Modulation of Apoptosis by Bovine Gammaherpesvirus 4 Infection in Bovine Endometrial Cells and the Possible Role of LPS in This Process.

The prevalent pathogens associated with bovine uterine infections are bacteria that appear to increase the host's susceptibility to secondary infections with other bacteria or viruses, among which BoGHV4 is the most frequently found. In this work, the study of the pathways of apoptosis induction was carried out on an experimental model of primary culture of endometrial cells, in order to know the implication of BoGHV4 and the presence of bacterial LPS in the pathogenesis of the bovine reproductive tract. For this, different staining techniques and molecular analysis by RT-PCR were used. The results obtained allowed us to conclude that the level of cell death observed in the proposed primary culture is directly related to the time of viral infection and the presence of LPS in BoGHV4 infection. The apoptosis indices in cells infected with BoGHV4 and BoGHV4 + LPS revealed a maximum that correlated with the appearance of cytopathic effects and the maximum viral titers in the model studied. However, morphological, biochemical, and molecular changes were evident during both early and late stages of apoptosis. These findings provide information on the factors that may influence the pathogenesis of BoGHV4 and help to better understand the mechanisms involved in virus infection.

Molecular Epidemiology of Rotavirus A in Calves: Evolutionary Analysis of a Bovine G8P[11] Strain and Spatio-Temporal Dynamics of G6 Lineages in the Americas.

Rotavirus A (RVA) causes diarrhea in calves and frequently possesses the G6 and P[5]/P[11] genotypes, whereas G8 is less common. We aimed to compare RVA infections and G/P genotypes in beef and dairy calves from major livestock regions of Argentina, elucidate the evolutionary origin of a G8 strain and analyze the G8 lineages, infer the phylogenetic relationship of RVA field strains, and investigate the evolution and spatio-temporal dynamics of the main G6 lineages in American countries. Fecal samples (n = 422) from diarrheic (beef, 104; dairy, 137) and non-diarrheic (beef, 78; dairy, 103) calves were analyzed by ELISA and semi-nested multiplex RT-PCR. Sequencing, phylogenetic, phylodynamic, and phylogeographic analyses were performed. RVA infections were more frequent in beef (22.0%) than in dairy (14.2%) calves. Prevalent genotypes and G6 lineages were G6(IV)P[5] in beef (90.9%) and G6(III)P[11] (41.2%) or mixed genotypes (23.5%) in dairy calves. The only G8 strain was phylogenetically related to bovine and artiodactyl bovine-like strains. Re-analyses inside the G8 genotype identified G8(I) to G8(VIII) lineages. Of all G6 strains characterized, the G6(IV)P[5](I) strains from "Cuenca del Salado" (Argentina) and Uruguay clustered together. According to farm location, a clustering pattern for G6(IV)P[5] strains of beef farms was observed. Both G6 lineage strains together revealed an evolutionary rate of 1.24 × 10-3 substitutions/site/year, and the time to the most recent common ancestor was dated in 1853. The most probable ancestral locations were Argentina in 1981 for G6(III) strains and the USA in 1940 for G6(IV) strains. The highest migration rates for both G6 lineages together were from Argentina to Brazil and Uruguay. Altogether, the epidemiology, genetic diversity, and phylogeny of RVA in calves can differ according to the production system and farm location. We provide novel knowledge about the evolutionary origin of a bovine G8P[11] strain. Finally, bovine G6 strains from American countries would have originated in the USA nearly a century before its first description.

Genomic evolution of bovine viral diarrhea virus based on complete genome and individual gene analyses.

Bovine viral diarrhea virus (BVDV) genome consists of a single-stranded, positive-sense RNA with high genetic diversity. In the last years, significant progress has been achieved in BVDV knowledge evolution through phylodynamic analysis based on the partial 5'UTR sequences, whereas a few studies have used other genes or the complete coding sequence (CDS). However, no research has evaluated and compared BVDV evolutionary history based on the complete genome (CG), CDS, and individual genes. In this study, phylodynamic analyses were carried out with BVDV-1 (Pestivirus A) and BVDV-2 (Pestivirus B) CG sequences available on the GenBank database and each genomic region: CDS, UTRs, and individual genes. In comparison to the CG, the estimations for both BVDV species varied according to the dataset used, pointing out the importance of considering the analyzed genomic region when concluding. This study may provide new insight into BVDV evolution history while highlighting the need to increase the available BVDV CG sequences to perform more comprehensive phylodynamic studies in the future.

Evolution of Animal South American RVA Told by the NSP4 Gene E12 Genotype.

Rotavirus A (RVA) possesses a genome of 11 double-stranded (ds) RNA segments, and each segment encodes one protein, with the exception of segment 11. NSP4 is a non-structural multifunctional protein encoded by segment 10 that defines the E-genotype. From the 31 E-genotypes described, genotype E12 has been described in Argentina, Uruguay, Paraguay, and Brazil in RVA strains infecting different animal species and humans. In this work, we studied the evolutionary relationships of RVA strains carrying the E12 genotype in South America using phylogenetic and phylodynamic approaches. We found that the E12 genotype has a South American origin, with a guanaco (Lama guanicoe) strain as natural host. Interestingly, all the other reported RVA strains carrying the E12 genotype in equine, bovine, caprine, and human strains are related to RVA strains of camelid origin. The evolutionary path and genetic footprint of the E12 genotype were reconstructed starting with the introduction of non-native livestock species into the American continent with the Spanish conquest in the 16th century. The imported animal species were in close contact with South American camelids, and the offspring were exposed to the native RVA strains brought from Europe and the new RVA circulating in guanacos, resulting in the emergence of new RVA strains in the current lineages' strongly species-specific adaption. In conclusion, we proposed the NSP4 E12 genotype as a genetic geographic marker in the RVA strains circulating in different animal species in South America.

Temporal and geographic dynamics of bovine viral diarrhea virus in American countries.

Bovine viral diarrhea virus (BVDV) is a worldwide distributed pathogen of livestock classified into three species, BVDV-1 (Pestivirus A), BVDV-2 (Pestivirus B), and HoBi-like pestivirus (HoBiPeV; Pestivirus H). Despite being considered endemic in several regions of the Americas, the spatiotemporal distribution of BVDV is scarcely known. This study aimed to reconstruct the population dynamics of BVDV in American countries. The analyses performed with the partial 5´UTR gene showed that BVDV-1 and -2 would have started their diversification in the 1670s and 1790s in the United States, whereas HoBiPeV probably emerged in the 1980s in Brazil. No evident geographic clustering was observed in the Bayesian trees, which may indicate that multiple introductions events would have occurred following the first introduction. This study provides new insights into BVDV dynamics, although further analyses including sequences from other American countries and continents will help to expand the knowledge of BVDV evolution and transmission.

Spatial-temporal trends and economic losses associated with bovine abortifacients in central Argentina.

The aims of this work are, firstly, to provide the geolocalization of cases of bovine abortion with definitive diagnosis and, secondly, to estimate the economic losses due to the most frequent abortifacients diagnosed agents in cattle in Buenos Aires province, Argentina. The total beef and dairy cattle population at risk of abortion is 8,358,186 and 538,076, respectively. In beef cattle, the overall risk of abortion was estimated at 4.5% for all pregnancies, where 27.9% are due to Campylobacter fetus, Neospora caninum, Leptospira spp., Brucella abortus, and bovine viral diarrhea virus with economic losses of US$ 440 per abortion, being the annual loss to the beef industry of US$ 50,144,101. In dairy cattle, there was an 8.0% risk of suffering abortion, 26.1% produced by the same abortigenic agents. The economic losses were estimated at US$ 1,415 per abortion, which equals a total loss of US$ 17,298,498 for the dairy industry in the region. The results of this study show that infectious causes are highly prevalent in Buenos Aires province, and they caused severe economic impacts in the dairy and beef industries. Furthermore, changes in temporal trends of infectious abortion occurrence were detected, probably related to the inclusion of molecular diagnostic techniques with more sensitivity or different epidemiological or husbandry conditions in the region analyzed.

Mycoplasma bovis-pneumonia and polyarthritis in feedlot calves in Argentina: First local isolation.

Bovine respiratory disease (BRD) is one of the most frequent clinical concerns in weaned calves after their arrival at the feedlot. This work reports the first local isolation of Mycoplasma bovis from feedlot calves with pneumonia and polyarthritis in Argentina. Twenty four out of 545 calves showed progressive, subacute to chronic respiratory distress, coughing, and fever. Thirty percent of the affected calves also showed lameness and swelling of elbow or carpal, and knee or tarsal joints. Five necropsies were performed and severe multifocal to coalescent pulmonary nodules, containing white-yellowish caseous exudate encircled by fibrous tissue, and fibrinonecrotic arthritis and tenosynovitis were detected. Mycoplasma was isolated from lung and joint samples. The 16S-23S rRNA ITS consensus sequence obtained from these isolates showed 100% similarity with the same region of M. bovis strains. Since there are no commercially available vaccines in the region for the prevention and control of M. bovis pneumonia and arthritis, surveillance is a priority to reduce the source of disease to naïve animals.

Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia.

Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::blaCTX-M-2) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.

Genomic diversity and phylodynamic of bovine viral diarrhea virus in Argentina.

Bovine viral diarrhea virus (BVDV) is an important pathogen of ruminants worldwide and is characterized by high genetic diversity and a wide range of clinical presentations. In Argentina, several studies have evaluated the genetic diversity of BVDV but no phylodynamic study has been published yet. In this study, a comprehensive compilation and update of Argentinean BVDV sequences were performed, and the evolutionary history of BVDV was characterized by phylodynamic analyses based on the 5´UTR. Although BVDV-1b and BVDV-1a were the most frequent subtypes, novel subtypes for Argentina, 1e and 1i, were identified. The phylodynamic analysis suggested that BVDV started its diversification in the mid-1650s with an exponential increase in viral diversity since the late 1990s, possibly related to the livestock expansion and intensification in the country. Evolutionary rate in the 5´UTR was faster for BVDV-1a than for BVDV-1b, and both subtypes presented an endemic nature according to the demographic reconstructions. The current study contributes to clarify the evolutionary history of BVDV in the main cattle region of the country and provides useful information about the epidemiology and future development of diagnostic and control tools in Argentina.

Frequency of bovine viral diarrhea virus (BVDV) in Argentinean bovine herds and comparison of diagnostic tests for BVDV detection in bovine serum samples: a preliminary study.

Bovine viral diarrhea (BVD) is a major worldwide disease with negative economic impact on cattle production. Successful control programs of BVD require the identification and culling of persistently infected (PI) animals with bovine viral diarrhea virus (BVDV). A variety of diagnostic tests are available to detect BVDV, but no comparison has been performed among those tests in Argentina. Sera collected from 2864 cattle, belonging to 55 herds from three Argentinean provinces, were analyzed by nested RT-PCR (RT-nPCR) to detect BVDV for diagnostic purposes. Additionally, this study evaluated the agreement of the RT-nPCR along with virus isolation, antigen-capture ELISA, and real-time RT-PCR for BVDV detection in archived bovine serum samples (n = 90). The RT-nPCR was useful for BVDV detection in pooled and individual serum samples. BVDV was detected in 1% (29/2864) of the cattle and in 20% (11/55) of the herds. The proportion of BVDV-positive sera was not statistically different among the tests. In addition, comparisons showed high agreement levels, with the highest values between both RT-PCR protocols. The frequency of BVDV infection at individual and herd level was lower than the reported values worldwide. Since follow-up testing was not performed, the frequency of PI cattle was unknown. Also, this study demonstrated that the four diagnostic tests can be used reliably for BVDV identification in individual serum samples. Further epidemiologically designed studies that address prevalence, risk factors, and economic impact of BVDV in Argentina will be necessary to implement effective control programs.

Detection methods and characterization of bovine viral diarrhea virus in aborted fetuses and neonatal calves over a 22-year period.

Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22 years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5'UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.

The first caprine rotavirus detected in Argentina displays genomic features resembling virus strains infecting members of the Bovidae and Camelidae.

Rotavirus group A (RVA) is a major cause of diarrhea in humans and young animals including small ruminants. The purpose of this study was to identify RVA in dairy goat kids, and to characterize the complete genomic constellation and genetic relatedness with other RVA strains. Four out of twenty fecal samples from diarrheic and non-diarrheic goat kids were positive for RVA by ELISA. A representative sample was selected for further genome analyses. The RVA strain RVA/Goat-wt/ARG/0040/2011/G8P[1] displayed the following genomic constellation: G8-P[1]-I2-R5-C2-M2-A3-N2-T6-E12-H3, reminiscent to guanaco and other bovine-like RVA strains detected in Argentina. Phylogenetic analyses revealed that most of the genome segments had a rather close relatedness with RVA strains typically obtained from cattle, sheep, South American camelids and goats. Interestingly, strain 0040 possessed the R5 and E12 genotypes which have up to date only been found in different animal species from Argentina. Overall, these findings suggest that strain 0040 could represent a typical goat RVA genome constellation similar to those previously found in other animal species within the order Artiodactyla.