The seasonality as a relevant aspect to be considered for differential diagnosis of Trypanosoma vivax infection and co-infections in female cattle.

Bovine Trypanosomiasis and other infectious diseases cause relevant loss for the livestock industry impacting productive/reproductive indices. This study intended to better understand the frequency, seasonality, and profile of infections associated with Bovine Trypanosomiasis. A total of 1443 serum samples were screened for T. vivax infection and other infectious diseases: Neosporosis, Leptospirosis, Bovine Leukosis Virus infection/(BLV), Infectious Bovine Rhinotracheitis/(IBR) or Bovine Viral Diarrhea/(BVD). Distinct methods were used for screening and diagnosis: immunofluorescence assay (Trypanosomiasis), ELISA (Neosporosis,BLV,IBR,BVD) and microscopic agglutination test (Leptospirosis). Our findings demonstrated that the seropositivity for Trypanosomiasis=57% was similar to Neosporosis=55%, higher than Leptospirosis=39% and BVL=34%, but lower than IBR=88% and BVD=71%. The seropositivity for Trypanosomiasis was higher in the autumn and lower in the winter. Regardless the season, the IBR seropositivity (min=73%;max=95%) was higher than Trypanosomiasis (min=48%;max=68%). Moreover, Neosporosis (min=71%;max=100%) and BVD (min=65%;max=76%) were more frequent than Trypanosomiasis in the summer, winter and spring. The diagnosis outcome revealed that Trypanosomiasis&IBR=43% and Trypanosomiasis&Neosporosis=35% were the most frequent co-infections with higher seropositivity in the autumn (58%) and summer (80%), respectively. Noteworthy, high seropositivity to Trypanosomiasis&BVD was registered in the autumn (46%). Together, our data re-enforce the relevance of differential diagnosis between Trypanosomiasis with other bovine infectious diseases and that differences in the seasonality profile is a relevant aspect to be considered while selecting the differential diagnosis to be applied.

A Rapid Nucleic Acid Visualization Assay for Infectious Bovine Rhinotracheitis Virus That Targets the TK Gene.

Infectious bovine rhinotracheitis virus (IBRV) can cause various degrees of symptoms in the respiratory system, reproductive system, and whole body of cattle. It also can lead to persistent and latent infection in cattle, posing a challenge to timely control of infectious bovine rhinotracheitis (IBR) in farms and causing large financial losses in the global cattle industry. Therefore, the goal of this study was to establish a rapid, simple, and accurate method that can detect IBRV in order to facilitate the control and eradication of IBR in cattle. We combined recombinant polymerase amplification (RPA) with a closed vertical flow visualization strip (VF) and established an RPA-VF assay that targets the thymidine kinase (TK) gene to rapidly detect IBRV. This method (reaction at 42°C for 25 min) was able to detect a minimum of 3.8 × 101 copies/µL of positive plasmid and 1.09 × 101 50% tissue culture infective dose (TCID50) of the IBRV. This assay has high specificity for IBRV and does not cross-react with other respiratory pathogens in cattle. The concordance between the RPA-VF assay and the gold standard was 100%. In addition, this assay was also suitable for the detection of DNA from clinical samples extracted by a simple method (heating at 95°C for 5 min), which can achieve the rapid detection of clinical samples in the field. Overall, the present sensitivity, specificity, and clinical applicability assessments indicated that the RPA-VF assay we developed can be utilized as a quick and accurate on-site test for IBRV detection in farms. IMPORTANCE IBRV causes different degrees of clinical symptoms in cattle and poses a great threat to the cattle industry. The infection is persistent and latent, and the elimination of IBRV in infected herds is difficult. A rapid, simple, and accurate method to detect IBRV is therefore vital to control and eradicate IBR. Combining RPA with an VF, we established an RPA-VF assay for the rapid detection of IBRV, which can complete the test of clinical samples in 35 min. The assay shows good sensitivity, specificity, and clinical applicability and can be used as an on-site test for IBRV in farms.

A retrospective evaluation of pooled serum ELISA testing in the frame of the French eradication program for infectious bovine rhinotracheitis.

Pooled serum testing using whole-virus indirect ELISA has been recently recognized as an official method for surveillance of bovine herpesvirus 1 (BoHV1) in cattle herds in Europe. In this study, a retrospective analysis of data from the French BoHV1 surveillance campaign 2018-2019, including 7434 BoHV1-free certified herds and 157 infected herds, was performed in order to evaluate the diagnostic specificity and sensitivity of two pooled serum indirect ELISAs (from IDEXX and IDVet), in comparison with individual testing by blocking ELISAs targeting the gB and gE proteins. Pooled serum testing showed a relative specificity higher than 97.5% and a detection rate of 100% since all gB+/gE+ samples were found in positive pools. At the herd level, no more than one false positive pool was observed in most of BoHV1-free certified herds, leading to a herd relative specificity of 85.1% and 86.0% for the IDEXX and IDVet pooled serum ELISAs, respectively. Among infected herds tested by pool sizes up to 10 sera (n = 122), 46% of herds were detected through pools of size 10 containing a single positive sample, 23% through pools of size 10 containing at least two positive samples, and 31% through pools of smaller sizes. A complementary study based on manually constituted pools revealed that at least one positive sample in 100% and 93.4% of herds could be detected individually by pools of size 10 with the IDEXX and IDVet ELISAs, respectively. However, pooled serum ELISAs were influenced by the level of individual reactivity, since pools composed of either one weak-positive sample or one gB+/gE- sample could yield negative results. Altogether, these results provided the first evidence that pooled serum testing (pool size up to 10) is a suitable strategy for surveillance of BoHV1-free cattle farms.

Use of pooled serum samples to assess herd disease status using commercially available ELISAs.

Pooled samples are used in veterinary and human medicine as a cost-effective approach to monitor disease prevalence. Nonetheless, there is limited information on the effect of pooling on test performance, and research is required to determine the appropriate number of samples which can be pooled. Therefore, this study aimed to evaluate the use of pooled serum samples as a herd-level surveillance tool for infectious production-limiting diseases: bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), enzootic bovine leukosis (EBL) and Neospora caninum (NC), by investigating the maximum number of samples one can pool to identify one positive animal, using commercial antibody-detection ELISAs. Four positive field standards (PFS), one for each disease, were prepared by pooling highly positive herd-level samples diagnosed using commercially available ELISA tests. These PFS were used to simulate 18 pooled samples ranging from undiluted PFS to a dilution representing 1 positive in 1,000 animals using phosphate-buffered saline as diluent. A 1:10 dilution of the PFS resulted in positive results for IBR, BVD and EBL. Moreover, for IBR and BVD, results were still positive at 1:100 and 1:30 dilutions, respectively. However, for NC, a lower dilution (8:10) was required for a seropositive result. This study indicates that, at herd-level, the use of pooled serum is a useful strategy for monitoring infectious diseases (BVD, IBR and EBL) but not NC, using readily available diagnostic assays.

Sero-diagnostic efficacy of various ELISA kits for diagnosis of infectious bovine rhinotracheitis (IBR) in cattle and buffaloes in India.

Bovine alphaherpesvirus-1 (BoHV-1), the causative agent of infectious bovine rhinotracheitis (IBR), is an economically important viral pathogen affecting cattle and buffaloes. Serological assays are mostly used for detection of the antibodies, but variation has been detected in the diagnostic performances of the individual assay. In the present study, four commercially available ELISA kits {two indirect ELISA (kits A and B) and two blocking ELISA (kits C and D)} were evaluated for the detection of antibodies against BoHV-1 in Indian cattle and buffaloes (fitness of purpose). The diagnostic sensitivity (dsn) and specificity (dsp) of these kits were determined by three ways; considering virus neutralization test (VNT) as gold standard test, using pre-test information of the samples, and majority of tests. Screening of 200 known negative sera (124 cattle, 76 buffaloes) sourced from IBR free farms revealed gB based ELISA kits are more specific than the indirect ELISA kits. Testing of 125 known positive sera (81 cattle, 44 buffaloes) suggests kit B be most sensitive followed by kit C, A and D. Interestingly, kit D was found to be most sensitive for detection of vaccination-induced BoHV-1 antibodies followed by kit B. Similar trend were also observed in the limit of dilution experiment performed using known infected and vaccinated sera. VNT was found to be the most specific test and its use as the gold standard test revealed all kits to have more than 99 % sensitivity. All the ELISA kits could detect BoHV-1 specific antibodies in the IBR vaccinated calves as early as 11 days post-vaccination. In Kappa statistics, an almost perfect agreement between the ELISA kits was recorded. The overall performance of the kits in serodiagnosis of IBR as determined by the area under curve in ROC analysis was good.

Field application of an indirect gE ELISA on pooled milk samples for the control of IBR in free and marker vaccinated dairy herds.

BACKGROUND: The aim of the present study was to assess the reliability of a new strategy for monitoring the serological response against Bovine Herpesvirus 1 (BoHV1), the causative agent of infectious bovine rhinotracheitis (IBR). Bulk milk samples have already been identified as cost effective diagnostic matrices for monitoring purposes. Nevertheless, most eradication programs are still based on individual standard assays. In a region of northwestern Italy (Piedmont), the voluntary eradication program for IBR has become economically unsustainable. Being the prevalence of infection still high, glycoprotein E-deleted marker vaccines are commonly used but gE blocking ELISAs are less sensitive on bulk milk samples compared to blood serum. RESULTS: A recently developed indirect gE ELISA showed high versatility when applied to a wide range of matrices. In this study, we applied a faster, cost effective system for the concentration of IgG from pooled milk samples. The IgG enriched fractions were tested using a gE indirect ELISA for monitoring purposes in IBR-positive and IBR-marker-vaccinated herds. Official diagnostic tests were used as gold standard. During a 3 years study, a total 250 herds were involved, including more than 34,500 lactating cows. The proposed method showed a very good agreement with official diagnostic protocols and very good diagnostic performances: only 37 positive animals were not detected across the entire study. CONCLUSIONS: The results highlighted the ability of the proposed method to support the surveillance of IBR in the Piedmont region, reducing the costs without affecting the diagnostic performances.

[Establishment and application of visual LAMP detection method of infectious bovine rhinotracheitis virus].

Three pairs of primers were designed according to the conserved region of IBRV gB gene published in GenBank(GenBank Accession No. DQ006857.1) using the software Primer Explorer V4. The loop mediated isothermal amplification (LAMP) assay was established by optimization of the reaction system and then evaluated through sensitivity and specificity tests. In total 393 clinical specimens were detected for IBRV using the established LAMP assay performed at 65℃ for 50 min, which produced a ladder-like pattern of amplification bands and the detection result could be judged by color change. The sensitivity of the assay was 10 copies/µL plasmid DNA which was 1000 times higher than that by PCR method and equivalent to nested-PCR. There was no cross-reactivity of the assay with bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and vesicular stomatitis virus (VSV). The positive rate of 301 nasal swabs and 92 serum specimens were 87.6% and 58.8%, respectively, which meant nasal swab specimen was more suitable for clinical IBRV detection by the method. The IBRV LAMP method established in this study has the advantages of visualization, quickness, specificity and sensitivity and be suitable for rapid detection of clinical IBRV detection on the spot.

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.

Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays.

BACKGROUND: Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV. METHODS: Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens. RESULTS: RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%. CONCLUSION: IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field.

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.

Infectious bovine rhinotracheitis in Scotland.

A cattle dashboard has recently been developed to share surveillance information gathered from submissions to the Great Britain veterinary diagnostic network. Data relating to Scotland come from the SAC C VS. This article, by Tim Geraghty, relates to cases of infectious bovine rhinotracheitis in Scotland, as summarised on the APHA Cattle Dashboard.

Characterization of three commercial ELISA kits for detection of BOHV-1 gE specific antibodies in serum and milk samples and applicability of bulk milk for determination of herd status.

Vaccination of animals with gE-deleted vaccine strains (gE- marker vaccines) and differential detection of vaccinated vs infected animals with antibody ELISA targeting the gE or the gB proteins have been proved to be useful tools in programs for control and eradication of the bovine herpesvirus 1 (BoHV-1) responsible for infectious bovine rhinotracheitis (IBR), a major pathogen of cattle. The diagnostic sensitivity (DSe) and specificity (DSp) of three commercial gE ELISA kits from IDEXX, IDVet and CIV-HIPRA were compared for serum and milk matrices. Limiting the analysis to 198 individual with concordant ELISA results in serum (91 naïve, 37 vaccinated and 70 infected) the DSe of gE kits was estimated to 0,97 for IDEXX, 0,93 for CIV-HIPRA and 0,53 for IDVet using milk samples and the DSp to 0,95 for IDEXX, 1,00 for IDVet and CIV-HIPRA. The applicability of gE ELISA for individual or bulk milk testing as an additional tool in control programs dedicated to the certification and control of vaccinated herds was evaluated. Two of the three evaluated gE ELISA kits presented substantial to good agreement individual milk and serum samples. The bulk-tank milk also proved to be suitable for the detection of BoHV-1 in vaccinated herds provided that gE prevalence is superior to 10% as false negative results are often observed at lower gE herd prevalence. This limitation could be reduced to 8% of prevalence when a prior concentration step was applied to bulk milk samples.

Determining bovine viral diarrhea and infectious bovine rhinotracheitis infections in dairy cattle using precolostral blood.

The objective of this study was to determine if precolostral blood samples are useful to detect apparent fetal infections with bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR) viruses. A convenience sample of 317 sera from 50 Canadian herds was used in the study. Antibody level was measured using 2 commercial IBR and BVD ELISA kits. Precolostral status of sera was confirmed on 304 samples using serum gamma-glutamyl transferase activity. Postcolostral serum samples yielded a higher proportion of positive results to IBR (OR = 86; 95% CI: 17.8 to 415.7) and BVD (OR = 199.3; 95% CI: 41.7 to 952.3) than did precolostral samples. All positive precolostral serum samples (n = 7 of 304) originated from calves born to vaccinated cows. Postcolostral positive serum samples (n = 11 of 13) originated mostly (60%) from calves born to non-vaccinated cows. Precolostral serum sampling can detect apparent fetal infections in a herd.

Utilisation du serum précolostral pour le dépistage de la diarrhée virale bovine (BVD) et rhinotracheite infectieuse bovine (IBR) dans les troupeaux laitiers. L'objectif de cette étude était d'évaluer l'utilité du prélèvement de sérum précolostral de nouveaux nés pour détecter des infections foetales apparentes par IBR et BVD dans un troupeau. Un échantillonnage de convenance de 317 sérums, prélevés de 50 troupeaux canadiens, a été utilisé. Les niveaux d'anticorps des sérums ont été mesurés en utilisant 2 trousses ELISA (IBR et BVD). Le statut précolostral a été confirmé pour 304 échantillons par la mesure de l'activité sérique des gamma glutamyl transférases. Une plus grande proportion de résultats positifs à IBR (RC = 86; IC 95%: 17,8 à 415,7) et BVD (RC = 199,3; IC 95 %: 41,7 à 952,3) a été observée parmi les échantillons postcolostraux que parmi les précolostraux. Tous les échantillons précolostraux positifs (n = 7/304) provenaient de veaux nés de mères vaccinées. Les échantillons postcolostraux positifs (n = 11/13) étaient majoritairement (60 %) prélevés à partir de veaux nés de mères non vaccinées. Le prélèvement de sérum précolostal peut détecter des infections foetales apparentes dans les troupeaux.(Traduit par les auteurs).

Surveillance of Infectious Bovine Rhinotracheitis in marker-vaccinated dairy herds: Application of a recombinant gE ELISA on bulk milk samples.

Infectious Bovine Rhinotracheitis (IBR) occurs worldwide, requiring significant resources for eradication programs or surveillance purposes. The status of infection is usually detected by serological methods using the virus neutralization test (VNT) or enzyme-linked immunosorbent assay (ELISA) on individual sera. The gE DIVA (Differentiating Infected from Vaccinated Animals) vaccines approach, adopted in order to reduce the virus circulation and prevent clinical signs, have tightened the range of available methods for the serological diagnosis. Different gE blocking ELISA could be performed to detect specific antibodies in sera of infected or whole virus-vaccinated animals but with less sensitivity if applied to bulk milk samples, especially in marker-vaccinated herds. A new rec-gE ELISA was recently developed in Italy and applied with good performances on blood serum samples. The present paper focuses on the application of a rapid protocol for purification/concentration of immunoglobulin G (IgG) from bulk milk and on the use of the new rec-gE indirect ELISA. The study involved three different partners and 225 herds (12,800 lactating cows) with different official IBR diagnostic statuses. The diagnostic specificity of the method was demonstrated closed to 100% while the diagnostic sensitivity was strictly related to the herd-seroprevalence. Considering 2.5% as the limit of detection of within-herd seropositivity prevalence, the diagnostic sensitivity showed by the proposed method was equal to 100%. A single reactivation of a whole strain vaccine in an old cow was detected inside a group of 67 lactating cows, showing the field applicability of the method.

Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle.

Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.

Identification of an epitope within the Bovine herpesvirus 1 glycoprotein E cytoplasmic tail and use of a monoclonal antibody directed against the epitope for the differentiation between vaccinated and infected animals.

We constructed a recombinant bovine herpesvirus type 1 triple mutant virus (BoHV-1 tmv) that lacks UL49.5 residues 30-32 and 80-96, gE cytoplasmic tail (gE CT) residues 452-575 and the entire 435 bp long Us9 ORF. To develop a gE CT-specific blocking ELISA test that is necessary to distinguish the BoHV-1 tmv vaccinated calves from the wild-type (wt) virus-infected calves, a mouse monoclonal antibody (mAb) 2H8F3 was generated by using the Escherichia coli expressed gE CT residues 452-575. Further, by performing a PEPSCAN analysis of 12 mer overlapping peptides spanning the entire gE CT, the epitope sequence recognized by the mAb2H8F3 was mapped within the gE CT residues 499SDDDGPASN507. A blocking ELISA test was then developed for detecting antibodies in wild-type BoHV-1 infected calves against the gE CT epitope specified by 499SDDDGPASN507. The assay is based on the use of HRP conjugated mAb2H8F3 and the E. coli expressed gE CT protein as an indicator antibody and a coating antigen, respectively. In this assay, serum from entire gE-deleted and BoHV-1 tmv-infected calves scored negative, whereas serum from calves infected with BoHV-1 wt scored positive. Therefore, the gE CT-ELISA, based on the mAb2H8F3 and E. coli expressed gE CT protein, is suitable for differentiating the wt virus-infected and BoHV-1 tmv-vaccinated cattle.

A potentiometric biosensor for rapid on-site disease diagnostics.

Quantitative point-of-care (POC) devices are the next generation for serological disease diagnosis. Whilst pathogen serology is typically performed by centralized laboratories using Enzyme-Linked ImmunoSorbent Assay (ELISA), faster on-site diagnosis would infer improved disease management and treatment decisions. Using the model pathogen Bovine Herpes Virus-1 (BHV-1) this study employs an extended-gate field-effect transistor (FET) for direct potentiometric serological diagnosis. BHV-1 is a major viral pathogen of Bovine Respiratory Disease (BRD), the leading cause of economic loss ($2 billion annually in the US only) to the cattle and dairy industry. To demonstrate the sensor capabilities as a diagnostic tool, BHV-1 viral protein gE was expressed and immobilized on the sensor surface to serve as a capture antigen for a BHV-1-specific antibody (anti-gE), produced in cattle in response to viral infection. The gE-coated immunosensor was shown to be highly sensitive and selective to anti-gE present in commercially available anti-BHV-1 antiserum and in real serum samples from cattle with results being in excellent agreement with Surface Plasmon Resonance (SPR) and ELISA. The FET sensor is significantly faster than ELISA (<10 min), a crucial factor for successful disease intervention. This sensor technology is versatile, amenable to multiplexing, easily integrated to POC devices, and has the potential to impact a wide range of human and animal diseases.

A New ELISA Using the ANANAS Technology Showing High Sensitivity to diagnose the Bovine Rhinotracheitis from Individual Sera to Pooled Milk.

Diagnostic tests for veterinary surveillance programs should be efficient, easy to use and, possibly, economical. In this context, classic Enzyme linked ImmunoSorbent Assay (ELISA) remains the most common analytical platform employed for serological analyses. The analysis of pooled samples instead of individual ones is a common procedure that permits to certify, with one single test, entire herds as "disease-free". However, diagnostic tests for pooled samples need to be particularly sensitive, especially when the levels of disease markers are low, as in the case of anti-BoHV1 antibodies in milk as markers of Infectious Bovine Rhinotracheitis (IBR) disease. The avidin-nucleic-acid-nanoassembly (ANANAS) is a novel kind of signal amplification platform for immunodiagnostics based on colloidal poly-avidin nanoparticles that, using model analytes, was shown to strongly increase ELISA test performance as compared to monomeric avidin. Here, for the first time, we applied the ANANAS reagent integration in a real diagnostic context. The monoclonal 1G10 anti-bovine IgG1 antibody was biotinylated and integrated with the ANANAS reagents for indirect IBR diagnosis from pooled milk mimicking tank samples from herds with IBR prevalence between 1 to 8%. The sensitivity and specificity of the ANANAS integrated method was compared to that of a classic test based on the same 1G10 antibody directly linked to horseradish peroxidase, and a commercial IDEXX kit recently introduced in the market. ANANAS integration increased by 5-fold the sensitivity of the 1G10 mAb-based conventional ELISA without loosing specificity. When compared to the commercial kit, the 1G10-ANANAS integrated method was capable to detect the presence of anti-BHV1 antibodies from bulk milk of gE antibody positive animals with 2-fold higher sensitivity and similar specificity. The results demonstrate the potentials of this new amplification technology, which permits improving current classic ELISA sensitivity limits without the need for new hardware investments.

Comparison of diagnostic tests for diagnosis of infectious bovine rhinotracheitis in natural cases of bovine abortion.

Rapid and precise diagnosis plays a pivotal role in implementing suitable control measures in natural field cases of bovine abortion due to infection with bovine herpesvirus (BHV)-1. In the present study, serology, virus isolation, histopathology, immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) for amplification of the gene encoding glycoprotein B were applied for diagnosis of infectious bovine rhinotracheitis (IBR) in cases of abortion. The seroprevalence of IBR in the population studied was 26.3% as determined by indirect enzyme-linked immunosorbent assay. BHV-1 abortions occurred between 4 and 8 months of gestation with an average gestational age of 6 months. Affected placentae showed necrosis of chorionic villi and of the endothelium of small villous blood vessels with characteristic intranuclear (IN) acidophilic inclusion bodies. Similar inclusions were also seen in most of the tissues examined. BHV-1 antigen was identified immunohistochemically in necrotic foci in the liver, the endothelium of placental blood vessels, the bronchial epithelium and hepatocytes. Lesions in the brain also had IN inclusion bodies that labelled positively by IHC. Eighteen samples (nine of stomach content, two of placental cotyledons, five of pooled fetal tissue and two of vaginal discharge) out of 84 tested were positive by real-time PCR for BHV-1.