Dynamics of Haematobia irritans irritans (Diptera: Muscidae) infestation on Nelore cattle in the Pantanal, Brazil

From June 1993 to May 1995, horn fly counts were conducted twice a month on untreated Nelore cattle raised extensively in the Pantanal. Horn fly population showed a bimodal fluctuation and peaks were observed every year after the beginning (November/December) and at the end (May/June) of the rainy season, which coincided with mid-late spring and mid-late fall, respectively. Horn flies were present on cattle throughout the year in at least 64% of the animals. Mean horn fly numbers on animals did not exceed 85 flies/cow during peaks and were under 35 flies/cow in most of the remaining periods. The highest infestations (population peaks) were short and dropped suddenly within two weeks. Less than 15% of the animals in both herds could be considered as "fly-susceptible" - showing consistently higher infestations, or "fly-resistant" - showing consistently lower infestations.

Cattle Dung Breeding Diptera in Pastures in Southeastern Brazil: Diversity, Abundance and Seasonallity

Diptera that breed in undisturbed cattle droppings in pastures present great diversity and abundance, and several species are of veterinary importance and may cause economic losses. To survey the diversity, abundance and seasonality of Diptera associated to this microhabitat, 83 samples of 10 dung pats each were taken from April 1992 to April 1994 in the vicinity of São Carlos, State of São Paulo, Southeastern Brazil. A total of 46,135 Diptera belonging to 20 families and at least 51 species were found to breed in the pats. The most abundant and diverse families were Sepsidae, Muscidae, Sarcophagidae and Sphaeroceridae. In general, the abundance was higher from October to March, the warm and wet months. The importance of some Diptera, both as horn fly enemies and as cattle dung decaying agents, is discussed.

Effect of the Contents and Form of Rabies Glycoprotein on the Potency of Rabies Vaccination in Cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.

The presence of Fasciola hepatica (Liver-fluke) in humans and cattle from a 4,500 Year old archaeological site in the Saale-Unstrut Valley, Germany

During an excavation of a site of the corded ware culture in the Saale-Unstrut-Valley (ca. 3000 BC) in Germany, a soil sample from the pelvis of a human skeleton was studied under palaeoparasitological aspects. Eggs of the trematode Fasciola hepatica and of the nematode genus Capillaria were found. This is the first case of a direct association of a F. hepatica-infestation to both a prehistoric human skeleton and domesticated animal remains. Sheep and cattle bones were present at the same site and F. hepatica eggs were found in bovine samples. This strongly points toward an existing infection cycle, involving humans as a final host.

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis - 63.7 and 56.4% and B. bigemina - 53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86).

Bovine papillomavirus type 2 detection in the urinary bladder of cattle with chronic enzootic haematuria

The bovine papillomavirus type 2 (BPV-2) involvement in the aetiology of chronic enzootic haematuria associated to bracken fern ingestion has been suggested for a long time. However, a few reports have shown the presence of the BPV-2 in urinary bladder tumors of cattle. The aim of this study was to investigate the presence of the BPV-2 infection in the urinary bladder of cattle with chronic enzootic haematuria in Brazilian cattle herds. Sixty-two urinary bladders were collected from adult cattle in beef herds from the north region of the state of Paraná, Brazil. According to clinical and pathological finds the specimens were distributed in three groups: the group A was constituted by 22 urinary bladders with macroscopic lesions collected at necropsy of cattle with clinical signs of chronic enzootic haematuria; the group B by 30 urinary bladders with macroscopic lesions collected in a slaughterhouse of cows coming from bracken fern-endemic geographical region; and the group C (control) by 10 urinary bladders without macroscopic lesions collected from asymptomatic cattle in a bracken fern-free geographical region. By a semi-nested polymerase chain reaction (PCR) assay, with an internal control, a fragment of the BPV-2 L1 gene with 386 bp length was amplified in 36 (58%) urinary bladder. The rate of BPV-2 positive urinary bladders was 50% (11/22) for group A, 80% (24/30) for group B, and 10% (1/10) for group C (control). The rate of the positive results found in groups A and B that included urinary bladder samples with macroscopic lesions was 67% (35/52) and the detection of the BPV-2 in both groups was significantly higher (P < 0.05) than in the control group. RFLP with Rsa I and Hae III enzymes evaluated the specificity of the BPV-2 amplicons. The PCR internal control that amplified a 626 bp fragment of the ND5 gene of the bovine mitochondrial genome was amplified in all analyzed samples and excluded false-negatives or invalid results in the semi-nested PCR. These results suggest the BPV-2 involvement in the chronic enzootic haematuria aetiology and open the perspective of the development of new strategies for the control of this disease that is the major cause of economical losses in beef herds from many Brazilian geographical regions.

Protein synthesis in jejunum and liver of neonatal calves fed vitamin A and lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus.Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts inR. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen.

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.

Use of microseaweeds (Chlorella pyrenoidosa) as a probiotic in dairy goats feeding

Ten Majorera dairy goats were divided in two groups in order to observe the effects of the Chlorella pyrenoidosaoral administration on the colostrum and milk quality and on the animals’ immune status. Treated animals received 5g/d of seaweed from 40 days before partum to 40 days after partum, and blood, colostrum and milk samples were obtained during the experimental period. No effects of the seaweed addition were observed on blood plasma IgG or Chitotriosidase activity, neither on colostrum/milk IgG, Chitotriosidase activity or fatty acid profile.