Suelee Robbe-Austerman

Introduction: Brucellosis, caused by a group of bacteria within the genus Brucella, is a zoonotic disease of significant public health concern. The RB51 strain vaccine is used to combat cattle brucellosis but has potential health consequences for exposed humans. Methods: A retrospective review of NVSL's records prior to 2021 allowed identification of six cases of RB51 strain Brucella in the milk of dairy cattle through culture, followed up by PCR and whole genome sequencing (WGS). Results: Six properly vaccinated dairy heifer calves actively shed strain RB51 in their milk as adults, years after vaccination. Two of these cows are linked to three human strain RB51 brucellosis cases (one cow to one human, one cow to two humans). All human cases were detected following consumption of unpasteurised (‘raw’) dairy products, highlighting the risk of raw dairy consumption. Conclusions: These cases demonstrate that milk‐borne RB51 strain Brucella has been responsible for multiple human cases of Brucella . Dairy products from cows vaccinated with RB51 should not be consumed raw. The continued and widespread use of the RB51 vaccine should be evaluated critically, and authorities and veterinarians should revisit regulations and vaccination practices to ensure they are epidemiologically justified, particularly in regions or herds without documented brucellosis risk.

African swine fever virus (ASFV), the etiologic agent of African Swine Fever (ASF), is a high-consequence pathogen requiring experiments to be conducted in containment in non-endemic countries, thereby restricting diagnostic development, the creation of reference standards, and proficiency testing (PT). Safe and reliable inactivation methods are essential to expand diagnostic capacity while preserving nucleic acid integrity for molecular assays in unaffected countries. This study employed gamma irradiation to achieve complete inactivation of ASFV without compromising downstream molecular detection, as gamma irradiation offers deep penetration and uniform dose delivery. ASFV-cell culture supernatants were subjected to gamma irradiation doses ranging from 2 to 50 kGy. Viral replication was evaluated using TCID₅₀ and serial passages, revealing a consistent dose‑dependent reduction in infectivity across increasing irradiation dose levels and a complete loss of ASFV infectivity at 30 and 50 kGy. Molecular detection remained unaffected at all of the tested doses as confirmed by qPCR Ct values and sequence identity of the p72 gene. Whole genome sequencing demonstrated >99% genome coverage and consistent read depth profiles across irradiated and non-irradiated samples, indicating preservation of genomic integrity at all tested doses. These findings demonstrate that gamma irradiation at 50 kGy fully inactivates ASFV-cell supernatants while maintaining nucleic acid quality suitable for molecular diagnostics. The resulting inactivated material meets quality assurance requirements for molecular reference standards and PT panels and can be safely distributed to laboratories outside high containment facilities, supporting broader diagnostic readiness and harmonization of ASFV testing.

Understanding the gross lesion distribution associated with bovine tuberculosis (bTB) and the relationship between antemortem test results is important for optimizing surveillance activities. Antemortem diagnostic test results and postmortem examination results from animals infected with bTB in the United States are routinely collected during surveillance and outbreak response. From 2017 to 2022, data were compiled and analyzed from 403 infected animals, representing both beef and dairy cattle from a variety of herds with different levels of disease prevalence. Overall, 95.3% of cattle infected with Mycobacterium bovis presented with gross lesions. Most cattle had lesions in one anatomic region. Lesions of the thorax and head were most common with 42.4% of infected cattle having lesions in only the thorax, 18.4% having lesions only in the head, and 15.6% having lesions in both the head and thorax. The most frequently affected tissues were the mediastinal, tracheobronchial, and medial retropharyngeal lymph nodes. Results of antemortem skin tests were not found to be associated with lesion count or location. This analysis presents an updated look at the current state and presentation of bTB in the U.S., makes use of data collected in the field, and can help guide future bTB surveillance and control strategies.

African swine fever virus (ASFV) is a highly transmissible pathogen affecting swine, causing a devastating disease with high mortality rates in naive populations. Given the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten the U.S. swine industry, given their proximity and involvement in movement to and from North America. Although surveillance programs are ongoing, limited point-of-care (POC) tests are available during outbreaks with the sensitivity and specificity standards of laboratory testing (e.g., real-time PCR [rtPCR]). However, the recently developed CRISPR-Cas–based testing systems may offer comparable high-quality results. We sought to develop a low-cost visual detection method for ASFV by employing a recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a POC assay. Our CRISPR-Cas12a assay had comparable sensitivity and specificity to rtPCR, both visually and when quantified using a fluorescence reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, our CRISPR assay achieved a sensitivity of 98.3% (10 ² DNA copies) and a specificity of 100%. Test results of our RPA-CRISPR assay can be visualized in as few as 7 min, with peak fluorescence at 40 min (RPA and CRISPR steps). Our results lay the groundwork for a large-scale POC assay assessment for ASFV detection and offer a robust workflow that works with commonly submitted diagnostic samples.

Highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus was identified in 4 cull dairy cows condemned by the U.S. Department of Agriculture because of pneumonia with accompanying systemic changes. Histologic findings were bronchopneumonia in 3 cows and embolic pneumonia and nephritis in 1 cow. In addition to detection of HPAI A(H5N1) virus by reverse-transcription real-time PCR in various formalin-fixed, paraffin-embedded tissues, influenza A virus was detected by immunohistochemistry and in situ hybridization in the pulmonary respiratory epithelium of 2 of the cows with bronchopneumonia and in renal medullary tubules of the cow with nephritis. A PCR panel screening for common bovine respiratory pathogens in the cows with bronchopneumonia revealed variable coinfections with Histophilus somni , Pasteurella multocida , Mannheimia haemolytica , Mycoplasmopsis bovis , and bovine coronavirus. We describe the distribution of HPAI A(H5N1) virus in naturally infected cows while highlighting the need for research on the roles of coinfection and immune response in influenza viral replication.

Highly pathogenic avian influenza (HPAI) A(H5N1) virus, clade 2.3.4.4b, genotype 3.13, was first confirmed in dairy cattle in March 2024 in a Texas dairy herd and has since spread to other states, likely via movement of subclinically affected cattle. On 2024 May 5, a backyard poultry farm in Idaho reported sick and dying chickens. This pasture-based farm included 1,100 chickens, 32 ducks, 18 alpacas, 13 yaks, 3 cats, 3 dogs, 1 llama, and 1 goat. Most animals had direct access to a pond filled by the stream bordering the property. Additional dairy premises in the same county had been confirmed with HPAI during the same timeframe. The poultry were depopulated on May 10, the same day the first alpaca abortion was observed. Overall, 4 abortions occurred among the alpacas. In one of the abortions, HPAI A(H5N1) virus was isolated from fetal tissues. Additional testing documented seroconversion in several alpacas and detection of HPAI A(H5N1) virus in milk from an alpaca with a cria. To our knowledge, HPAI A(H5N1) virus, clade 2.3.4.4b, has not been reported previously in alpacas.

Described within this protocol are the preparation, performance, and interpretation of a real-time polymerase chain reaction (rtPCR) assay for African Swine Fever Virus (ASFV) surveillance and diagnostic testing using the Applied Biosystems™ QuantStudio™ 5 (ABI QS 5) platform and TaqMan™ chemistry. To monitor extraction efficiency and PCR inhibition an exogenous internal extraction control (Xeno™ RNA) is incorporated. Successful execution of this assay is expected to produce sigmoidal amplification curves with Ct's below 40 for valid controls and positive samples. To better increase laboratory flexibility three commercial mastermix options are presented: VetMAX™ Fast Multiplex Master Mix (with ROX) and UltraPlex® 1-Step ToughMix® Low ROX™ (4X) and TaqMan® Fast Virus 1-Step Master Mix.

Described and outlined within this protocol is the standardized methodology for the propagation, processing, and titration of Vero cell adapted African Swine Fever Virus (ASFV) to produce viral stocks for experimental use. Utilizing the Vero cell line CCL-81, ASFV Lisbon-60 is propagated until 70% of the monolayer exhibit cytopathic effects (CPE), generally within 5-8 days. To maximum viral harvest yields, infected flasks undergo a freeze-thaw cycle followed by centrifugation to obtain clarified viral supernatant. The virus is then titered using TCID50 assays and stored at −80°C for future use.

This protocol describes a general methodology for inactivating viruses using PrimeStore® Molecular Transport Media (PSMTM) to allow for the safe transfer of diagnostic samples from high containment laboratories (BSL3/4) to lower biosafety levels (BSL1/2). PSMTM eliminates the need for standard cold chain transport, enabling ambient temperature shipment, and reduces stringent transport conditions while ensuring rapid viral nucleic acid stabilization. Proper inactivation is achieved by maintaining specific media to sample ratios and incubation time conditions. Upon successful execution complete viral inactivation is expected and may be confirmed through TCID50 titration assays and polymerase chain reaction (PCR) testing. For cytopathic effect (CPE) producing viruses’ success is shown through the absence of any CPE and can also been seen by increases in cycle thresholds (Ct) values across a minimum of three serial passages. For non CPE producing viruses Ct values must be seen increasing across three serial passages.