Natto extract, a Japanese fermented soybean food, directly inhibits viral infections including SARS-CoV-2 in vitro.

Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.

A molecular survey using a validated real-time PCR assay finds no evidence of bovine alphaherpesvirus 2 in samples from animals with suspected vesicular disease in Brazil between 2014 and 2017.

Bovine alphaherpesvirus 2 (BoHV-2) is the etiologic agent of bovine mammillitis (BM) and pseudo-lumpy skin disease. BM is also important because its clinical presentation can be confused with foot-and-mouth disease (FMD), making it necessary to establish differential diagnoses and perform additional laboratory tests. The objective of this work was to use a validated real-time PCR assay to test for the presence of BoHV-2 in samples from cattle and buffalo with suspected vesicular disease in Brazil. The method could detect the virus at a concentration of 0.5 fg/µL and had 99.4% amplification efficiency, a repeatability error of only 4.1%, and good reproducibility with other reagents. No evidence of BoHV-2 causing vesicular disease in cattle and buffalo was found in this work. This study was able to validate a new methodology for detection of BoHV-2 and evaluate its usefulness for investigating outbreaks of vesicular disease Brazil. The importance of BoHV-2 in cases involving other clinical signs should still be studied using the qPCR developed in this work.

Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1.

Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.

Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation.

UNLABELLED: VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status. IMPORTANCE: In this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other alphaherpesviruses. Interestingly, lack of phosphorylation of VP8 by CK2 and US3 resulted in reduced incorporation of viral DNA into capsids during mutant BoHV-1 infection, as well as lower numbers of extracellular virions. Furthermore, mutant VP8 remained nuclear throughout infection, in contrast to WT VP8, which is nuclear at early stages and Golgi apparatus associated late during infection. This correlates with smaller amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, with the latter dependent on phosphorylation.

First report of bovine herpesvirus 1 isolation from bull semen samples in China.

Bovine herpesvirus 1 (BoHV-1) infection causes substantial economic losses to the cattle industry worldwide. So far, the isolation of BoHV-1 field virus has not been reported in China. Here, for the first time we report that two isolates of BoHV-1 designated as NJ16-1 and NJ16-2 were obtained from semen samples from breeding bulls in China. Typical cytopathic effect in MDBK cells, detection of viral protein VP16 in western blot analysis, PCR detection of BoHV-1 gB gene proved BoHV-1 infection and subsequent nucleotide sequence analysis showed a 99% identity with BoHV-1 Cooper strain. These results suggest that these isolated viruses are BoHV-1.

Application of loop-mediated isothermal amplification (LAMP) assays for the detection of bovine herpesvirus 1.

Bovine herpesvirus-1 (BoHV-1), a causative agent of Infectious Bovine Rhinotracheitis (IBR), is responsible for high economic losses in cattle farming industry. The use of testing methods that allow early detection of BoHV-1-infected animals is a key element of each program of IBR eradication. The aim of the study was to design and evaluate two variants of LAMP isothermal tests with SYBR Green fluorescence probes, specific to the genes encoding gD and gE glycoproteins of BoHV-1. LAMP gE BoHV-1 assay was able to distinguish between gE- and gE+ strains of the virus. Both LAMP gD and gE assays were specific to BoHV-1 and did not react with other related to BoHV-1 alphaherpesviruses. Sensitivity of LAMP gD was 2x104 copies of the viral genome whereas for LAMP gE it was 2x105. Diagnostic sensitivity calculated for LAMP gD was 64.7% whereas for LAMP gE it was 80%. Diagnostic specificity for LAMP gD and LAMP gE was 78.9% and 89.3%, respectively. LAMP assay can be a rapid and simple method of diagnosis of acute BoHV-1 infections and discrimination of gE- strains. However, relatively low diagnostic sensitivity of the method can limit its use in routine diagnostics.

Complete genomic sequence and comparative analysis of four genital and respiratory isolates of bovine herpesvirus subtype 1.2b (BoHV-1.2b), including the prototype virus strain K22.

Bovine herpesvirus subtype 1.2b (BoHV-1.2b) is associated primarily with bovine infectious pustular vulvovaginitis. We report here the complete genomic sequence of four BoHV-1.2b isolates. The DNA sequence identity of the four genomes is 98.9 %. Differences were primarily in regions containing direct repeats, specifically gene UL36 and the terminal repeat regions immediately flanking gene BICP22. BoHV-1.2b and BoHV-1.1 genomes are similar in size (~135 kb), completely orthologous with respect to regional structure and gene location, and have a 97.5 % DNA sequence homology. The most notable difference is the structure of the DNA replication origin of the two viruses.

Association between antibody status to bovine herpesvirus 1 and quality of milk in dairy herds in Poland.

Bovine herpesvirus 1 (BoHV1) is one of the most important pathogens of cattle; however, its effect on somatic cell count and milk components is not completely understood. The aim of the current study was to examine the effect of BoHV1 infection on quality of bovine bulk tank milk (BTM). A total of 1,790 individual blood samples collected at 28 dairy farms were used to determine the BoHV1 infection status of the herds with ELISA tests. The quality parameters of milk were evaluated by instrumental methods with BTM samples collected at monthly intervals from May 2011 to May 2012. The statistical analysis was performed to study the associations between BoHV1 herd status, quality of BTM, and herd-specific parameters. The risk factors influencing bulk milk somatic cell count (BMSCC) were estimated using the multivariable mixed-effects maximum likelihood regression model. The true prevalences of BoHV1 infection at the animal and herd levels were 49.3 and 64.6%, respectively. The average BMSCC differed significantly between the herds grouped accordingly to their BoHV1 infection status. Interestingly, the highest BMSCC was observed in the vaccinated herds (240.3×10(3) cells/mL). Additionally, the BoHV1 herd status had a significant effect on the fat content of BTM. The largest herds that were investigated had a BoHV1 seroprevalence over 30%. The herd status was considerably influenced by the numbers of cows in the herds. Besides, no significant differences in total bacterial count or protein content in milk from BoHV1-infected und uninfected herds were observed. An increase in BMSCC was observed during summer compared with the winter months regardless of the BoHV1 status of the herds. In the final multivariable regression model, the main risk factors associated with BMSCC were BoHV1 herd status, the percentage of BoHV1 infected animals in a herd, the number of cows in a herd, and the season. Our study suggests that BoHV1 infection may influence BMSCC levels, which are key parameters of BTM quality and a reference for subclinical mastitis in a herd. In conclusion, BoHV1 infection may cause economic losses by decrease both of quantity and quality of milk.

Pathogens at the livestock-wildlife interface in Western Alberta: does transmission route matter?

In southwestern Alberta, interactions between beef cattle and free-ranging elk (Cervus elaphus) may provide opportunities for pathogen transmission. To assess the importance of the transmission route on the potential for interspecies transmission, we conducted a cross-sectional study on four endemic livestock pathogens with three different transmission routes: Bovine Viral Diarrhea Virus and Bovine Herpesvirus 1 (predominantly direct transmission), Mycobacterium avium subsp. paratuberculosis (MAP) (indirect fecal-oral transmission), Neospora caninum (indirect transmission with definitive host). We assessed the occurrence of these pathogens in 28 cow-calf operations exposed or non-exposed to elk, and in 10 elk herds exposed or not to cattle. We characterized the effect of species commingling as a risk factor of pathogen exposure and documented the perceived risk of pathogen transmission at this wildlife-livestock interface in the rural community. Herpesviruses found in elk were elk-specific gamma-herpesviruses unrelated to cattle viruses. Pestivirus exposure in elk could not be ascertained to be of livestock origin. Evidence of MAP circulation was found in both elk and cattle, but there was no statistical effect of the species commingling. Finally, N. caninum was more frequently detected in elk exposed to cattle and this association was still significant after adjustment for herd and sampling year clustering, and individual elk age and sex. Only indirectly transmitted pathogens co-occurred in cattle and elk, indicating the potential importance of the transmission route in assessing the risk of pathogen transmission in multi-species grazing systems.

Rapid detection of bovine herpesvirus 1 in bovine semen by loop-mediated isothermal amplification (LAMP) assay.

Bovine herpesvirus 1 (BoHV-1) is the most common viral pathogen found in bovine semen, causing numerous reproductive disorders leading to economic losses to the cattle industry. For rapid detection of BoHV-1 in bovine semen, in this study, we applied a loop-mediated isothermal amplification (LAMP) assay. The assay could be completed within 90 min, including total DNA isolation, target amplification, and visual interpretation of positive or negative results with the naked eye. The assay detected as little as 10 fg of BoHV-1 DNA per reaction. The analytical sensitivity of the assay was 0.2 TCID50 BoHV-1 per reaction, which was 100 times more sensitive than conventional PCR and comparable to TaqMan real-time PCR. The applicability of the assay was assessed by analysing 118 semen samples collected from breeding bulls. On comparison with TaqMan real-time PCR, the LAMP assay had a diagnostic sensitivity of 97 %, specificity of 100 %, and accuracy of 99.2 % for detection of BoHV-1 in bovine semen. The LAMP assay developed in this study is a rapid, sensitive, and cost-effective alternative for detection of BoHV-1 in bovine semen.

Genetic diversity of 3' region of glycoprotein D gene of bovine herpesvirus 1 and 5.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses of cattle. While BoHV-1 is mainly associated with respiratory/genital disease and rarely associated with neurological disease, BoHV-5 is the primary agent of meningoencephalitis in cattle. The envelope glycoprotein D of alphaherpesviruses (BoHV-1/gD1 and BoHV-5/gD5) is involved in the early steps of virus infection and may influence virus tropism and neuropathogenesis. This study performed a sequence analysis of the 3' region of gD gene (gD3') of BoHV-1 isolates recovered from respiratory/genital disease (n = 6 and reference strain Cooper) or from neurological disease (n = 7); and from seven typical neurological BoHV-5 isolates. After PCR amplification, nucleotide (nt) sequencing, and aminoacid (aa) sequence prediction; gD3' sequences were compared, identity levels were calculated, and selective pressure was analyzed. The phylogenetic reconstruction based on nt and aa sequences allowed for a clear differentiation of BoHV-1 (n = 14) and BoHV-5 (n = 7) clusters. The seven BoHV-1 isolates from neurological disease are grouped within the BoHV-1 branch. A consistent alignment of 346 nt revealed a high similarity within each viral species (gD1 = 98.3 % nt and aa; gD5 = 97.8 % nt and 85.8 % aa) and an expected lower similarity between gD1 and gD5 (73.7 and 64.1 %, nt and aa, respectively). The analysis of molecular evolution revealed an average negative selection at gD3'. Thus, the phylogeny and similarity levels allowed for differentiation of BoHV-1 and BoHV-5 species, but not further division in subspecies. Sequence analysis did not allow for the identification of genetic differences in gD3' potentially associated with the respective clinical/pathological phenotypes, yet revealed a lower level of gD3' conservation than previously reported.

Microarray chip based identification of a mixed infection of bovine herpesvirus 1 and bovine viral diarrhea 2 from Indian cattle.

Bovine herpesvirus 1 (BHV1) and bovine viral diarrhea virus 2 (BVD2) are endemic in India although no mixed infection with these viruses has been reported from India. We report first mixed infection of these viruses in cattle during routine screening with a microarray chip. 62 of the 69 probes of BHV1 and 42 of the 57 BVD2 probes in the chip gave positive signals for the virus. The virus infections were subsequently confirmed by RT-PCR. We also discuss the implications of these findings.

Investigation of an outbreak of infectious pustular balanoposthitis in cattle breeding bulls at a frozen semen bank.

Infectious pustular balanoposthitis (IPB) is one of the reproductive disorders caused by bovine herpesvirus 1 (BoHV1) that can be transmitted through artificial insemination. A herd of 63 breeding bulls at a frozen semen bank in Odisha state in India experienced a suspected outbreak of IPB, with 11 bulls showing clinical signs of the infection. Clinical signs were noticed in two bulls initially and a few days after in the other nine animals. Serum samples from 53 bulls were examined for anti-BoHV1 antibodies using a virus neutralisation test (VNT) and a competitive enzyme-linked immunosorbent assay (cELISA); the remaining ten bulls were not included in the study because it was difficult to restrain them at that time. Paired serum samples were collected 21 days apart from ten clinically affected bulls (the eleventh clinically affected bull was not included in the study for the reason stated above). In the neutralisation test, the paired serum samples showed a two- to fourfold increase in anti-BoHV1 antibody titre; in the cELISA, the paired samples were also found positive for anti-BoHV1 antibodies. Serum samples from 43 in-contact bulls were collected about day 22 after the first observation of clinical infection in the herd. Among these serum samples, a total of 30 were found positive for anti-BoHV1 antibodies in the VNT and a total of 30 were found positive in cELISA. Ten samples were positive in one test but not the other and 25 tested positive in both tests. In all, 35 serum samples from in-contact bulls tested positive in either one or both of the two types of test. An overall agreement of 76.74% was found in detection of anti-BoHV1 antibodies in the two tests. Sensitivity was higher than specificity in detection of anti-BoHV1 antibodies in the serum samples. The glycoprotein C region of the genomic DNA of BoHV1 was amplified from semen samples by polymerase chain reaction. The findings from the outbreak indicate that continuous monitoring of breeding bulls at frozen semen banks is warranted to avoid the risks associated with artificial insemination.

Simultaneous detection of bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) using recombinase polymerase amplification.

Bovine viral diarrhea virus (BVDV) is considered to be the most common agent of severe diarrhea in cattle worldwide, causing fever, diarrhea, ulcers, and abortion. Bovine herpesvirus 1 (BoHV-1) is also a major bovine respiratory disease agent that spreads worldwide and causes extensive damage to the livestock industry. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method with the advantages of high efficiency, rapidity and sensitivity, which has been widely used in the diagnosis of infectious diseases. A dual RPA assay was developed for the simultaneous detection of BVDV and BoHV-1. The assay was completed at a constant temperature of 37 °C for 30 min. It was highly sensitive and had no cross-reactivity with other common bovine viruses. The detection rate of BVDV RPA in clinical samples (36.67%) was higher than that of PCR (33.33%), the detection rate of BoHV-1 RPA and PCR were equal. Therefore, the established dual RPA assay for BVDV and BoHV-1 could be a potential candidate for use as an immediate diagnostic.

Presence of co-infection between bovine leukemia virus and bovine herpesvirus 1 in herds vaccinated against bovine respiratory complex.

The aim of this study was molecular identification of bovine leukemia virus and possible co-infection with bovine respiratory disease complex (BRDC) viral agents in Mexican dairy herds. We collected 533 blood samples from cattle vaccinated against the BRDC virus in 9 states across Mexico. Peripheral blood leukocytes were removed and genetic material was extracted to detect bovine leukemia virus (BLV), bovine herpesvirus 1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV-3), and bovine respiratory syncytial virus (BRSV) infection using polymerase chain reaction. We identified high BLV infection rates in 270 cattle (50.65%). One hundred and thirty-three cows (24.95%) tested positive for BoHV-1, of which 65 samples were positive for both viruses (BoHV-1 and BLV) and 68 were only positive for BoHV-1. Only 4 samples tested positive for BPIV-3 and no sample was positive for BVDV or BRSV. Relative risk and odds ratio analyses did not identify that the presence of BLV infection favors BoHV-1 co-infection in vaccinated herds.

Le but de cette étude était l'identification moléculaire du virus de la leucémie bovine et une éventuelle co-infection par des agents viraux du complexe des maladies respiratoires bovines (BRDC) dans des troupeaux laitiers mexicains. Nous avons recueilli 533 échantillons de sang de bovins vaccinés contre le virus BRDC dans neuf états du Mexique. Les leucocytes du sang périphérique ont été prélevés et le matériel génétique a été extrait pour détecter le virus de la leucémie bovine (BLV), le virus de l'herpès bovin 1 (BoHV-1), le virus de la diarrhée virale bovine (BVDV), le virus parainfluenza bovin 3 (BPIV-3), et le virus respiratoire syncytial bovin (BRSV) par réaction d'amplification en chaîne par la polymérase. Nous avons identifié des taux élevés d'infection par le BLV chez 270 bovins (50,65 %). Cent trente-trois bovins (24,95 %) ont été testés positifs pour le BoHV-1, desquels 65 échantillons étaient positifs pour les deux virus (BoHV-1 et BLV) et 68 étaient uniquement positifs pour le BoHV-1. Seuls quatre échantillons ont été testés positifs pour le BPIV-3 et aucun échantillon n'a été positif pour le BVDV ou le BRSV. Les analyses du risque relatif et des rapports de cotes n'ont pas identifié que la présence d'une infection par le BLV favorise la co-infection par le BoHV-1 dans les troupeaux vaccinés.(Traduit par les auteurs).

One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3.

BACKGROUND: Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD. RESULTS: A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT). CONCLUSIONS: The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

Cost-effectiveness of bulk-tank milk testing for surveys to demonstrate freedom from infectious bovine rhinotracheitis and bovine enzootic leucosis in Switzerland.

In Switzerland, annual surveys to substantiate freedom from infectious bovine rhinotracheitis (IBR) and enzootic bovine leucosis (EBL) are implemented by a random allocation of farms to the respective survey as well as blood sampling of individual animals at farm level. Contrary to many other European countries, bulk-tank milk (BTM) samples have not been used for active cattle disease surveillance for several years in Switzerland. The aim of this project was to provide a financial comparison between the current surveillance programme consisting of blood sampling only and a modified surveillance programme including BTM sampling. A financial spreadsheet model was used for cost comparison. Various surveillance scenarios were tested with different sample sizes and sampling frequencies for BTM samples. The costs could be halved without compromising the power to substantiate the freedom from IBR and EBL through the surveillance programme. Alternatively, the sensitivity could be markedly increased when keeping the costs at the actual level and doubling the sample size. The risk-based sample size of the actual programme results in a confidence of 94,18 % that the farm level prevalence is below 0,2 %. Which the doubled sample size, the confidence is 99,69 % respectively.

Seroprevalence of BHV-1 (bovine herpesvirus type 1) among non-vaccinated dairy cattle herds with respiratory disorders.

The objective of this study was to estimate a herd-level seroprevalence of bovine herpesvirus type 1 (BHV-1) in herds with clinical symptoms of the respiratory tract. Eighty-three herds with suspected BHV-1 infection were selected and divided into two categories with respect to their size: small (n = 27) and large herds (n = 56). Samples were collected from calves, heifers and cows older than 24 months. Seroprevalence was determined using the gB ELISA test. The herd level seroprevalence was estimated as 53% (44/83) in the tested herds, 11.1% (3/27) in the small herds and 73.2% (41/56) in the large herds. Our study suggests that the current biosecurity measures still warrant improvement.

Limitations of bacterial culture, viral PCR, and tulathromycin susceptibility from upper respiratory tract samples in predicting clinical outcome of tulathromycin control or treatment of bovine respiratory disease in high-risk feeder heifers.

A cross-sectional prospective cohort study including 1026 heifers administered tulathromycin due to high risk of clinical signs of bovine respiratory disease (BRD), measured poor association between BRD clinical outcomes and results of bacterial culture and tulathromycin susceptibility from BRD isolates of deep nasopharyngeal swabs (DNS) and adequate association with viral polymerase chain reaction (PCR) results from nasal swabs. Isolation rates from DNS collected on day-0 and at 1st BRD-treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected exclusively at 1st BRD-treatment were: bovine parainfluenza virus type-3 (bPIV-3) 34.1%; bovine viral diarrhea virus (BVDV) 26.3%; bovine herpes virus type-1 (BHV-1) 10.8%; and bovine respiratory syncytial virus (BRSV) 54.1%. Increased relative risk, at 95% confidence intervals, of 1st BRD-treatment failure was associated with positive viral PCR results: BVDV 1.39 (1.17-1.66), bPIV-3 1.26 (1.06-1.51), BHV-1 1.52 (1.25-1.83), and BRSV 1.35 (1.11-1.63) from nasal swabs collected at 1st BRD-treatment and culture of M. haemolytica 1.23 (1.00-1.51) from DNS collected at day-0. However, in this population of high-risk feeder heifers, the predictive values of susceptible and resistant isolates had inadequate association with BRD clinical outcome. These results indicate, that using tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at 1st BRD-treatment to evaluate tulathromycin clinical efficacy, is unreliable.

Using scenario tree modelling for targeted herd sampling to substantiate freedom from disease.

BACKGROUND: In order to optimise the cost-effectiveness of active surveillance to substantiate freedom from disease, a new approach using targeted sampling of farms was developed and applied on the example of infectious bovine rhinotracheitis (IBR) and enzootic bovine leucosis (EBL) in Switzerland. Relevant risk factors (RF) for the introduction of IBR and EBL into Swiss cattle farms were identified and their relative risks defined based on literature review and expert opinions. A quantitative model based on the scenario tree method was subsequently used to calculate the required sample size of a targeted sampling approach (TS) for a given sensitivity. We compared the sample size with that of a stratified random sample (sRS) with regard to efficiency. RESULTS: The required sample sizes to substantiate disease freedom were 1,241 farms for IBR and 1,750 farms for EBL to detect 0.2% herd prevalence with 99% sensitivity. Using conventional sRS, the required sample sizes were 2,259 farms for IBR and 2,243 for EBL. Considering the additional administrative expenses required for the planning of TS, the risk-based approach was still more cost-effective than a sRS (40% reduction on the full survey costs for IBR and 8% for EBL) due to the considerable reduction in sample size. CONCLUSIONS: As the model depends on RF selected through literature review and was parameterised with values estimated by experts, it is subject to some degree of uncertainty. Nevertheless, this approach provides the veterinary authorities with a promising tool for future cost-effective sampling designs.