Mapping the sites of latency and reactivation by bovine herpesvirus 5 (BoHV-5) and a thymidine kinase-deleted BoHV-5 in lambs

A thymidine kinase (tk)-deleted bovine herpesvirus 5 (BoHV-5tkΔ) was previously shown to establish latent infection and reactivate - even poorly - in a sheep model (Cadore et al. 2013). As TK-negative alphaherpesviruses are unlike to reactivate in neural tissue, this study investigated the sites of latency and reactivation by this recombinant in lambs. For this, groups of lambs were inoculated intranasally with the parental BoHV-5 strain (SV-507/99) or with the recombinant BoHV-5tkΔ. During latent infection (40 days post-inoculation, pi), the distribution of recombinant virus DNA in neural and non-neural tissues was similar to that of the parental virus. Parental and recombinant virus DNA was consistently detected by PCR in trigeminal ganglia (TGs); frequently in palatine and pharyngeal tonsils and, less frequently in the retropharyngeal lymph nodes. In addition, latent DNA of both viruses was detected in several areas of the brain. After dexamethasone (Dx) administration (day 40pi), the recombinant virus was barely detected in nasal secretions contrasting with marked shedding of the parental virus. In tissues of lambs euthanized at day 3 post-Dx treatment (pDx), reverse-transcription-PCR (RT-PCR) for a late viral mRNA (glycoprotein D gene) demonstrated reactivation of parental virus in neural (TGs) and lymphoid tissues (tonsils, lymph node). In contrast, recombinant virus mRNA was detected only in lymphoid tissues. These results demonstrate that BoHV-5 and the recombinant BoHV-5tkΔ do establish latent infection in neural and non-neural sites. Reactivation of the recombinant BoHV-5tkΔ, however, appeared to occur only in non-neural sites. In anyway, the ability of a tk-deleted strain to reactivate latent infection deserves attention in the context of vaccine safety.

Stereological study of the elastic fiber and smooth muscle cell system in the bovine and buffalo penis

Samples of ten penises of Mediterranean buffaloes and ten penises of Red Sindhi cattle were used. The thickness of the tunica albuginea (TA), distribution of smooth muscle cells (SMC) and volume density (Vv) of elastic system fibers in TA, corpus cavernosum (CC) and corpus spongiosum (CS) were evaluated. The Vv of elastic system fibers in buffalo and bovine penis was respectively 4.07% ±0.88% and 3.36% ±1.21% in TA; 17.32% ±2.21% and 13.14% ±1.27% (CC), 26.58% ±4.31% and 31.36% ±3.67% (CS). The CC of buffalo presented higher Vv of elastic fibers than bovine, while in the CS the Vv of elastic fibers in buffaloes was smaller than in cattle. The TA thickness showed a significant difference among the species studied. The arrangement of SMC in the bovine penises and in the water buffalo suggests that this pattern is common to animals that have fibroelastic penises.

Insights on bovine genetic engineering and cloning

Transgenic technology has become an essential tool for the development of animal biotechnologies, and animal cloning through somatic cell nuclear transfer (SCNT) enabled the generation of genetically modified animals utilizing previously modified and selected cell lineages as nuclei donors, assuring therefore the generation of homogeneous herds expressing the desired modification. The present study aimed to discuss the use of SCNT as an important methodology for the production of transgenic herds, and also some recent insights on genetic modification of nuclei donors and possible effects of gene induction of pluripotency on SCNT.

Assessing bovine babesiosis in Rhipicephalus (Boophilus) microplus ticks and 3 to 9-month-old cattle in the middle Magdalena region, Colombia

Babesia sp. is a protozoan hemoparasite that affects livestock worldwide. The Colombian Middle Magdalena is an enzootic region for babesiosis, but there is no previous research providing detail on its transmission cycle. This study aims to assess some Babesia sp. infection indicators in cattle and ticks from the area, by using direct microscopic and molecular techniques to detect the infection. In the cattle, 59.9% and 3.4 % positivity values for B. bigemina and mixed infection (B. bovis + B. bigemina) were found respectively. In ticks, the positivity of B. bigemina reached 79.2% and 9.4% for the mixed infection. The degree of infestation in the region was 3.2 ticks per bovine. There was positive correlation between tick control acaricide frequencies and infestation in bovines. This leads us to infer that control periodicity greater than 90 days, in stable zones, is an abiotic factor that benefits the acquisition of protective immunity in calves, the natural control of the infection and eventual disease absence. It is necessary to monitor the disease by applying new entomological and parasitological indicators showing the complexity of this phenomenon.

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Severe diarrhea outbreak in beef calves (Bos indicus) caused by G6P[11], an emergent genotype of bovine rotavirus group A

The episodes of diarrhea caused by neonatal bovine rotavirus group A (BoRVA) constitute one of the major health problems in the calf rearing worldwide. The main G (VP7) and P (VP4) genotypes of BoRVA strains involved in the etiology of diarrhea in calves are G6P[1], G10P[11], G6P[5], and G8P[1]. However, less frequently, other G and P genotypes have been described in BoRVA strains identified in diarrheic fecal samples of calves. This study describes the identification and molecular characterization of an emerging genotype (G6P[11]) in BoRVA strains involved in the etiology of a diarrhea outbreak in beef calves in a cattle herd of high production in extensive management system. The diarrhea outbreak, which showed high morbidity (60%) and lethality (7%) rates, occurred in calves (n= 384) Nelore (Bos indicus) up to 30-day-old from the State of Mato Grosso do Sul, Brazil. BoRVA was identified in 80% (16/20) of the fecal samples analyzed by polyacrylamide gel electrophoresis (PAGE) technique. In all PAGE-positive fecal samples were amplified products with 1,062-bp and 876-bp in the RT-PCR assays for VP7 (G type) and VP4 (VP8*) (P type) of BoRVA, respectively. The nucleotide sequence analysis of VP7 and VP4 genes of four wild-type BoRVA strains showed G6-III P[11]-III genotype/lineage. The G6P[11] genotype has been described in RVA strains of human and animal hosts, however, in calves this genotype was only identified in some cross-sectional studies and not as a single cause of diarrhea outbreaks in calves with high morbidity and lethality rates as described in this study. The monitoring of the G and P genotypes of BoRVA strains involved in diarrhea outbreaks in calves is important for both animal and public health by allowing the identification of the most frequent genotypes, the characterization of novel genotypes and to identify reassortments with genotypes described in animal and human hosts. The results of this study show the importance of the monitoring of the genotypes of BoRVA strains involved in episodes of bovine neonatal diarrhea as for characterization of frequency of occurrence and pathogenic potential of uncommon genotypes as for monitoring of the emergency of different BoRVA genotypes not included in commercial vaccines.

Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle

Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.

Risk factors associated with the antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis

The objective of this study was to evaluate herd management practices and mastitis treatment procedures as risk factors associated with Staphylococcus aureus antimicrobial resistance. For this study, 13 herds were selected to participate in the study to evaluate the association between their management practices and mastitis treatment procedures and in vitro antimicrobial susceptibility. A total of 1069 composite milk samples were collected aseptically from the selected cows in four different periods over two years. The samples were used for microbiological culturing of S. aureus isolates and evaluation of their antimicrobial susceptibility. A total of 756 samples (70.7%) were culture-positive, and S. aureus comprised 27.77% (n=210) of the isolates. The S. aureus isolates were tested using the disk-diffusion susceptibility assay with the following antimicrobials: ampicillin 10mg; clindamycin 2μg; penicillin 1mg; ceftiofur 30μg; gentamicin 10mg; sulfa-trimethoprim 25μg; enrofloxacin 5μg; sulfonamide 300μg; tetracycline 30μg; oxacillin 1mg; cephalothin 30μg and erythromycin 5μg. The variables that were significantly associated with S. aureus resistance were as follows: the treatment of clinical mastitis for ampicillin (OR=2.18), dry cow treatment for enrofloxacin (OR=2.11) and not sending milk samples for microbiological culture and susceptibility tests, for ampicillin (OR=2.57) and penicillin (OR=4.69). In conclusion, the identification of risk factors for S. aureus resistance against various mastitis antimicrobials is an important information that may help in practical recommendations for prudent use of antimicrobial in milk production.

Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis

This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.

Increased expression of Interleukin-6 related to nephritis in chickens challenged with an Avian infectious bronchitis virus variant

A Brazilian field isolate (IBV/Brazil/PR05) of avian infectious bronchitis virus (IBV), associated with development of nephritis in chickens, was previously genotyped as IBV variant after S1 gene sequencing. The aim of this study was to evaluate the levels of IL-6 in kidneys and trachea of birds vaccinated and challenged with IBV/Brazil/PR05 strain, correlating these results with scores of microscopic lesions, specific IBV antigen detection and viral load. The up-regulation of IL-6 and the increased levels of viral load on renal and tracheal samples were significantly correlated with scores of microscopic lesions. Reduced levels of viral load were detected in kidneys of birds previously vaccinated and challenged, compared to non-vaccinated challenged group, although markedly microscopic lesions were observed for both groups. The expression of IL-6, present both in the kidney and in the tracheas, was dependent on the load of the virus present in the tissue, and the development of lesions was related with IL-6 present in the tissues. These data suggest that variant IBV/Brazil/PR05 can induce the expression of proinflammatory cytokines in a manner correlated with viral load and increased IL-6 is involved in the tissue with the influx of inflammatory cells and subsequent nephritis. This may contribute with a model to the development of immunosuppressive agents of IL-6 to prevent acute inflammatory processes against infection with IBV and perhaps other coronaviruses, as well as contribute to the understanding of the immunopathogenesis of IBV nephropatogenic strains.

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil

Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predominantly from North America, that is caused by Histophilus somniwith sporadic descriptions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations characteristic of brain dysfunction. Hematological and cerebrospinal fluid analyses were not suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis with vasculitis and thrombosis of small vessels that contained discrete neutrophilic and/or lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal nerve ganglion of all animals. All tissues from the central nervous system used during this study were previously characterized as negative for rabies virus by the direct immunofluorescence assay. PCR and RT-PCR assays investigated the participation of infectious agents associated with bovine neurological disease by targeting specific genes of H. somni, Listeria monocytogenes, bovine herpesvirus -1 and -5, bovine viral diarrhea virus, and ovine herpesvirus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA gene of H. somni from brain sections of all animals with histopathological diagnosis of TME; all other PCR/RT-PCR assays were negative. These findings confirmed the participation of H. somni in the neuropathological disease observed in these animals, extend the geographical distribution of this disease, and support previous findings of H. somni from Brazil.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Morphoquantitative description of bovine digital cushion

Abstract The digital cushion is characterized as a modified subcutaneous tissue that absorbs the shock during gait, assists venous return of the hoof and supports a considerable part of body weight. Digital cushions have particular importance in the pathogenesis of the hoof, since they need to properly work in order to prevent compression and traumas in soft tissues. This study aimed to measure and determine how is the arrangement of these structures, and for this it was established the proportions of connective, adipose, vascular tissues and collagen fibers and collagen types found in palmar and plantar digital cushion of bovine using fore and hindlimbs of twelve adult zebu cattle of both sexes, 11 male and one female, with 269kg average carcass weight and without limb disorders. Fragments of cushions were subjected to conventional histology, cut to a thickness of 4µm and stained with Red Picrosirius. With digital optical microscope, the quantification of the connective tissue and differentiation of types of collagen used the Image Pro Plus® software, and of adipose and vascular tissue, the test point system. The mean and standard error were estimated with the GraphPad Prism 5.0 software, and then data were subjected to Kolmogorov-Smirnov normality test and Student's t-test with significance level set at 5% for determining the amount of different tissues between fore and hindlimbs of studied animals. In forelimbs the mean and standard error of the connective tissue proportion was 50.10%+1.54, of the adipose tissue was 21.34%+1.44, and of vascular tissue was 3.43%+0.28. Hindlimbs presented a proportion of connective tissue of 61.61%+1.47, 20.66%+1.53 of adipose tissue, and 3.06%+0.20 of vascular tissue. A significant difference (p<0.001) was detected in the connective tissue proportion between fore and hindlimbs. Types I and II collagen fibers have presented, respectively, a proportion of 31.89% and 3.9% in forelimbs and 34.05% and 1.78% in hindlimbs. According to the used methodology, digital cushions had a clear differentiation relative to adipose tissue between fore and hindlimbs.

Presence of Porphyromonas and Prevotella species in the oral microflora of cattle with periodontitis

Abstratc: Bovine periodontitis is a progressive purulent infectious process associated with the presence of strictly and facultative anaerobic subgingival biofilm and epidemiologically related to soil management in large geographic areas of Brazil. This study aimed to detect species of the genera Porphyromonas and Prevotella, which occurr in periodontal pockets of cattle with lesions deeper than 5mm (n=26) and in gingival sulcus of animals considered periodontally healthy (n=25). Presence of the microorganisms was evaluated by independent-culture medium diagnostic method, using polymerase chain reaction (PCR) with specific primers of Porphyromonas asaccharolytica, P. endodontalis, P. gingivalis, P. gulae, Prevotella buccae, P. intermedia, P. loescheii, P. melaninogenica, P. nigrescens, P. oralis and P. tannerae. The species P. endodontalis (80.7%), P. melaninogenica (73.1%) and P. intermedia (61.5%) were the most predominant in samples of cattle with periodontitis. Regarding non-injured gingival sulcus of cattle, P. endodontalis (40%) and P. loeschei (40%) prevailed. Porphyromonas gingivalis, P. gulae and Prevotella tannerae were not detected in the 51 samples studied. Data evaluation by T test, enabled to verify that ocorrence of Porphyromonas asaccharolytica (p=0.000003), P. endodontalis (p=0.0023), Prevotella buccae (p=0.0017), P. intermedia (p=0.0020), P. melaninogenica (p=0.00006) and P. oralis (p=0.0028) is correlated with bovine periodontitis.

Equine infectious anemia on Marajo Island at the mouth of the Amazon river

Abstract: Equine infectious anemia (EIA) is a transmissible and incurable disease caused by a lentivirus, the equine infectious anemia virus (EIAV). There are no reports in the literature of this infection in Equidae on Marajo Island. The objective of this study was to diagnose the disease in the municipalities of Cachoeira do Arari, Salvaterra, Santa Cruz do Arari and Soure, on Marajó Island, state of Pará, Brazil. For serological survey samples were collected from 294 horses, over 5-month-old, males and females of puruca and marajoara breeds and from some half-breeds, which were tested by immunodiffusion in Agar gel (AGID). A prevalence of 46.26% (136/294) positive cases was found. EIA is considered endemic in the municipalities studied, due to the ecology of the region with a high numbered population of bloodsucking insect vectors and the absence of official measures for the control of the disease.