Bovine Blood Samples Research Articles

Edoxaban pharmacokinetics during in vitro continuous renal replacement therapy

BackgroundTo evaluate the clearance of edoxaban during modeled in vitro continuous renal replacement therapy (CRRT), assess protein binding and circuit adsorption, and provide initial dosing recommendations.MethodsEdoxaban was added to the CRRT circuit and serial pre-filter bovine blood samples were collected along with post-filter blood and effluent samples. All experiments were performed in duplicate using continuous veno-venous hemofiltration (CVVH) and hemodialysis (CVVHD) modes, with varying filter types, flow rates, and point of CVVH replacement fluid dilution. Concentrations of edoxaban and urea were quantified via liquid chromatography-tandem mass spectrometry. Plasma pharmacokinetic parameters for edoxaban were estimated via noncompartmental analysis. Two and three-way analysis of variance (ANOVA) models were built to assess the effects of mode, filter type, flow rate, and point of dilution on CLCRRT. Linear regression was utilized to provide dosing estimations across CRRT effluent flow rates from 0.5 to 5 L/h. Optimal edoxaban doses were suggested using CLCRRT and population non-renal clearance (CLNR) to estimate total clearance and match the systemic AUC associated with efficacy in the treatment of venous thromboembolism.ResultsEdoxaban clearance from the CRRT circuit occurred primarily via hemofilter adsorption to the HF1400 and M150 filters at 74% and 65%, respectively, while mean percent protein binding was 41%. Multivariate analyses confirmed the lack of influence of CRRT mode, filter type, and point of dilution on the CLCRRT of edoxaban allowing dosing recommendations to be made based on effluent flow rate. Edoxaban doses of 30-45 mg once daily were estimated to achieve target the AUC threshold for flow rates from 0.5 to 5 L/h.ConclusionFor CRRT flow rates most employed in clinical practice, an edoxaban dose of 45 mg once daily is predicted to achieve target systemic exposure thresholds for venous thromboembolism treatment. The safety and efficacy of this proposed dosing warrants further investigation in clinical studies.

Designing an experimental method for assessing biocompatibility of circuit coatings using biomarkers for platelet activation during cardiopulmonary bypass.

Cardiopulmonary bypass is an essential component of cardiothoracic surgeries. However, significant complications such as systemic inflammatory response syndrome (SIRS) resulting from cardiopulmonary bypass (CPB) are a common occurrence due to contact between circulating blood and foreign surfaces that leads to platelet activation. It is suggested that different available CPB circuit coatings can potentially reduce platelet activation. However, there have been no published evidence-based reports confirming these claims. In addition, there is no well-established protocol for studying platelet activation biomarkers during CPB in vitro in a laboratory setting. CPB was simulated in the laboratory using bovine blood in two different types of coated CPB circuits: Trillium® Biosurface by Medtronic, and XcoatingTM Surface by Terumo. Fresh bovine blood samples were collected and circulated through the CPB circuit following the standard protocol used in the operation rooms. Blood samples were then collected at 5min, 30min, and 55min during the circulation. Blood plasmas were separated and subjected to enzyme-linked immunosorbent assay to measure most established platelet activation markers P-selectin, Platelet Factor 4 (PF4), Glycoprotein IIb/IIIa (GPIIb/IIIa), and β-thromboglobulin (β-TG) at different time points. The biomarker values at 30min and 55min were compared to the base values at 5min for each type of CPB circuit. The results of the means from all measured biomarkers showed data measurements that indicated no significant variability within each coating. All collected data points fell within±2 SD of the means, which was considered acceptable variations across technical replicates. Conclusion: In this study, we were able to establish an in vitro protocol in the laboratory setting that is precise and reliable with minimum intra-variability. This established protocol will allow for future studies in which different coated CPB circuits can be compared for their effectiveness in blocking platelet activation during the CPB.

Screening of mRNA markers in early bovine tuberculosis blood samples.

Bovine tuberculosis (bTB) is a chronic zoonotic disease caused by Mycobacterium bovis. A large number of cattle are infected with bTB every year, resulting in huge economic losses. How to control bTB is an important issue in the current global livestock economy. In this study, the original transcriptome sequences related to this study were obtained from the dataset GSE192537 by searching the Gene Expression Omnibus (GEO) database. Our differential gene analysis showed that there were obvious biological activities related to immune activation and immune regulation in the early stage of bTB. Immune-related biological processes were more active in the early stage of bTB than in the late. There were obvious immune activation and immune cell recruitment in the early stage of bTB. Regulations in immune receptors are associated with pathophysiological processes of the early stage of bTB. A gene module consisting of 236 genes significantly related to the early stage of bTB was obtained by weighted gene co-expression network analysis, and 18 hub genes were further identified as potential biomarkers or therapeutic targets. Finally, by random forest algorithm and logistic regression modeling, FCRL1 was identified as a representative mRNA marker in early bTB blood. FCRL1 has the potential to be a diagnostic biomarker in early bTB.

Diagnostic advancements: Isolating Mycobacterium avium ssp. paratuberculosis and unveiling its molecular identity with nested-PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis, which is currently prevalent in many parts of Iran and produces severe economic loss. It is hence necessary to identify and isolate the animals infected with this bacterium, so this research aimed to isolate MAP from milk and fecal samples of ELISA-positive animals and determine the molecular identity of isolates. After performing ELISA on 3,700 bovine blood samples, 115 samples of milk and feces were taken from ELISA-positive cattle and were cultured on Herald's egg yolk medium with and without mycobactin-J and then the acid-fastness of positive samples was determined using Ziehl-Neelsen staining. The 16S rRNA-PCR test was performed after DNA extraction to determine the molecular identity of isolates. Primers IS6110 and IS901 were employed to ensure that the isolates were not related to members of M. tuberculosis complex and M. avium, respectively. Primer IS900 was also used to determine the molecular identity of MAP isolates. Also, expression levels of MAP-related genes (IS900, ISMAP02, F57, MAP2191, MAP4027) were evaluated via qPCR. Finally, positive samples were confirmed based on the Nested-PCR. Results showed that a total of 9 isolates were obtained from the culture of 90 ELISA-positive samples. The results revealed that all grown samples were positive for acid-fastness. The 16S rRNA-PCR test revealed the 543 bp band, which confirms the presence of Mycobacterium in the samples. The PCR test with Primer IS900 generated the 398 bp fragment in the first step and the 298 bp fragment in the second step, indicating the presence of MAP in samples. Also, relative expression analysis revealed that MAP-related genes were significantly higher in ELIZA-positive samples than in negative ones. Based on the study findings, it can be concluded that MAP-infected animals can be identified by ELISA. In addition, mycobacterium can be isolated by culturing the samples on appropriate media and then its molecular identity can be determined by using nested-PCR.

The First Report on the Complete Sequence Characterization of Bluetongue Virus Serotype 3 in the Republic of Korea.

The bluetongue virus (BTV) is a significant animal pathogen with economic implications in the ruminant industry. Despite global reports on BTV detection and epidemiologic investigations, limited studies have focused on the virus in the ROK. In this study, BTV epidemiological research was conducted on blood samples from cattle and goat farms across nine regions during 2013-2014. The results showed that 3.33% of bovine blood samples (194/5824) and 0.19% of goat blood samples (2/1075) tested positive for BTV antibodies using ELISA. In Jeju-do, BTV RNA amplification occurred in 51 of 422 samples (12.1%) using real-time reverse transcription (RT-qPCR). The isolation of one sample revealed it as serotype 3, as indicated by the sequence of segments 2 (Seg-2) and 6 (Seg-6), associated with the eastern BTV topotype. However, based on Seg-1, -3, -4, -5, -7, -8, -9, and -10 analyses, the BTV-3/JJBB35 strain is more closely related to distinct BTV strains. These findings imply BTV circulation and that the Korean-isolated BTV might originate from Asian BTV strains due to multiple reassortment events. This study provides foundational data for ongoing BTV monitoring and disease-control policies in the ROK.

Trypanosome Infection in Cattle and Associated Vectors in Etang District of Gambella, Ethiopia.

Bovine trypanosomosis produces significant economic losses due to anemia, loss of body condition, and emaciation. The disease is transmitted biologically by tsetse flies and mechanically by biting flies such as Tabanus and Stomoxys. Therefore, this research is aimed at estimating the prevalence of bovine trypanosomosis and the apparent density of its vectors in the Etang Special District. A cross-sectional study was employed from November 2016 to April 2017 for the prevalence and entomological survey. Trypanosoma species were identified using buffy coat and Giemsa staining techniques. Besides, the entomological surveys were conducted using NGU, pyramidal, biconical, and monoconical traps. The vectors were identified to their genus level based on their morphological features like size, color, wing venation, and proboscis. A total of 457 bovine blood samples were collected and tested, of these 16 (3.50%) animals were positive for trypanosomosis. Similarly, 13 T. vivax (81.25%) and 3 T. congolense (18.75%) were the trypanosome species detected. The prevalence in the young (2.56%) and adult (3.99%) age groups was not statistically significant (P > 0.05). However, there were significant (P < 0.05) differences in prevalence between body condition scores, where poor is 6.31%, medium is 1.30%, and good is 0%. Moreover, the difference in mean PCV values between the parasitemic (20.97) and aparasitemic (28.58) groups was statistically significant (P < 0.05). Glossina flies were not found, although 1756 Tabanus and 52 Stomoxys biting flies were gathered. Accordingly, the overall apparent density of Glossina flies was zero (0), with biting flies (20.54) recorded per trap per day. Moreover, the apparent density of Tabanus and 52 Stomoxys was 39.01 and 1.18, respectively. This study confirmed that trypanosomosis and abundant mechanical vectors continue to be problems in the study area, resulting in cattle productivity losses. As a result, strategic management and prevention methods for trypanosomosis and associated vectors should be prioritized. Further investigation of vector needs to be conducted to clear out tsetse presence.

The Characteristic Function of Blood-Derived Exosomes and Exosomal circRNAs Isolated from Dairy Cattle during the Dry Period and Mid-Lactation.

Exosomes are key mediators of intercellular communication. They are secreted by most cells and contain a cargo of protein-coding genes, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), which modulate recipient cell behavior. Herein, we collected blood samples from Holstein cows at days 30 (mid-lactation) and 250 (dry period) of pregnancy. Prolactin, follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone levels showed an obvious increase during D250. We then extracted exosomes from bovine blood samples and found that their sizes generally ranged from 100 to 200 nm. Further, Western blotting validated that they contained CD9, CD63, and TSG101, but not calnexin. Blood-derived exosomes significantly promoted the proliferation of mammary epithelial cells, particularly from D250. This change was accompanied by increased expression levels of proliferation marker proteins PCNA, cyclin D, and cyclin E, as detected by EdU assay, cell counting kit-8 assay, and flow cytometric cell cycle analysis. Moreover, we treated mammary epithelial cells with blood-derived exosomes that were isolated from the D30 and D250 periods. And RNA-seq of two groups of cells led to the identification of 839 differentially expressed genes that were significantly enriched in KEGG signaling pathways associated with apoptosis, cell cycle and proliferation. In bovine blood-derived exosomes, we found 12,747 protein-coding genes, 31,181 lncRNAs, 9374 transcripts of uncertain coding potential (TUCP) candidates, and 460 circRNAs, and 32 protein-coding genes, 806 lncRNAs, 515 TUCP candidates, and 45 circRNAs that were differentially expressed between the D30 and D250 groups. We selected six highly expressed and four differentially expressed circRNAs to verify their head-to-tail splicing using PCR and Sanger sequencing. To summarize, our findings improve our understanding of the key roles of blood-derived exosomes and the characterization of exosomal circRNAs in mammary gland development.

Molecular identification of Trypanosoma theileri (Laveran, 1902) in cattle from two slaughterhouses in Ecuador and its relation with other haemotropic agents.

Trypanosoma theileri is a worldwide distributed haemoparasite that has been reported throughout the American continent in various species, including bovines, buffaloes and bats. In bovines, high incidence of T. theileri can be harmful when associated with other infections or under stress situations. There is little information on this hemoflagellate in Ecuador, which prompted the study and molecular identification of the trypanosomes collected in two slaughtering centers. Between February and April 2021, a total of 218 samples of bovine blood were collected in abattoirs located in the Andean region of Quito (n = 83) and in the coastal region, in Santo Domingo (n = 135). Quito public Slaughterhouse is the biggest in Ecuador, and for that, they receive animals from all country; on the other hand, Santo Domingo's Slaughterhouse is a small one where mainly females from the region are sacrificed and some males. The samples were evaluated using two molecular tests, the PCR cathepsin L-like (CatL) specific for T. theileri and for the positive samples, a Nested PCR that targets the ITS of the 18S gene. The corresponding PCR products were sequenced, analyzed by BLAST/NCBI and the sequences were used to build a concatenated phylogenetic tree, using the MEGA XI software. Overall, 34 out of the 218 samples, (15.6%) were positive to T. theileri by PCR CatL, resulting from 20/83 (24.1%) positives from the Quito abattoir and 14/135 (10.4%) from the Santo Domingo slaughterhouse. These prevalence rates were found to be significantly different (p = 0.006). According to the phylogenetic tree based on the CatL and ITS concatenated sequences (n = 13), the two novel Equatorial T. theileri isolates, ThI (n = 7) and ThII (n = 6) are closely related and associated to the IC, IB and IIB genotypes, present in Brazil, Venezuela and Colombia. Thirty-one out of the thirty-four T. theileri-positive bovines were co-infected with other haemotropic pathogens, Anaplasma marginale Babesia spp and T. vivax. This coinfection could be responsible for additional pathologies and harmful effects on the affected cattle. This study presents the molecular identification and genotypification of T. theileri isolated from cattle in Ecuador through the analysis of CAtL and ITS sequences, and the high frequency of coinfection of this hemoflagellate with other blood haemotropic organisms.

Construction and comparison of Prussian blue analogs nanomaterials with tailorable coordination metal ions as novel adsorbents for efficient structure-selective protein adsorption

Efficient and selective purify histidine (His)-rich proteins from complex biological sample for disease initiation and progression diagnostic is crucial, but it is still a challenge. Herein, for the first time, nanomaterials composed of Prussian blue analogues (PBAs) with tailorable coordination metal ions were synthesized and operated as adsorbents for selective separation of His-rich proteins. Benefiting from the strong metal affinity forces between the His residual in protein and the immobilized Cu2+, Ni2+ and Co2+ in CuFe, NiFe and CoFe PBAs, the obtained adsorbents exhibit highly selective adsorption performance for structure-selective proteins toward bovine hemoglobin (BHb). As a results, the maximum adsorption capacities of CuFe, NiFe and CoFe PBAs for BHb are 3365.8 mg g−1, 2421.3 mg g−1 and 1791.2 mg g−1, respectively. Moreover, the high-performance CuFe, NiFe and CoFe PBAs can be easily regenerated and used to effectively separation of BHb from real bovine blood samples, permitting their excellent practicality in the complex biological sample pretreatment, and further expanding the application scope of the traditional classical coordination compound in biochemical analysis fields.

Blood from Horses and Cows In Vitro Exposed to Quaternium-15 and Thiacloprid: Haematology and Erythrocyte Osmotic Fragility Alterations

Blood cells are constantly exposed to several pollutants, including xenobiotics, and they can be considered a useful target for pollution exposition of the animal. The aim of this study was to investigate the effects of two xenobiotics (Quaternium-15, a preservative used in personal care products, and Thiacloprid, a neonicotinoid pesticide) on the haematological profile and erythrocyte osmotic fragility (EOF) of equine and bovine blood samples. Ten blood samples from horses and cows were exposed for 24 h to Quaternium-15 at a concentration of 0.1 and 1 mg/L and to Thiacloprid at a concentration of 30 and 60 µg/mL. A decrease in the values of the red blood cells, white blood cells, haematocrit, haemoglobin, mean corpuscular volume, and platelets, and an increase of EOF were found in blood samples exposed to xenobiotics compared to the control. According to the results gathered in the current study, the two xenobiotic compounds herein tested negatively affect the haematological indices causing haemolysis both in cattle and horse blood. This study, despite being preliminary, emphasizes the concept that blood cells are an excellent target for evaluating the effects of xenobiotics.

Diagnosis and phylogenetic analysis of bovine leukemia virus in dairy cattle in northeastern Brazil.

Enzootic bovine leukosis (EBL) is a chronic viral disease of wide distribution in cattle herds and may take several years for the first manifestation of clinical signs. Most animals do not present clinical signs. However, the economic losses are underestimated due to this disease. Thus, this work aimed to detect and characterize BLV in dairy cattle in the Maranhão state, northeastern Brazil. Blood samples were collected from 176 animals from 8 municipalities in the southeastern state of Maranhão. Bovine blood samples were subjected to DNA extraction and molecular diagnosis using nested PCR assays for BLV, targeting gp51 gene. Positive samples were then sequenced and then subjected to phylogenetic inferences. BLV DNA was detected in 16 cattle (16/176, 9.09%) in 4 municipalities. Phylogenetic analyzes showed that the sequence obtained clustered in a clade containing BLV sequences classified as genotype 6, with a high degree of support. Our data shows BLV occurrence in the Northeast of Brazil and the identification of genotype 6 in this region. These findings contribute to the molecular epidemiology of this agent in Brazil.

Molecular identification of Trypanosoma theileri in cattle from the Ecuadorian Amazon.

Trypanosoma theileri is a cosmopolitan opportunistic haemoparasite described in wild and domestic ruminants, and also in arthropod vectors. The presence of this parasite has been reported in several South American countries, including Amazonian regions. Despite the importance of livestock production, Ecuador possesses scarce studies about trypanosomosis and no T. theileri reports in its territory. Here, we showed molecular evidences of the presence of T. theileri in cattle from a province located in the Ecuadorian Amazon. Bovine blood samples were collected from 2014 to 2019, during campaigns to detect haemoparasites in the Ecuadorian provinces of Orellana and Sucumbíos. DNA was extracted from the buffy coat and used in PCR assays with three different molecular markers, ITS1, 18S and Cathepsin L-like. T. theileri was detected only in the Sucumbíos province, with a specific molecular prevalence of 8.6% (3/35) using the three primers and an additional animal detected as positive (11.4% prevalence) only by the ITS1 marker. DNA sequences derived from the generated amplicons were subjected to phylogenetics maximum parsimony and maximum likelihood analysis, which indicate the presence of TthI and TthII genotypes circulating in the evaluated animals. Molecular surveillance should be continually implemented in Ecuador in order to deepen the epidemiological and evolutionary knowledge about T. theileri as well other haemoparasites in the amazon parts of the country.

Screen printing and laser-induced flexible sensors for the simultaneous sensitive detection of uric acid, tyrosine, and ascorbic acid in sweat.

A wearable sweat sensor, which could continuously monitor biomolecules related to the human physiological state, is emerging as a promising piece of health surveillance equipment. However, current sensors cannot simultaneously achieve a detection performance that equates to that of traditional sensors and satisfactory mechanical strength. Herein, a wearable sweat sensor with excellent detection performance and mechanical stability is designed and fabricated. Based on the integration of laser-induced graphene electrodes and a screen printing technique, this wearable sweat sensor could realize both the separate and simultaneous detection of uric acid (UA), tyrosine (Tyr), and ascorbic acid (AA) with high sensitivity. Good UA sensing performance in artificial sweat could be maintained even after 20 000 bends. In addition, the sensor can operate well in the wearing state or in a complex bovine whole blood sample. For the detection of human sweat, the changes in UA concentration after a purine-rich meal are continuously monitored and the results are in accordance with the corresponding serum UA detection results tested with a commercial serum UA meter. These results suggest its application potential in health monitoring for both gout patients and healthy humans.

Identifying Hypocalcemia in Dairy Cattle by Combining 3D Printing and Paper Diagnostics

This paper describes the design, fabrication, and validation of a paper-based diagnostic device for the rapid diagnosis of hypocalcemia in dairy cattle at the point-of-care (POC). The device incorporates a 3D printed calcium ion-selective membrane (ISM) as the sensing element for free—unbound—calcium in real bovine whole blood samples. With a linear response range of 100 mM to 97.7 μM, the sensor covers the clinically relevant concentrations of Ca2+ associated with both healthy cattle as well as those suffering from hypocalcemia. The components of the Ca2+ ion-selective electrodes were successfully translated to a paper-based device to provide a sensing platform that is simple to use, disposable, and low-cost, and is therefore well-situated for applications at the POC. The paper-based calcium sensor showed a Nernstian response between 10 mM and 100 μM and required only 12 μl of sample to perform a measurement, which can be accomplished in less than two minutes without the need for time-consuming separation steps. The performance of the paper-based Ca2+ sensor was validated using the commercially available epoc® Blood Analysis System, which provided results within 5% of the data obtained with 3D printed Ca2+-ISM integrated paper-based device.

The Upregulation of Cathepsin G Is Associated with Resistance to Bovine Paratuberculosis.

An in silico genomic-transcriptomic combined approach allowed the identification of a polymorphism (cis-eQTL-rs41976219) in the Bos taurus genome associated with the CTSG mRNA expression in bovine blood samples, which suggests that individual genetic variation might modulate the CTSG transcriptional response. In the current study, a sandwich ELISA is used to measure the CTSG protein levels in supernatants of monocyte-derived macrophages (MDMs) isolated from cows with the AA (n = 5) and AC (n = 11) genotypes for the rs41976219 and infected ex vivo with MAP. Cows with the AC genotype have significantly higher CTSG protein levels (1.85 ng/mL) in the supernatants of enriched CD14+-MDMs after 2 h of infection when compared with infected CD14+-MDMs from cows with the AA genotype (1.68 ng/mL). Statistically significant differences in the intracellular MAP load at 7 d p.i. are observed between animals with the AA (2.16 log CFUs) and AC (1.44 log CFUs) genotypes. Finally, the association between the rs41976219 allelic variants and resistance to PTB is tested in a larger cattle population (n = 943) classified according to the presence (n = 442) or absence (n = 501) of PTB-associated lesions. The presence of the two minor alleles in the rs41976219 (CC) is more frequent among healthy cows than in cows with PTB-associated lesions in gut tissues (2.2% vs. 1.4%, OR = 0.61). In agreement with this, the CTSG levels in plasma samples of cows without lesions in gut tissues and with the CC (n = 8) genotype are significantly higher than in the plasmas of cows with the AA + AC (n = 36) genotypes.

Use of bovine plaque-rich plasma in piglets after the castration process

Platelet-rich plasma (PRP) is a platelet concentrate obtained from whole blood centrifugation, widely used in human and veterinary medicine because it contains growth factors that enhance the tissue repair process. The PRP of autologous origin is the most used in the routine, however, the impossibility of obtaining autologous blood must be considered, proceeding with a PRP of homologous or heterologous origin. To evaluate the efficiency of heterologous PRP in wound healing and possible reactions caused by the application, 24 piglets were submitted to the surgical castration process and divided into 2 groups, with G1 the control group and G2 the group that received topical application at the site of heterologous PRP incision. Bovine blood samples were used to obtain PRP, which were processed following a double centrifugation protocol. The wounds were evaluated for 3 days and showed no significant difference in the onset of infection, swelling, redness and formation and crusting. The presence of pruritus was more significant on the first day of infection, which may be related to heterologous PRP. Thus, it was concluded that the evaluated signs are compatible with the physiological healing of the lesions, with no adverse reactions observed.

Comparative Performance and Evaluation of Two Molecular Assays and Conventional Detection of Theileria annulata in Bovines

Background: Theileria annulata is predominant and of utmost economical importance tick borne pathogen of bovines in the region which is routinely diagnosed based on the microscopic examination of Romanowsky stained thin blood smears. Present study was intended to evaluate and analyze the detection efficacy of a commercial polymerase chain reaction kit based assay in comparison to conventional PCR assay and classical microscopy for detection of T. annulata from blood samples of bovines from Punjab state. Methods: In this comparative study 360 bovine blood samples from various districts of agro-climatic zones in Punjab were first screened for T. annulata by Giemsa-stained thin blood smear (GSTBS) examination. The same panel of blood samples was tested for T. annulata by a commercial PCR kit Bovi-TheiDX Theileria annulata (Genext Genomics) (PCR1) and established conventional PCR assay targeting merozoite piroplasm surface antigen (Tams1) gene of T. annulata (PCR2). Result: Out of 360 samples screened, positivity of T. annulata by GSTBS was found to be 12.5% (45/360) with a sensitivity of 37.20% and specificity of 96.00% when compared with commercial kit (PCR1), the difference was statistically significant (p less than .0001). The detection prevalence by PCR1 29.70% (107/360) and PCR2 assay 33.90% (122/360) showed significant (p less than .0001) difference. The conventional PCR targeting the Tams1 gene (PCR2) was found to be more sensitive (84.6%) (p less than 0.001) than PCR1 (69.7%).

Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Bosnia and Herzegovina

Crimean-Congo haemorrhagic fever virus is a tick-borne zoonotic agent of the genus Orthonairovirus, and the causal agent of Crimean-Congo haemorrhagic fever in humans. The study is aimed at investigating the presence of CCHFV RNA in ticks and cattle in Bosnia and Herzegovina (B&H), as well as the exposure of cattle to CCHFV in this country. Only one pool of three male Hyalomma marginatum ticks tested positive for viral RNA (3/760, 0.39%). No viral RNA was detected in 206 bovine blood samples using real-time reverse-transcriptase PCR (RT-qPCR). The overall seroprevalence of CCHFV in the cattle population was estimated as 14.97% (72/481). The results indicate the presence and the established circulation of CCHFV in B&H. Considering the endemic character of CCHFV in the Balkans as well as our results, the possibility that CCHFV is endemic in the territory of Bosnia and Herzegovina is considered to be very high.

Parasitological and molecular identification of Trypanosoma species circulating in cattle and tsetse flies in Matutuine District, Maputo Province, Mozambique

Tsetse flies (Diptera: Glossinidae) are haematophagous insects of medical and veterinary importance due to their role in the transmission of trypanosomes. Tsetse-transmitted trypanosomosis is one of the major constraints to agriculture and livestock development in sub-Saharan Africa. Thus, the importance of the assessment of the distribution of both disease and vector in areas where cattle are kept, as is the case of Matutuíne district, where the last longitudinal study was conducted in 2000. From March 2009 to December 2012, 2427 bovine blood samples were collected in five administrative areas of Matutine district (Bela Vista, Zitundo, Catuane, Catembe and Machangulo). For each animal, the packed cell volume (PCV) was determined and blood examined for the presence of motile trypanosomes using microscopy. At the same time, tsetse flies were also captured. Additionally, DNA was extracted from both cattle blood and tsetse flies. The samples were further analysed using the 18S PCR–RFLP. Trypanosome infections in bovines were detected in all Matutuíne´s administrative areas. The overall prevalence of trypanosome infections was 15.62% by buffy coat and 47.34% by molecular testing. A total of 1379 tsetse flies (1379 Glossina brevipalpis and 150 G. austeni) were captured. From this, 408 were analysed by 18S PCR for the presence of trypanosomes. The results showed a percentage of infection of 12.74% and 3.19% for G. brevipalpis and G. austeni, respectively. With these results, it is concluded that tsetse-transmitted trypanosomosis is a major threat to cattle in Matutuine and control actions need to be implemented.

Uses of Modified Buffy Coat Method As a Tool for Diagnosis of Bovine Hemoparasites

Modified buffy coat method (BCM) using microhematocrit tubes and acridine orange (AO) staining permitsquick(~10 min/sample) and accurate diagnosis of commonbovine hemoparasites compared to the light microscopy(LM) of Giemsa stained smears.Babesia, Theileria,Anaplasma, Ehrlichia and Trypanosoma parasites were detected. It was easy to see Babesia merozoites, trypomastigotes and epimastigotes of Trypanosoma parasite. Similarly, fluorescent microscopy (FLM), offers a method for detecting low parasitemiathat are undetectable by light microscopy. Under LM, the agreement between the twodiagnostic techniquesusing Cohen’s Kappa index was perfect for Trypanosoma (1.000), substantial for Anaplasma(0.615), moderate for Theileria, and Ehrlichia (0.459, 0.433 respectively). BCM was able to detect all positive cases (100%) of Babesia infections using LM and FLM. Modified buffy coat method usingdark field microscopy (DFM) is recommended as a fast and dependable tool to detect Babesiaand Trypanosomaparasites. BCMusing AO stain can be set up directlywitha low cost using anordinary LM.BCM is a valuable tool for diagnosinghemoparasitesmixed infection. Buffy coatmethodcan be used as an alternative technique for the dry stained smear for quickly screening of bovine blood samples.