Auger-type granular fertylizer distributor: matemathical model and dynamic simulation

The objective of this study was to model mathematically and to simulate the dynamic behavior of an auger-type fertilizer applicator (AFA) in order to use the variable-rate application (VRA) and reduce the coefficient of variation (CV) of the application, proposing an angular speed controller θ' for the motor drive shaft. The input model was θ' and the response was the fertilizer mass flow, due to the construction, density of fertilizer, fill factor and the end position of the auger. The model was used to simulate a control system in open loop, with an electric drive for AFA using an armature voltage (V A) controller. By introducing a sinusoidal excitation signal in V A with amplitude and delay phase optimized and varying θ' during an operation cycle, it is obtained a reduction of 29.8% in the CV (constant V A) to 11.4%. The development of the mathematical model was a first step towards the introduction of electric drive systems and closed loop control for the implementation of AFA with low CV in VRA.

New Type Nuclear Reactors (NETNUC) 2008-2011 Final Report

This report summarizes the work done by a consortium consisting of Lappeenranta University of Technology, Aalto University and VTT Technical Research Centre of Finland in the New Type Nuclear Reactors (NETNUC) project during 2008–2011. The project was part of the Sustainable Energy (SusEn) research programme of the Academy of Finland. A wide range of generation IV nuclear technologies were studied during the project and the research consisted of multiple tasks. This report contains short articles summarizing the results of the individual tasks. In addition, the publications produced and the persons involved in the project are listed in the appendices.

A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Histopathological aspects of Bovine Enzootic Hematuria in Brazil

The bladder lesions of 59 cattle, from the States of Rio de Janeiro, São Paulo, Minas Gerais, Espírito Santo, Rio Grande do Sul, Santa Catarina, Paraná and Amazon, affected by Bovine Enzootic Haematuria (BEH), were studied histologically. The objective of this study was to describe and reclassify neoplastic and non-neoplastic alterations not yet reported, according to the more complete current nomenclature used in human medicine. There was an almost complete identity with alterations observed in the bladder of man. Due to the occurrence of two or more neoplasms in the same animal, differences in the methodology and in the concept of classification, a more precise comparison was not possible. Coexistence of different types of epithelial and/or mesenchymal tumour growth was frequently seen. Rare neoplasms or differentiations not previously described were found in the bladder of some animals affected by BEH. These were trabecular carcinoma with Paneth cells differentiation, mesonephroid adenoma, mesonephroid adenocarcinoma, "signet ring" cell carcinoma, plasmocytoid carcinoma, chromophobe cell carcinoma and nested type of transitional cell carcinoma. Haemangiosarcomas originating from haemangiomas were also observed. This study also revealed the occurrence of many tumors with anaplasia and pronounced infiltrative features, but which did not metastasize. The elucidation of the cause of this "barrier against metastases" and its relationship with chemical carcinogenesis induced by the ptaquiloside, the active principle of bracken fern (Pteridium aquilinum), could be of interest to future research on the control ofneoplasia in man and animals.

Prevalence and geographical distribution of bovine eurytrematosis in cattle slaughtered in northern Paraná, Brazil

A retrospective study of cattle slaughtered in northern Paraná during 2000 was performed to determine the prevalence and geographical distribution of bovine eurytrematosis (BE), as identified by the Federal Inspection Service (SIF). The cattle was from different regions of the State of Paraná; all regions had cattle parasitized by Eurytrema spp. BE was identified in 12.1% (12,534/103,411) of the total number of cattle inspected. Prevalence of animals parasitized by Eurytrema spp varied from 8.3% (Region G, São João do Caiuá, 1,069/12,914) to 40.5% (Region R, Ponta Grossa, 225/555). BE was more prevalent during the month of March (1.6) and markedly reduced during May (-2). A possible seasonal predominance of BE was identified: comparatively fewer cases occurred from April to August, while there was a peak from December to March. The study indicates that bovine eurytrematosis is hypoendemic and occurs in almost all geographical regions of the State of Paraná. The prevalence within this State is variable and may be directly related to factors of the biological cycle of the trematode, particularities of each region, and environmental conditions.

Immunoprophylactic strategies against enterotoxemia caused by Clostridium perfringens type D in goats

The serological response to an experimental vaccine against Clostridium perfringens type D enterotoxemia was evaluated in four groups of goats. Group 1 received colostrum from unvaccinated cows and no vaccine. Groups 2, 3 and 4 received colostrum from vaccinated cows. In addition, Groups 3 and 4 received a vaccine dose at 80 days of age, and Group 4 received a second vaccine dose at 120 days of age. Serum antibody levels were determined by ELISA in cows before and after calving, and in goats at 3, 80, 120 and 160 days of age. No significant difference in serum antibody levels was observed between vaccinated and unvaccinated cows, or between the four groups of goats evaluated at 3 days of life. Groups 3 and 4 presented mean antibody titers of 0.6 and 1.1 IU/ml, respectively, 40 days after first vaccination. The vaccine response of Group 4 was 1.8 IU/ml 40 days after the booster dose and was higher than that observed for Group 3 (0.2 IU/ml). Thus, in the proposed regimen the use of heterologous colostrum did not induce passive immunization in goat kids. However, first vaccination and a booster dose after 40 days triggered satisfactory antibody levels.

Monitoring bovine viral diarrhea virus (BVDV) infection status in dairy herds

This study was designed to assess the relationship between antibodies against bovine viral diarrhea virus (BVDV) determined in the bulk tank milk (BTM) and the within-herd seroprevalence. We also assessed the efficiency of measuring antibody levels in BTM samples to monitor BVDV infection status in a herd. In the 81 farms included in the study, BTM samples were obtained and blood samples withdrawn from all cattle older than one year. The infection status was then determined in serum and milk using a commercial blocking ELISA based on the detection of anti-p80 antibodies. Apart from these baseline serum and milk samples, another BTM sample was collected from each herd 9 months later, and a third BTM sample obtained 9 months after this. In these second and third milk samples, anti-BVDV antibodies were determined using the same ELISA kit. Statistical tests revealed good agreement between herd seroprevalences (% seropositive animals in the herd) and the antibody levels detected in the BTM samples. During the 18 months of follow-up, the farms with persistently infected cattle at the study outset (14.8% of the herds) showed a significant decrease in BTM antibody titers after virus clearance. Conversely, a significant increase in BTM antibody levels was observed in the herds infected with BVDV during the follow-up period. Our findings indicate that monitoring antibody levels in the BTM is a useful method of identifying changes in the BVDV infection status of a herd.

Analysis of Escherichia coli isolated from bovine mastitic milk

Mastitis has been recognized for some time as the most costly disease in dairy herds. From February to November 2004, 670 samples of bovine mastitic milk from which 231 Escherichia coli strains were isolated, were collected from two Brazilian states. The strains were screened for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twenty (8.6%) strains were detected by PCR to harbor the Shiga toxin genes (8 the stx 1 gene, 12 the stx 2 gene and none both of them). Two (0.8%) of the Escherichia coli strains studied were eae positive non Shiga toxin-producing. The strains were also examined for resistance to 12 antimicrobial agents. The predominantly observed resistance was to tetracycline (92.2%), streptomycin (90.4%), nalidixic acid (88.3%), amikacin (86.5%) and cephalothin (84.8%). Multidrug resistance was found among 152 isolates (65.8%).

Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5

The serum neutralization (SN) test is the gold standard method to measure neutralizing antibodies to bovine herpesviruses. However, in view of the further subdivisions of bovine herpesviruses in types/subtypes, defining which virus to use at challenge in SN tests may be difficult. In view of that, this study was carried out to re-evaluate (SN) sensitivity with different types/subtypes of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) as challenge viruses. Bovine sera (n=810) were collected from two distinct geographic regions and tested by SN with three type 1 viruses (BoHV-1.1 strains "Los Angeles" and "EVI123/98"; BoHV-1.2a strain "SV265/96") and three type 5 viruses (BoHV-5a strain "EVI88/95"; BoHV-5b strain "A663" and BoHV-5c "ISO97/95"). SN tests were performed with a 1 hour incubation of the serum-virus mixtures at 37ºC against 100 TCID50 of each of the viruses. SN sensitivity varied greatly depending on the challenge virus used in the test. The highest sensitivity (327 positive/810 total sera tested; 40.37%) was attained when the positive results to the six viruses were added together. No association could be found between any particular type or subtype of virus and the sensitivity of the test. When positive results to each single strain were considered, SN sensitivity varied from 41.7% to 81.7%, depending on the virus and the geographic region of origin of the sera. Variation was detected even when challenge viruses belonged to the same subtype, where disagreement between positive results reached 41%. These results indicate that one hour incubation SN tests against single viruses, as performed here, may display a significantly low sensitivity (p=0.05); performing SN tests against a number of different viruses may increase considerably SN sensitivity. Furthermore, the choice of virus used for challenge is critical in SN tests. In addition, sera from different geographic regions may give rise to disagreeing results with different strains of BoHV-1 and BoHV-5. This might be particularly relevant for control programs and in international trade, were maximum sensitivity should be targeted.

Effect of medroxy-progesterone acetate on follicular growth and endometrial cycloxygenase-2 (COX-2) expression during the bovine estrous cycle

The objective of this study was to evaluate the effect of medroxy-progesterone acetate (MAP) with or without estradiol benzoate (EB) on follicular growth during the estrous cycle in cattle. In the first experiment, Hereford cows were synchronized with a synthetic analogue of PGF2 alpha and were treated with two different doses of MAP (250 or 500 mg) with or without EB for 7 days starting on day 8 of the estrous cycle. Follicular growth was inhibited (P<0.05) in all cows except controls and those receiving 250mg MAP without EB. Seventy-five percent of the animals (15/20) showed estrus on days 21 and 22 of the cycle rather than at MAP withdrawal, demonstrating that these treatments did not induce estrus. To determine whether the EB treatment altered endometrial sensitivity to oxytocin and thus the luteolytic cascade, multiparous pre-synchronized cows received 5 mg of EB followed 6 hours later with 50 IU of oxytocin (OT; n=9). Eight hours after EB injection, endometrial fragments were collected from the cows on days 4, 13 and 17 of the estrous cycle and COX-2 gene expression was measured by PCR. EB increased COX-2 mRNA levels only on day 17 of the estrous cycle (P<0.05). In conclusion, MAP alone or associated with EB is able to suppress bovine follicular growth. However, EB in the presence of MAP is not efficient to induce luteolysis in cows when injected on day 8 of the estrous cycle.

Polymerase chain reaction for the diagnosis of bovine genital campylobacteriosis

Bovine genital campylobacteriosis is a common venereal disease of cattle; the prevalence of this disease can be underestimated mostly because of the nature of the etiological agent, the microaerobic Campylobacter fetus subspecies venerealis. The purpose of the current study was to evaluate the utilization of polymerase chain reaction (PCR) in the diagnosis of genital campylobacteriosis in samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents of aborted fetuses, collected into enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lysis with proteinase K, lysis with guanidine isothiocyanate, lysis with DNAzol, and lysis with hexadecyltrimethylammonium bromide (CTAB). The specificity, sensitivity, and technical application of the PCR assay were also evaluated with clinical samples and compared to bacterial isolation by standard culture. DNA extraction by the CTAB protocol provided better results in PCR, and it was able to detect 63 colony-forming units per ml of C. fetus. Out of 277 clinical samples tested, 68 (24%) were positive for Campylobacter fetus using PCR, while only 8 (2.8%) of the samples were positive by bacterial isolation in solid medium, proving the superiority of the PCR technique when compared to the standard isolation method, and providing evidence for its usefulness as a better screening test in cattle for the diagnosis of bovine genital campylobacteriosis.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 10(7.5)TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (10(7.5)TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 10(7.3)TCID50). All vaccinated animals developed VN titers from 2 to 16 at day 30 pv. A boost vaccination performed at day 240 pv resulted in a rapid and strong anamnestic antibody response, with VN titers reaching from 16 to 256 at day 14 post-booster. Again, serum samples remained negative for gE antibodies. Selected serum samples from vaccinated animals showed a broad VN activity against nine BoHV-5 and eight BoHV-1 field isolates. These results show that the recombinant virus is attenuated, immunogenic for calves and induces an antibody response differentiable from that induced by natural infection. Thus, the recombinant BoHV-5gE/TKΔ is an adequate candidate strain for a modified live vaccine.

A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection

Mutant viral strains deleted in non-essential genes represent useful tools to study the function of specific gene products in the biology of the virus. We herein describe an investigation on the phenotype of a bovine herpesvirus 5 (BoHV-5) recombinant deleted in the gene encoding the enzyme thymidine kinase (TK) in rabbits, with special emphasis to neuroinvasiveness and the ability to establish and reactivate latent infection. Rabbits inoculated with the parental virus (SV-507/99) (n=18) at a low titer (10(5.5)TCID50) shed virus in nasal secretions in titers up to 10(4.5)TCID50 for up to 12 days (average: 9.8 days [5-12]) and 5/ 16 developed neurological disease and were euthanized in extremis. Rabbits inoculated with the recombinant BoHV-5TKΔ at a high dose (10(7.1)TCID50) also shed virus in nasal secretions, yet to lower titers (maximum: 10(2.3)TCID50) and for a shorter period (average: 6.6 days [2-11]) and remained healthy. PCR examination of brain sections of inoculated rabbits at day 6 post-infection (pi) revealed a widespread distribution of the parental virus, whereas DNA of the recombinant BoHV-5TKΔ-was detected only in the trigeminal ganglia [TG] and olfactory bulbs [OB]. Nevertheless, during latent infection (52pi), DNA of the recombinant virus was detected in the TGs, OBs and also in other areas of the brain, demonstrating the ability of the virus to invade the brain. Dexamethasone (Dx) administration at day 65 pi was followed by virus reactivation and shedding by 5/8 rabbits inoculated with the parental strain (mean duration of 4.2 days [1 - 9]) and by none of seven rabbits inoculated with the recombinant virus. Again, PCR examination at day 30 post-Dx treatment revealed the presence of latent DNA in the TGs, OBs and in other areas of the brain of both groups. Taken together, these results confirm that the recombinant BoHV-5TKΔ is highly attenuated for rabbits. It shows a reduced ability to replicate in the nose but retains the ability to invade the brain and to establish latent infection. Additional studies are underway to determine the biological and molecular mechanisms underlying the inability of BoHV-5TKΔ to reactivate from latency.

Assessing the histopathology to depict the different stages of bovine tuberculosis infection in a naturally infected herd

The standard method for detection of bovine tuberculosis (TB) is the single intradermal tuberculin test (SITT). Nevertheless, current studies suggest that a single test is not enough to detect all cattle infected by TB, particularly when animals present different stages of infection. A dairy herd comprised of 270 cows was studied and 15 were reactive to SITT plus nine inconclusive animals. Blood samples (for IFN and ELISA) were collected from these 24 cows. At 30 days after injection of PPD, all the cows that were reactive to any of the employed tests were slaughtered, and tissues were processed by Bacteriology, Histopathology (HP) and PCR. According to HP 33.4% of the animals were positive, 45.8% inconclusive and 20.8% were negative. The inconclusive samples came from IFN positive animals, signalizing recent infection. Regarding the animals that were negative to HP, all of them were identified by IFN while ELISA was negative. Immune responses are different in recent and advanced infections, what supports the identification between chronically or recently infected animals. This multidisciplinary approach is mandatory for the interpretation of the various tools that are frequently employed for the diagnosis of TB and mainly to identify all infected animals.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.