heifers in in vitro fertilization procedures. Seven BVDV PI Aberdeen Angus and British crossbred heifers ranging from 18 to 36 months of age were spayed and their ovaries used for viral isolation, microscopic examination, and in vitro fertilization procedures. Bovine Viral Diarrhea Virus was detected from the follicular fluid and sera of all PI heifers. Microscopic examination of the ovaries from PI heifers showed a significant drop in the number of follicles cortical regions, compared with controls. A comparative analysis of the stages of follicular development showed a significant decrease in the number of primordial and tertiary follicles in the cortical regions of ovaries from PI heifers. Viral antigen was detected by immunohistochemistry, and was widely distributed throughout the ovarian tissues. There were differences in the rate of cleavage and embryo development between oocytes obtained from the ovaries of control animals and PI heifers. Furthermore, two developed embryos obtained from oocytes from one of the PI heifers were positive to BVDV, as well as two media from in vitro fertilization (IVF) procedures. The results of this study demonstrate that BVDV PI heifers exhibit alterations in follicular population through of the early interaction between the virus and germ cell line affecting directly the mechanisms involved in the ontogenesis of the ovary.

Search for the genome of bovine herpesvirus types 1, 4 and 5 in bovine semen.

Bovine herpesvirus type 1 (BoHV-1) causes respiratory and reproductive disorders in cattle. Recently, bovine herpesvirus type 5 (BoHV-5) and bovine herpesvirus type 4 (BoHV-4) have been identified to be associated with genital disease. In this study, the presence of the genome of BoHV-1, BoHV-4 and BoHV-5 in bovine semen of Argentinean and international origin was analyzed by PCR assays. The most important finding of this study is the detection of the genome of BoHV-1 and BoHV-4 in semen of bulls maintained at artificial insemination centers. It is particularly relevant that BoHV-1 DNA was also identified in one sample of international origin suggesting the need for extensive quality control measures on international transport of bovine semen.

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.

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.

Immune response to Neospora caninum native antigens formulated with immune stimulating complexes in calves.

The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P<0.05). Analysis of the subisotype specific antibodies in experimentally infected calves revealed a predominant IgG(2) response; however, a predominant IgG(1) response was observed in animals inoculated with N. caninum native antigens formulated with ISCOM-matrix. Control calves remained seronegative until challenge infection. The pattern of bands by Western blot was similar when testing sera from animals in Groups A and B. The levels of IFN-γ production after respective immunization schedules were similar between Groups A and B. Neospora-DNA was detected in plasma samples shortly after intravenous challenge in calves from all groups including those receiving the experimental vaccine formulation. The duration of the parasitemia was similar in all groups.

Variation in the immuno-pathological responses of lambs after experimental infection with different strains of Mycobacterium avium subsp. paratuberculosis.

Ruminant infection by Mycobacterium avium subsp. paratuberculosis (MAP) causes a granulomatous inflammatory response in the intestine and associated lymph nodes. Differences either in the affected organs or in the inflammatory infiltrate were observed between species and individuals. Such differences are usually attributed to variations in host immune responses or to inconsistent effects among different MAP strains. To evaluate if different MAP strains induce different immuno-pathological responses in lambs, 28 one-month-old individuals were divided into six groups and inoculated with different MAP strains. Groups 1 and 2 were inoculated with two bovine strains isolated in Argentina that showed different genetic patterns after BstEII-IS900-RFLP (hereafter strains E and A respectively). Group 3 was inoculated with a bovine strain isolated in Spain obtained after a previous step of culture (patterns C1). Group 4 was inoculated with a homogenate of intestinal mucosa of a clinical case affected by the same bovine strain as that of group 3. Group 5 was inoculated with an ovine strain that was directly purified from the intestinal mucosa of a clinical case, and group 6 was kept as control (i.e. no inoculation). Peripheral immune responses were assessed until 150 days post-infection (dpi), when lambs were humanely killed. Pathological studies were performed in tissues from the intestine and lymph nodes. Lesion types and inflammatory infiltrates were examined as indicators of pathogenicity. All the lambs infected with bovine MAP strains showed a common lesion pattern regardless of the strain type. Such pattern was characterized by focal lesions mainly in the mesenteric lymph nodes, the presence of fibrous tissue, and, occasionally, necrosis in the granulomas as well as the presence of numerous giant cells. Differences in lesion severity were observed among groups: lambs from groups 1 and 2 had the highest number of granulomas and the largest lymph node area affected. Lesions in animals from group 5 (infected with an ovine strain) were more severe and occurred mostly in the intestinal lymphoid tissue; necrosis, fibrosis or giant cells were never detected in this group. These results indicate that the MAP strain type induces different pathological responses in lambs.

Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study.

In the present study, we compared the utility of immunohistochemistry with serological and histological results for the characterization of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) in tissues of affected red deer. Bacterial isolation was considered the standard reference. Samples were taken from seven clinically affected animals with typical macroscopic lesions. The enzyme linked immunosorbent assay (ELISA) and the gel diffusion tests (GD) were used for serological determinations. Samples from intestine and mesenteric lymph nodes were processed for bacterial isolation and histology. M. paratuberculosis was isolated from all the animals. Histologically, lymph nodes displayed necrosis and mineralization at the cortical and medullar areas. Ziehl-Neelsen stained bacteria were numerous inside macrophages and Langhans-type giant cells. Giant and epithelioid cells and lymphocytes were prominent at the ileal mucous membrane. The immunostaining of M. paratuberculosis was very clear inside epithelioid and giant cells. Image analysis was carried out to determine the immunostained area. There was total agreement among the methods employed. Immunohistochemistry can be very useful when the microorganism cannot be recovered from tissues or faeces.