Bovine Parainfluenza Virus Research Articles

Isolation of a Divergent Strain of Bovine Parainfluenza Virus Type 3 (BPIV3) Infecting Cattle in China.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important known viral respiratory pathogens of both young and adult cattle. It is also named “heat stress in transport”, causing morbidity and mass death. New variants of BPIV3 have been detected or isolated in China since 2008. Here, we isolate one BPIV3 strain (named BPIV3 BJ) in Madin-Darby bovine kidney (MDBK) cells from nasal samples collected in China. Phylogenetic analysis showed that our isolate is related to BPIV3 of the genotype A. The comparison of BPIV3-BJ and the reference Chinese isolate NM09 showed that these strains are highly divergent. We found many differences in the amino acid composition in the nucleocapsid (NP) protein among these genotype A strains. Since the NP protein has been implicated in immunization studies, our BPIV3 isolate will be useful for the development of immune assays and vaccine studies. The diversity of BPIV3 lineages that we found in China indicated ongoing evolution for immune escape. Our study highlights the importance of genetic surveillance for determining the effect of BPIV3 variability on pathogen evolution and population-scale immunity.

Profiling of local disease-sparing responses to bovine respiratory syncytial virus in intranasally vaccinated and challenged calves

Bovine and human respiratory syncytial viruses (BRSV, HRSV) are primary causes of pneumonia in calves and children respectively, with vaccination offering protection via antibody and cellular immune responses. However, with no vaccines currently licensed for human use, evaluation of local responses to BRSV vaccination may provide insights to aid the design of effective safe HRSV vaccines. Calves received intranasal single component BRSV vaccine or “3-Way” vaccine (BRSV, Bovine Herpes Virus-1 (BHV-1), Bovine Parainfluenza Virus Type-3 (BPIV-3)), and were BRSV-challenged 42 days post-vaccination. All vaccinates exhibited reduced pulmonary lesioning with elevated anti-BRSV serum IgG, and higher nasal anti-BRSV IgA in 3-Way vaccinates. Thirty-nine proteins associated with homeostatic and immune processes were altered in vaccinates, with enhanced 3-Way vaccinate group proteins associated with Th1/Th2 balance and immunoglobulin class switching. Proteins altered in the pharyngeal tonsil of animals euthanized early related to anti-inflammatory responses and lymphoid tissue remodeling. These findings indicate that multivalent vaccines distinctly modulate local immune responses, with clear correlation between the pharyngeal tonsil proteome profile and resulting immune protection and disease-sparing. This suggests that the efficacy of low-antigenic subunit vaccine components for problematic pathogens such as HRSV could be enhanced by use in combination with existing safe live vaccines. SignificanceThis study demonstrates that vaccine valency can alter post-challenge proteome responses within the pharyngeal tonsil, a sentinel site of primary immune responses, with the magnitude of response dependent on antigen formulation. Observed differential responses can be attributed to antigenic material and viral nucleic acid from multivalent formulations providing additional T-cell epitopes and PAMPS. These findings indicate that incorporation of subunits proteins within multivalent formulations containing live virus has the potential to induce/skew a favorable immune response, utilising the natural adjuvanting effects of safe proven live vaccines.

Development of a nanoparticle-assisted PCR assay for detection of bovine respiratory syncytial virus

BackgroundBovine respiratory syncytial virus (BRSV) is a common pathogen causing respiratory disease in cattle and a significant contributor to the bovine respiratory disease (BRD) complex. BRSV is widely distributed around the world, causing severe economic losses. This study we established a new molecular detection method of BRSV pathogen NanoPCR attributed to the combination of nano-particles in traditional PCR (Polymerase chain reaction) technology.ResultsIn this study, the BRSV NanoPCR assay was developed, and its specificity and sensitivity were investigated. The results showed that no cross-reactivity was observed for the NanoPCR assay for related viruses, including the infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza virus type 3 (BPIV3), and the assay was more sensitive than the conventional PCR assay, with a detection limit of 1.43 × 102 copies recombinant plasmids per reaction, compared with 1.43 × 103 copies for conventional PCR analysis. Moreover, thirty-nine clinical bovine samples collected from two provinces in North-Eastern China, 46.15% were determined BRSV positive by our NanoPCR assay, compared with 23.07% for conventional PCR.ConclusionsThis is the first report to demonstrate the application of a NanoPCR assay for the detection of BRSV. The sensitive and specific NanoPCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of BRSV infection.

Enhancement of Immunogenicity and Protective Efficacy against Bovine Respiratory Disease Viruses (BVD, BoHV1, PI3, and BRS) Vaccine.

A highly potent pneumo-4 vaccine of combined inactivated Bovine Viral Diarrhea Virus, Infectious Bovine Rhinotracitis Virus, Parainfluenza-3 Virus and Bovine Respiratory Syncytial Virus could play a valuable role in respiratory disease control program in calves. In this study we determined the influence of virus concentration by polyethylene glycol (PEG) precipitation, and the use of montanide oil & gel as adjuvants on the immune response of calves to the inactivated Pneumo-4 vaccine. Two batches of concentrated viruses’ vaccine were prepared, one adjuvanted with aluminum hydroxide gel and the other adjuvanted with oil. Both batches administered twice to six-month-old seronegative calves through intramuscular (IM) route. Different doses, 2ml, 2.5ml, and 3ml of the concentrated gel adjuvanted & 2.5ml,3ml and 3.5ml of concentrated oil vaccine were tested compared with 5ml for the commercial un-concentrated Pneumo-4 vaccine. The induced BVDV, IBRV, PI-3V and BRSV antibodies were followed up in the sera of vaccinated calves up to12M post vaccination (M.P.V.) using Serum Neutralization Test (SNT). The result revealed that concentration of viruses by PEG improve the quality of antigen used in the vaccine formulation to produce a good quality combined inactivated vaccine. Also, both concentrated adjuvanted vaccines gave a significant effect on all vaccinated animals but the oil adjuvanted vaccine was particularly better and effective than gel adjuvanted where oil adjuvanted vaccine induced early protective immune response and showed prolongation of antibody secretion in comparison to the Aluminum Hydroxide gel.

Detection of influenza D virus in respiratory disease samples from Northern Irish cattle

Influenza D virus (IDV) is a newly described member of the Orthomyxoviridae family, initially identified during a 2011 outbreak of respiratory disease in North American pigs. Cattle were subsequently shown to be the main reservoir of the virus and accumulating evidence suggests a role for IDV in bovine respiratory disease complex. During the winter of 2017/2018, cattle submitted to the Agri-Food and Biosciences Institute for post-mortem with confirmed respiratory disease were tested for the presence of IDV by real-time RT-PCR. Virus isolation was performed in Swine Testes cells and full-genome sequence determined. Of 104 cattle with confirmed respiratory disease, 9 tested positive for IDV (8.7 % prevalence). Virus was detected in both the upper and lower respiratory tract. Lung tissues from IDV positive samples were negative for the presence of bovine herpesvirus 1, bovine respiratory syncytial virus, bovine viral diarrhea virus and parainfluenza virus 3. Of the 9 cattle which tested positive for IDV, 3 tested positive for coronavirus. Histological analysis of lungs from IDV positive samples revealed pathological features including necrosis, neutrophil infiltration of alveolar spaces, fibrosis, congestion, oedema and haemorrhage. Sequenced isolates were shown to cluster with European isolates of the D/swine/Oklahoma/1334/2011 clade. To date, IDV has been detected in North America, Mexico, Japan, China, France, Italy and the Republic of Ireland. This study is the first to identify IDV in UK cattle herds. The presence of IDV in respiratory disease samples supports a role for this virus in bovine respiratory disease complex.

Comparative Evaluation of the Immune Responses in Cattle Mammary Tissues Naturally Infected with Bovine Parainfluenza Virus Type 3 and Bovine Alphaherpesvirus-1.

Bovine parainfluenza virus type 3 (BPIV-3) and Bovine alphaherpesvirus-1 (BoHV-1) lead to severe diseases in domesticated animals, such as Bovine, sheep, and goats. One of these diseases is mastitis, whose signs may not be observable in cases of viral infection due to the dominance of other clinical symptoms. This may lead to failure to predict viral agents in subclinical Bovine cases. Since viral infections have not been substantially investigated in mastitis studies, information about immune response to BPIV-3 and BoHV-1 infected Bovine mammary tissues may be inadequate. The present study aimed to determine the presence and prevalence of BPIV-3 and BoHV-1 agents in Bovine mammary tissues, and the immune response of such tissues against BPIV-3 and BoHV-1 infection. For this purpose, we first detected these viruses with qRT-PCR in mammary tissues. Then, we determined the expression profiles of interferon-γ (IFN-γ), CD4, and CD8 genes with qRT-PCR. Lastly, we performed immunohistochemistry staining to identify the presence of IFN-γ, CD4, and CD8 proteins in the mammary tissues. We found that 26, 16, and five of the 120 samples were BPI3-, BoHV1-, and BPIV-3 + BoHV-1 infected, respectively. Moreover, the gene expression levels of IFN-γ and CD4 were strongly up-regulated in the virus-infected tissues, whereas the CD8 gene expression level was only moderately up-regulated. Immunohistochemistry staining results were consistent with qRT-PCR results. Overall, our findings showed a high prevalence of BPIV-3 and BoHV-1 and indicated that cell-mediated immune response plays an important role against BPIV-3 and BoHV-1 infection in Bovine mammary tissues. Meanwhile, IFN-γ is an important cytokine for antiviral immunity against such infection.

Screening and Identification of ssDNA Aptamers against HN Protein for Detection of Bovine Parainfluenza Virus Type 3 Antibodies in Serum.

Bovine Parainfluenza Virus type 3 (BPIV3) is a major but often overlooked pathogen that causes respiratory disease in cattle, especially during transportation and in feedlot situations. There is a demand for the rapid detection and serological diagnosis of BPIV3 to monitor the presence of the virus and its antibodies in cattle, which is critical in designing suitable interventions and control. In the present study, ssDNA aptamers with high affinity and specificity against the HN protein of BPIV3 were selected using microplates as the matrix. After eleven rounds selection, thirty-four different DNA sequences were obtained in total, wherein w-32, w-33, and w-34 were repeated seven, eleven, and nine times, and with Kd values of 56.57 ± 2.7 nM, 24.64 ± 2.84 nM, and 31.3 ± 3.32 nM, respectively. Two-dimensional structural analysis showed that the three aptamers had several loop structures that were probably more energetically favorable for target binding. Of the three candidates, aptamer w-33 showed the best affinity in an indirect enzyme-linked aptamer assay (ELAA). The percent inhibition cutoff value of the ELAA, assessed using twenty negative sera, was 31%. In a comparative study with commercial ELISA kits, the positive detection rate of the ELAA was slightly higher than that of the commercial ELISA kits, and the coincidence rate of ELAA and ELISA was 88%. Further optimization of the ELAA method with more serums is needed.

Fetal Pathology in an Aborted Holstein Fetus Infected With Bovine Parainfluenza Virus-3 Genotype A.

Bovine parainfluenza virus-3 (BPIV-3) is a recognized respiratory pathogen of cattle, and it has also been identified in aborted fetuses. However, little is known of this agent as a reproductive pathogen and detailed descriptions of fetal pathology on natural cases are lacking in the scientific literature. This article describes and illustrates lesions in a fetus spontaneously aborted by a first-calving Holstein heifer, naturally infected with BPIV-3 genotype A, broadening the current knowledge on fetal pathology by this virus. Fetal autopsy revealed diffusely reddened, rubbery and unexpanded lungs. Histologically, there was necrotizing bronchiolitis/alveolitis with intraluminal fibrin exudate and syncytial cells in the bronchiolar/alveolar spaces, and non-suppurative peribronchiolitis and perivascular interstitial pneumonia. In the small intestine there was multifocal necrotizing cryptitis and occasional necrotic syncytial enterocytes. Intralesional and extralesional BPIV-3 antigen was detected by immunohistochemistry in the lung and small intestine, and BPIV-3a was identified in fetal tissues by RT-PCR and sequencing.

A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the bovine respirovirus 3

Bovine respirovirus 3 also known as Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory agents of both young and adult cattle. Rapid diagnosis could contribute greatly in containing epidemics and thus avoid economic losses. However, the lack of robust isothermal visual method poses difficulty. In this study, a novel isothermal assay for detecting BPIV3 was established. The method includes a lateral flow dipstick (LFD) assay combined with reverse transcription recombinase polymerase amplification (RT-RPA). First, the analytical sensitivity and specificity of BPIV3 LFD RT-RPA were tested. The LFD RT-RPA assay has a detection limit of up to 100 copies per reaction in 30 min at 38 °C. Then the performance of LFD RT-RPA was evaluated using 95 clinical samples. Compared to qPCR, the LFD RT-RPA assay showed a clinical sensitivity of 94.74%, a clinical specificity of 96.05% and 0.8734 kappa coefficient. These results have demonstrated the efficiency and effectiveness of the method to be developed into a point of care protocol for the diagnosis of BPIV3.

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED. The BRDC multiplex PCR-microarray assay was able to detect and differentiate four bacteria (Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis) and five viruses [bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV)] associated with BRDC. The BED multiplex PCR- microarray- assay was able to detect and differentiate four bacteria (Clostridium perfringens, Escherichia coli, Salmonella enterica Dublin, and Salmonella enterica Typhimurium), three protozoa (Eimeria zuernii, Eimeria bovis, and Cryptosporidium parvum), and four viruses (bovine torovirus, bovine rotavirus, BCoV, and BVDV) associated with the BED. Both assays detected their respective targets individually or in combination when present. The limit-of-detection of each assay at the PCR amplification and DNA microarray levels was determined using previously titrated laboratory amplified target pathogens or using quantified synthetic nucleotides. Both assays showed very high analytical sensitivity and specificity, and were validated using a limited number of clinical samples. The BRDC and BED multiplex PCR- microarray-assays developed in this study, with further clinical validation, could be used in veterinary diagnostic laboratories for the rapid and simultaneous identification of pathogens to facilitate quick and accurate decision making for the control and treatment of these two economically important disease complexes. Furthermore, these assays could be very effective tools in epidemiological studies as well as for screening of healthy animals to identify carriers that may potentially develop BRDC or BED.

HEALTH STATUS OF REINTRODUCED WOOD BISON (BISON BISON ATHABASCAE): ASSESSING THE CONSERVATION VALUE OF AN ISOLATED POPULATION IN NORTHWESTERN CANADA

A central goal for reintroduced populations of threatened wood bison ( Bison bison athabascae) is to maintain them free of diseases of concern, particularly bovine tuberculosis (caused by Mycobacterium bovis) and brucellosis (caused by Brucella abortus). A wood bison population in southwestern Yukon, Canada was reintroduced into the wild in 1988, but no health assessment has been done since then. To provide an initial assessment of the health status and, hence, the conservation value of this population, we serologically tested 31 wood bison (approximately 3% of the population) for pathogens of interest and obtained histopathology results for select tissues. We found no evidence of exposure to M. bovis or Brucella spp., but antibodies were present to bovine parainfluenza virus 3, bovine coronavirus, Leptospira interrogans, and Neospora caninum, with seroprevalences of 87, 7, 61, and 7% of the tested animals, respectively. Reintroduced wood bison in southwestern Yukon may be of high value for wood bison recovery because it is a large and geographically isolated population with no bacteriologic, histopathologic, or serologic evidence of exposure to Brucella spp. or M. bovis.

Comparative health assessment of urban and non-urban free-ranging mule deer (Odocoileus hemionus) in southeastern British Columbia, Canada.

BackgroundThe provincial wildlife management agency, British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development, performed a translocation to control the urban mule deer (Odocoileus hemionus; uMD) overpopulation and supplement the declining non-urban mule deer (nuMD) population in the Kootenay region, British Columbia, Canada. The objectives of this cross-sectional study were to evaluate the health of the urban and nuMD populations by comparing pathogen exposure, body condition scores (BCS) and pregnancy rates, to characterize the health risks associated with the translocation and to investigate the role of infectious diseases in the decline of the nuMD deer population.MethodsTwo hundred free-ranging mule deer were captured in urban and non-urban environments in the Kootenay region from 2014 to 2017. BCS and morphometric examinations were performed for each deer. Blood samples collected from each deer were tested for exposure to selected pathogens and pregnancy status.ResultsBody condition scores averaged 3.4 on a five-point scale, was greater in nuMD, and significantly differed between years. Antibodies were detected for adenovirus hemorrhagic disease virus (38.4% (uMD 43.7%, nuMD 33.3%)), bluetongue virus (0.6% (uMD 1.2%, nuMD 0%)), bovine respiratory syncytial virus (8.4% (uMD 4.6%, nuMD 12.1%)), bovine viral diarrhea virus (1.1% (uMD 0%, nuMD 2.2%)), bovine parainfluenza-3 virus (27.0% (uMD 27.6%, nuMD 26.4%)), Neospora caninum (22.1% (uMD 24.4%, nuMD 19.7%)) and Toxoplasma gondii (8.2% (uMD 12.3%, nuMD 3.9%)). No antibodies against epizootic hemorrhagic disease virus were detected. Pregnancy rates did not differ between the two deer populations (90.7% (uMD 90.6%, nuMD 90.9%)). Exposure to N. caninum was associated with a reduced probability of being pregnant. uMD were more likely to be exposed to T. gondii than nuMD.DiscussionComparison of BCS, pregnancy rates and pathogen exposure of uMD and nuMD showed that the health of the two populations did not significantly differ, suggesting uMD translocations do not pose a severe risk of pathogen transmission between mule deer populations and that these selected pathogens do not factor in the decline of the nuMD population. However, inclusion of additional health indicators and creation of a robust predictive disease model are warranted to further characterize the health of mule deer and the health risks associated with uMD translocations. These results should be considered as part of a formal risk assessment for future uMD translocations in southeastern British Columbia.

DIVA metabolomics: Differentiating vaccination status following viral challenge using metabolomic profiles.

Bovine Respiratory Disease (BRD) is a major source of economic loss within the agricultural industry. Vaccination against BRD-associated viruses does not offer complete immune protection and vaccine failure animals present potential routes for disease spread. Serological differentiation of infected from vaccinated animals (DIVA) is possible using antigen-deleted vaccines, but during virus outbreaks DIVA responses are masked by wild-type virus preventing accurate serodiagnosis. Previous work by the authors has established the potential for metabolomic profiling to reveal metabolites associated with systemic immune responses to vaccination. The current study builds on this work by demonstrating for the first time the potential to use plasma metabolite profiling to differentiate between vaccinated and non-vaccinated animals following infection-challenge. Male Holstein Friesian calves were intranasally vaccinated (Pfizer RISPOVAL®PI3+RSV) and subsequently challenged with Bovine Parainfluenza Virus type-3 (BPI3V) via nasal inoculation. Metabolomic plasma profiling revealed that viral challenge led to a shift in acquired plasma metabolite profiles from day 2 to 20 p.i., with 26 metabolites identified whose peak intensities were significantly different following viral challenge depending on vaccination status. Elevated levels of biliverdin and bilirubin and decreased 3-indolepropionic acid in non-vaccinated animals at day 6 p.i. may be associated with increased oxidative stress and reactive oxygen scavenging at periods of peak virus titre. During latter stages of infection, increased levels of N-[(3α,5β,12α)-3,12-dihydroxy-7,24-dioxocholan-24-yl]glycine and lysophosphatidycholine and decreased enterolactone in non-vaccinated animals may reflect suppression of innate immune response mechanisms and progression to adaptive immune responses. Levels of hexahydrohippurate were also shown to be significantly elevated in non-vaccinated animals from days 6 to 20 p.i. These findings demonstrate the potential of metabolomic profiling to identify plasma markers that can be employed in disease diagnostic applications to both differentially identify infected non-vaccinated animals during disease outbreaks and provide greater information on the health status of infected animals.

Terminal sequences in the M protein of bovine parainfluenza virus type 3 is important for virus egress

Bovine parainfluenza virus type 3 (BPIV3), which is one of the most important respiratory pathogens in cattle, impairs the global cattle industry. To gain insights into the virus particle formation of BPIV3, we focused on the membrane (M) protein, which is thought to interact with the host cell membrane and virus ribonucleoprotein (RNP) complex. First, we generated M‐gene‐deficient BPIV3 (ΔM) that can propagate in cells transfected with M protein‐expressing plasmids. With ΔM and modified M gene, we found that 10‐ amino‐acid or 4‐amino‐acid deletion from its N terminal or C terminal completely abrogated virus infectivity. Furthermore, vero cells transfected with M genes produced and released virus‐like particle (VLP) even without virus infection. VLP production was also induced with an M gene in which the terminal sequence was deleted for a few amino acids. These results suggest that the terminal structures of M protein interact with RNP, which affects virus infectivity. Though the actual three‐dimensional structures of both terminals are still unclear, these findings demonstrate the importance of a few amino acids in the terminal of M protein for virus infection. These results can contribute to further research on virus M protein function and antiviral development.Support or Funding InformationOhkura T, Minakuchi M, Sagai M, Kokuho T, Konishi M, Kameyama K, Takeuchi K. Infection of the upper respiratory tract of hamsters by the bovine parainfluenza virus type 3 BN‐1 strain expressing enhanced green fluorescent protein. Virology. 476:134–40; 2015This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RT-PCR for detection of bovine parainfluenza virus type 3 (bPIV-3)

The RT-PCR technique has been frequentely used for detection of the human parainfluenza virus type 3 (hPIV-3) but the literature is scarce in relation to the bovine parainfluenza virus type 3 (bPIV-3). The aim of this study was to describe a reverse transcriptase polymerase chain reaction (RT-PCR) for detection of bovine parainfluenza virus type 3 (bPIV-3) using degenerate oligonucleotides targeting a conserved region of hemagglutinin-neuraminidase (HN) gene. Reference strain SF-4 and three different brazilian bPIV-3 isolates, besides five viral strains from different sources, were included in this study. Viruses were cultured in MDBK cells under standard conditions. Hemagglutination (HA) test was used for viral titration and a direct immunofluorescence test (DFAT) for isolate screening. In RT-PCR all bPIV-3 isolates showed amplification of an expected 1009 bp fragment of HN gene, as oposed to non PIV-3 viral samples where no amplification was detected. Using SF-4 as positive control, sensitivity of 95 pg cDNA wasachieved. In spite of the low number of bPIV-3 isolates tested, the results obtained in this study point out the potential use of this technique for detection of bPIV-3 in bovine clinical specimens.

Besi sığırlarında solunum sistemi virüslerinin seroprevalansı ve persiste BVD virüs enfeksiyonu tespiti

Sigir solunum sistemi enfeksiyonlari tum dunyada besi ve sut sigiri isletmelerindeki en onemli problemlerden birisidir. Bu calismada, buyuk kapasiteli bir besi sigiri isletmesinde bulunan uc farkli yas grubundaki (6-8 aylik; 9-11 aylik; 12-15 aylik) hayvanlarda solunum sistemi viruslerinin serolojik dagilimi incelenmistir. Bu kapsamda orneklenen hayvanlarda, bovine viral diarrhoea virus (BVDV), bovine herpesvirus-1 (BHV-1), bovine respiratorik sinsityal virus (BRSV) ve bovine parainfluenza -3 (PI-3) virusleri icin spesifik antikor varligi arastirilarak, bireysel ve coklu solunum sistemi enfeksiyonlarinin degerlendirmesi yapildi. Arastirma kapsaminda ele alinan her yas grubundan 80 adet olmak uzere toplam 240 adet kan serumu ornegi toplandi. Virus notralizasyon yontemiyle yapilan analizler sonucunda BVDV, BHV-1, BRSV ve PI-3 icin seropozitiflik degerleri sirasiyla 6-8 aylik grup icin %96.3; %98.8; %100; %100; 9-11 aylik grup icin %88.8; %100; %97.5; %98.8 ve 12-15 aylik grup icin %71.3; %95; %93.8; %96.3 olarak tespit edildi. Elde edilen verilere gore 6-8 aylik ve 9-11 aylik hayvanlarin test edilen tum etkenlere karsi yuksek seropozitiflige sahip oldugu gorulurken 12-15 aylik hayvanlarda bu oranin azalmaya basladigi belirlendi. Ayrica ornekleme yapilan besi sigirlarinin tumunun test edilen viruslerden en az iki veya daha fazlasina karsi seropozitif bulunmasi coklu enfeksiyon varliginin yuksek oranda seyrettigini gosterdi. Seropozitif hayvanlarin yas gruplari arasindaki dagiliminin BVDV enfeksiyonu icin 6-8 aylik ile 12-15 aylik ve 9-11 aylik ile 12-15 aylik yaslar arasinda istatistiki olarak onemli duzeyde farkli oldugu goruldu (P<0.05)

Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus

Bovine ephemeral fever virus (BEFV), identified as the causative pathogen of bovine ephemeral fever (BEF), is responsible for increasing numbers of epidemics/outbreaks and has a significant harmful effect on the livestock industry. Therefore, a rapid detection assay is imperative for BEFV diagnosis. In this study, we described the development of lateral-flow dipstick isothermal recombinase polymerase amplification (LFD-RPA) assays for detection of BEFV. RPA primers and LF probes were designed by targeting the specific G gene, and the amplification product can be visualized on a simple lateral flow dipstick with the naked eyes. The amplification reaction was performed at 38 °C for 20 min and LFD incubation time within 5 min. The detection limit of this assay was 8 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses such as bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine vesicular stomatitis virus. In addition, the assay was performed with total 128 clinical specimens and the diagnostic results were compared with conventional RT-PCR, real-time quantative(q) PCR. The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.

Detecting the Effect of Umckaloabo/EPs®7630 Liquid Extract after its Therapeutic Purposed Usage in Calves Showing Symptoms of Respiratory Tract Infection

Background: Respiratory tract diseases are commonly seen in beef cattle. Young calves are affected with many respiratory pathogens. Viral pathogens are particularly seen. There are many causative factors, e.g. environmental conditions, immune system of calves. Therefore, alternative treatments are needed for viral respiratory infections. The purpose of the current study was to investigate effectiveness of Umckaloabo/EPs®7630 liquid extract in some bovine viral pathogens of young beef calves.Materials, Methods &amp; Results: Antibody presence in terms of bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV) and bovine adenovirus type 3 (BAV-3) was searched in blood serum samples of 40 Holstein calves aged 6 months and over showing respiratory tract infection symptoms. All animals were found seronegative in terms of other factors except BRSV. Out of 20 BRSV seropositive calves, 10 of them were classified as control group and the other ten as testing group. BRSV antibody titers were also detected in blood samples of both groups on day 0. Umckaloabo/EPs®7630 liquid extract was given through oral route to animals in testing group according to their weights for 14 days morning, noon and night. No application was performed on animals in control group. BRSV antibody titers were detected in blood samples of animals in both groups taken on days 1st, 3rd, 5th, 7th, 10th and 14th. At the end of day 14th, BRSV antibody titer increased in 9 out of 10 animals (90%) in testing group that were given Umckaloabo/EPs® 7630 liquid extract while one of them (10%) showed no variability. BRSV antibody titer increased in 6 out of 10 animals (60%) in control group while it decreased in one of them (10%) and 3 of them (30%) showed no variability. In testing group, BRSV antibody titer started to increase on day 3rd in 6 out of 10 animals (60%), on day 5th in one (10%), on day 10th in one (10%) and on day 14th in another (10%). In control group, BRSV antibody titer started to increase on day 3rd in 3 out of 10 animals (30%) and on day 5th in 3 of them (30%). When haematologic values of blood samples taken from animals in testing and control groups on day 14th were studied comparatively, no statistical importance (P &lt; 0.01 or P &lt; 0.05) and difference was detected. As a result, in stock calves showing respiratory tract infection symptoms, applying Umckaloabo/EPs® 7630 liquid extract helped BRSV antibody titer to increase and symptoms decreased. The increase in antibody titers started from day 3rd especially after applying Umckaloabo/EPs® 7630 liquid extract.Discussion: Respiratory system infections are contagious and fatal in calves. Respiratory tract bacterial and viral vaccinations are applied in order to prevent losses of calves due to these problems. However, these vaccinations are not sufficient most of the time and antibiotics+supportive treatment applications follow afterwards. Therefore, we advise to use a herbal product, Umckaloabo/ EPs®7630 liquid extract, that has both an antiviral efficiency and a feature of supporting immune system in order to prevent mortalities in stock calves, to stop the infection from spreading on all the herd and to decrease the symptoms of respiratory system. We believe that the obtained data should be supported with more examples and more wide-ranging studies.

Seroprevalence of viral and bacterial diseases among the bovines in Himachal Pradesh, India.

Aim:The study was designed to measure the seroprevalence of viral and bacterial diseases: Infectious bovine rhinotracheitis, bovine viral diarrhea, bovine leukemia, bovine parainfluenza, bovine respiratory syncytial disease, brucellosis, and paratuberculosis among bovine of Himachal Pradesh during the year 2013-2015.Materials and Methods:The serum samples were collected from seven districts of state, namely, Bilaspur, Kangra, Kinnaur, Lahul and Spiti, Mandi, Sirmour, and Solan. The samples were screened using indirect ELISA kits to measure the seroprevalence of viral and bacterial diseases.Results:The overall seroprevalence of infectious bovine rhinotracheitis was 24.24%, bovine viral diarrhea 1.52%, bovine leukemia 9.09%, bovine parainfluenza 57.58%, bovine respiratory syncytial disease 50%, brucellosis 19.69%, and paratuberculosis 9.09% in Himachal Pradesh. The seroprevalence of bovine rhinotracheitis, bovine leukemia, bovine parainfluenza, bovine respiratory syncytial disease, and paratuberculosis in the state varied significantly (p<0.01) while was insignificant for bovine viral diarrhea and brucellosis (p>0.01). Multiple seropositivity has been observed in this study. Bovine parainfluenza virus 3 was observed commonly in mixed infection with almost all viruses and bacteria under study.Conclusion:The viral and bacterial diseases are prevalent in the seven districts of Himachal Pradesh investigated in the study. Therefore, appropriate management practices and routine vaccination programs should be adopted to reduce the prevalence of these diseases.

Molecular survey of infectious agents associated with bovine respiratory disease in a beef cattle feedlot in southern Brazil.

We investigated the occurrence of infectious pathogens during an outbreak of bovine respiratory disease (BRD) in a beef cattle feedlot in southern Brazil that has a high risk of developing BRD. Nasopharyngeal swabs were randomly collected from steers ( n = 23) and assessed for the presence of infectious agents of BRD by PCR and/or RT-PCR assays. These included: Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoHV-1), and bovine parainfluenza virus 3 (BPIV-3). Pulmonary sections of one steer that died with clinical BRD were submitted for pathology and molecular testing. The frequencies of the pathogens identified from the nasopharyngeal swabs were: H. somni 39% (9 of 23), BRSV 35% (8 of 23), BCoV 22% (5 of 23), and M. haemolytica 13% (3 of 23). PCR or RT-PCR assays did not identify P. multocida, M. bovis, BoHV-1, BVDV, or BPIV-3 from the nasopharyngeal swabs. Single and concomitant associations of infectious agents of BRD were identified. Fibrinous bronchopneumonia was diagnosed in one steer that died; samples were positive for H. somni and M. haemolytica by PCR. H. somni, BRSV, and BCoV are important disease pathogens of BRD in feedlot cattle in Brazil, but H. somni and BCoV are probably under-reported.