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Current state and future directions for veterinary antimicrobial resistance research

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Antimicrobial resistance (AMR) is a critical One Health concern with implications for human, animal, plant, and environmental health. Antimicrobial susceptibility testing (AST), antimicrobial resistance testing (ART), and surveillance practices must be harmonized across One Health sectors to ensure consistent detection and reporting practices. Veterinary diagnostic laboratory stewardship, clinical outcomes studies, and training for current and future generations of veterinarians and laboratorians are necessary to minimize the spread of AMR and move veterinary medicine forward into an age of better antimicrobial use practices. The purpose of this article is to describe current knowledge gaps present in the literature surrounding ART, AST, and clinical or surveillance applications of these methods and to suggest areas where AMR research can fill these knowledge gaps. The related Currents in One Health by Maddock et al, JAVMA , March 2024, addresses current limitations to the use of genotypic ART methods in clinical veterinary practice.

Antimicrobial resistance (AMR) is a One Health challenge with far-reaching implications for human, animal, plant, and environmental health. 1 In 2019, 1.27 million human deaths were attributed to bacterial AMR, and the impact is expected to continue to grow if substantive changes or interventions are not made across all health sectors. 2 The American Society for Microbiology and National Action Plan for Combating Antibiotic-Resistant Bacteria (2020–2025) advocate for a One Health approach to AMR, particularly in regard to research funding, to improve diagnostic methods, prevention strategies, and public health surveillance of AMR. 3 , 4

Veterinary diagnostic laboratories (VDLs) are critical One Health partners in combatting AMR through the identification of pathogenic bacteria, through the performance of antimicrobial susceptibility testing (AST), and as a source of expertise for veterinarians regarding resistance mechanisms and therapeutics. VDLs are also critical in the communication of AMR data to human and animal public health practitioners. To maintain capacity and minimize the spread of AMR, diagnostic veterinary medicine requires resources, education, and advocacy to support appropriate antimicrobial use (AMU) and characterization of AMR challenges in animals. In this review, we will highlight areas of research and diagnostic support needed for standardization of laboratory testing, surveillance across the One Health spectrum, laboratory stewardship, clinical outcomes, and true One Health partnership. We will also suggest areas of opportunity for interdisciplinary collaboration as well as training and education needs for veterinarians and laboratorians.

One Health Harmonization

AMR-centric studies with the greatest impact will consider the problem through an interdisciplinary lens ( Figure 1 ) . The battle against AMR requires a robust, interdisciplinary approach, but the current system lacks harmonization in many areas. AMR phenotypes and genotypes of One Health concern are commonly discussed, but there is little consensus across the One Health spectrum regarding detection, reporting, and action based on the detection of AMR. 5 – 7 Nosocomial outbreaks with antimicrobial-resistant bacteria are predicted to become more common in veterinary medicine; these bacteria have zoonotic potential and reporting should be clear and unified. 5 , 8 , 9 For example, extensive AMR surveillance systems are in place for human medicine at the state level in the US, but often it is unclear whether an animal with a resistant infection of One Health concern, such as a carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE), should be reported to state public health officials or if reporting is at the discretion of the respective Board of Animal Health or state animal health regulators. 5 – 9

Research domains through the One Health antimicrobial resistance (AMR) lens. To minimize the adverse impact of AMR development, it is necessary to consider critically important antimicrobials and their use in human and veterinary medicine. ART = Antimicrobial resistance testing. AST = Antimicrobial susceptibility testing. Created with BioRender.com.

Citation: American Journal of Veterinary Research 85, 3; 10.2460/ajvr.23.12.0294

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Additionally, there are no One Health-oriented criteria for classifying a bacterium tested as resistant to an antimicrobial or what constitutes a multidrug-resistant organism (MDRO). Several authors 10 , 11 have independently proposed criteria, but lack of consensus results in inconsistencies in what is resistant and ultimately defines an MDRO classification, leading to differences in perceived rates of resistance to multiple drugs between human and veterinary medicine. For example, a veterinary clinical breakpoint may call a bacterium resistant whereas the human breakpoint would call it susceptible due to differences in pharmacokinetics entirely unrelated to whether the bacterium does or does not possess an expressed resistance mechanism. To complicate matters further, the epidemiologic cut-off value, a better measure of acquired resistance, may not align with either veterinary or human clinical breakpoints.

Limitations in available test methods pose an additional challenge in the coordination of definitions of resistance across sectors. For example, recommended phenotypic methods for the detection of CP-CRE, which are resistant to all β-lactams approved for use in veterinary medicine, are harmonized between Clinical and Laboratory Standards Institute (CLSI) human and veterinary testing criteria. However, animal species-specific breakpoints do not exist for carbapenems and their use in veterinary medicine is illegal in some jurisdictions. Therefore, testing, recognition of public health importance, and reporting practices vary across laboratories. Several commonly used commercial veterinary MIC methods test only imipenem, rather than meropenem, which is considered by CLSI, the CDC, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) to represent the best balance of sensitivity and specificity when screening for the presence of carbapenamases. 12 – 14 While CP-CRE remains more commonly encountered in human medicine, reports among veterinary species are increasing globally, including documented nosocomial outbreaks within the US. 9 , 15 The lack of standardization for veterinary testing and/or reporting of carbapenems and lack of public health oversight for veterinary isolates are strong indicators that cases go unnoticed, furthering the potential for zoonotic transmission and global dissemination.

Due to the One Health significance of AMR and inconsistencies between sectors, collaboration, and adequate resources are necessary at the intersection of human, animal, and environmental health. Failure to collaborate and harmonize will undermine individual efforts in these areas. Public health reporting methods and standards should be harmonized to enable the appropriate interventions and ensure consistency of communication when it comes to AMR. 5 , 6

Veterinary Diagnostic Laboratories as Stewards

VDLs play a critical role in antimicrobial stewardship, although the power of laboratory communication to impact clinician behavior has not been fully explored in veterinary medicine. Often, VDLs report all antimicrobials tested on a susceptibility panel, regardless of their clinical appropriateness or the intrinsic resistance of an organism to a particular antimicrobial or antimicrobial class. Such practices should be discouraged, and veterinary clients should be aware that more results on an AST report are not indicative of better value or quality of test results. Several studies 16 , 17 in human medicine have demonstrated that laboratory reports directly impact physician treatment choices, termed Nudging in Microbiology Laboratory Evaluation (NIMBLE). Studies 17 – 19 of prescribers in human medicine have found that alterations to AST reports can improve the appropriateness of antimicrobial prescribing and that drugs listed on an AST report are more likely to be selected than those that are not included. A similar study of these reporting practices for both phenotypic AST and genotypic antimicrobial resistance testing (ART) in veterinary medicine may provide insight into ways that laboratories can help steer the reduction of and response to AMR in our animal populations.

AST Standardization and Optimization

Phenotypic AST is the current gold standard for determining which antimicrobials are most appropriate for clinical use. Phenotypic AST is performed by broth microdilution or gradient diffusion, resulting in MIC values, or by disk diffusion, resulting in zone diameter (ZD) data. The MIC or ZD data provide information on the concentration of antimicrobials that have an inhibitory effect on bacterial growth, but these values alone are not indicative of clinical efficacy and must be interpreted using clinical breakpoints such as those developed by the CLSI or the EUCAST. 20 , 21 The clinical breakpoint allows the laboratory to provide the veterinarian with clinically predictive information on the likelihood of successful treatment of the patient’s infection with a particular antimicrobial.

Genotypic test methods, such as whole genome sequencing (WGS), targeted sequencing, and metagenomic sequencing, are increasingly suggested as alternatives to traditional phenotypic methods. 22 , 23 Genotypic ART is the use of molecular methods (PCR, targeted sequencing, WGS, or metagenomic sequencing) to predict the presence of AMR genes. 22 In a clinical context, genotypic ART is used to determine which antimicrobials should not be used, whereas phenotypic AST guides the clinician on which antimicrobials could be used. Genotypic ART methods are increasingly used in human laboratory medicine in conjunction with phenotypic AST, and it would be best practice for veterinary medicine to adopt the same model if genotypic methods are used. Still, the incorporation of these methods in veterinary diagnostic medicine is unclear due to the high cost associated with routine use and lack of outcome studies.

Although widely used as part of the diagnostic testing algorithm, there are still many gaps in veterinary phenotypic AST. Commercially produced AST panels used in human medicine are stringently regulated by the FDA. In contrast, there is no regulation of veterinary AST panels, so the level of rigor and proven performance standards required for human AST panels is not required. While the potential harms of this are not well documented, the onus of verification and/or validation of test methodology is the sole responsibility of the VDL, which may have few resources for such activities. Recently, users of commercial AST panels were made aware of interlaboratory methodological discrepancies due to a lack of clarity in device instructions for use. 24 This highlights an additional need for more thorough evaluations of AST panels used in veterinary medicine to determine if resistance is being accurately detected. It also suggests more extensive training may be necessary for those performing veterinary AST, which might be accomplished through a variety of mechanisms, including proficiency testing, live training events, and webinars.

MIC-producing AST methods are often thought to be more desirable because of the numerical value and pharmacokinetic-pharmacodynamic (PK-PD) relationship that can be assigned to the result. However, more data are not always better, and no single method will perform best for every species of bacteria. Disk diffusion testing continues to play an important role in veterinary AST and is frequently used in human laboratories. There is a need for additional research and data to expand veterinary disk diffusion antimicrobial options. Interpretive criteria are required to use ZD and MIC data to make predictions about clinical outcomes in patients.

A greater diversity of commercial AST methods is desirable to fit the needs of the veterinary diagnostic community. Currently, most VDLs perform AST using disk diffusion or broth microdilution. 7 Disk diffusion is highly standardized and has been widely used for AST for many decades; however, many veterinary-specific MIC breakpoints have been published in CLSI VET01S without corresponding disk diffusion breakpoints, making MIC methods the only option available for laboratories seeking to utilize veterinary-specific breakpoints. A mixture of available AST methodologies is necessary when testing diverse bacterial species, and careful consideration must be given to which reference methods are most appropriate for use.

Some fastidious bacteria (e.g., Pasteurellaceae ) grow better on a solid medium, such as those used in disk diffusion, agar dilution, or gradient diffusion methodologies. Bacterial isolates that are growing well will produce more accurate, reproducible results. Manufacturer claims of utility for fastidious bacteria do not ensure the appropriateness of the method recommended for use. 25 Solid medium data for more fastidious bacterial species are just as powerful as broth microdilution methods, because the method is more appropriate and reproducible for that bacterium. Such test methods can then be easily and affordably implemented in a diagnostic setting, and AST findings generated from these studies can be used to help develop consensus data for standards development. In many cases, only a few antimicrobials are necessary for routine testing due to predictable susceptibility patterns of an organism in line with recommended first-line treatment recommendations. In such cases, an entire broth microdilution panel is not necessary and the flexibility to choose a small number of antimicrobials for a disk diffusion method will be more affordable. Limiting the number of antimicrobials tested and reported directly supports stewardship by focusing attention on the most appropriate narrow-spectrum antimicrobial. 16

Phenotypic to genotypic correlation studies are powerful and potentially impactful for diagnostic medicine. Initial genotypic research studies should include both phenotypic and genotypic profiling. Agreement between testing will corroborate results, and disagreements will identify limitations of each test method or identify areas requiring additional research. Currently, there are 2 national veterinary programs geared at phenotypic to genotypic correlation studies. 26 – 28 Beyond phenotypic to genotypic correlation, these programs help laboratories compare methods and may help find areas of test method variability. 24 It should be noted that while important, surveillance data may be biased toward bacterial species with acquired resistance mechanisms (non-wild–type populations) due to isolation from clinically ill animals, which may skew resistance rates.

AST in Research

Quality control (QC) should be woven into all tasks performed to ensure consistency, quality of test results, and competency of personnel. QC must be included in research studies with procedures intended to be used in a diagnostic setting to ensure that results are consistent, reproducible, and directly translatable to practice in the laboratory and clinical setting. QC is necessary regardless of whether a genotypic or phenotypic approach is used. 21 , 29 In the case of phenotypic AST, users must refer to the interpretive criteria of choice (CLSI and EUCAST) to determine which QC organisms are appropriate for a particular antimicrobial and organism. 21 Appropriate QC for genotypic methods will depend on the approach used. For PCR, positive and negative controls along with a dilution series and tests of sensitivity and specificity are required. 30 WGS and metagenomic approaches will have different requirements for QC. 29 , 31 , 32 No international standards have been developed for quality assurance, but many proposed guidelines are available and should be evaluated for use in these genotypic ART studies. 29 , 31 , 32

Researchers must be aware that the methods used for phenotypic AST should only be used per manufacturer instructions for use and/or CLSI or EUCAST standards. Once altered, the methods are unstandardized and cannot be accurately interpreted by the interpretive criteria chosen (CLSI and EUCAST). 21 Whether contracting research to a diagnostic laboratory or performing the work in a traditional research setting, all parties should be aware of the exact methods used for testing and report any modifications to the manufacturer’s instructions or other standardized methods. 21

When mining retrospective data, it is important to review the precise methods used for AST performance, ideally comparing the laboratory protocol to the manufacturer’s instructions for use and interpretive standards used. CLSI and EUCAST clinical breakpoints are updated periodically as more literature and clinical outcome data are generated. Some of the breakpoint changes are major and will dramatically affect the number of organisms that are classified as resistant going forward. For example, the 2013 introduction of feline-specific amoxicillin-clavulanate breakpoints for Escherichia coli isolated from skin and soft tissue or urine resulted in all E coli isolated from cats being categorized as resistant. 33 Previous data may have relied on extrapolation of human-specific breakpoints that more closely predict the presence of AMR genes in E coli but do not adequately predict the clinical success of using amoxicillin-clavulanate to treat systemic infections in cats. In 2020, based on new data and a reanalysis of existing data, UTI-specific feline breakpoints were revised to align with the human-specific breakpoints and dog-specific urinary tract infection breakpoints. 34 A hypothetical retrospective study analyzing resistance trends over a 10-year period from 2012 to 2021 would, therefore, exhibit wild variation in the resistance rates. Depending on the time range of data collection, it might be necessary to harmonize the interpretation of the MIC or ZD results using a single edition of the interpretive criteria standard. 21 In addition, AST system manufacturers periodically update the dilution ranges on commercially available MIC plates, and older MIC results may not include test dilutions that correlate with current breakpoints. It is important to note that current and recently archived editions of CLSI VET01S and CLSI M100 are publicly available for free online at the CLSI website, so all researchers have open access to the most current standards.

Clinical Breakpoints

Clinical breakpoints are expressed as susceptible, susceptible dose-dependent, intermediate, resistant, or nonsusceptible. 12 , 35 Animal species-specific clinical breakpoints have yet to be established for many antimicrobials while some existing breakpoints require re-examination using current methods for breakpoint determination. Modern breakpoint evaluation is primarily informed by 3 types of data: PK-PD data, microbiological AST data, and clinical outcomes data. 36 , 37 Detailed information about wild-type distributions, PK-PD modeling, and clinical cutoffs have been described in-detail elsewhere. 21 , 38 – 40

Clinical cutoffs, determined by clinical outcome studies, play an important role in the establishm ent of CLSI breakpoints for humans but are infrequent in veterinary medicine. In the absence of outcome data, PK-PD data become increasingly important for setting veterinary speciesspecific breakpoints. However, PKPD data are sparse or incomplete for some antimicrobial drugs and in many species of animals, leaving laboratories to rely on extrapolation of breakpoints between animal species or humans. Additionally, based on a label or commonly used dose, PK-PD data may indicate a breakpoint that would split the wild-type population of bacteria, meaning that bacteria without an acquired resistance mechanism would be categorized as resistant to an antimicrobial due to the pharmacokinetic limitations in a particular animal species. 38 However, setting a breakpoint in the wild-type results in poor AST reproducibility, with isolates lacking acquired resistance mechanisms yielding variable results of susceptible or resistant if replicate testing were to be performed. Such reproducibility and accuracy issues have negative impacts on patients, AST method validation, and surveillance efforts. One solution to this problem may be through dose optimization that would allow for breakpoints that categorize all isolates within the wild-type population as Susceptible. Although frequently considered in human breakpoint evaluations and clinical practice, dose optimization is yet to be a widely accepted approach in veterinary medicine due to concerns about off-label use, especially in food animals. An additional limitation is the increased difficulty posed by administering drugs more frequently or parenterally. MIC and ZD distributions are also relied on but sometimes sufficient microbiological data have not been generated to determine the range of MICs demonstrated by the wild-type population of a bacterial species, making dose optimization and breakpoint determination a challenge.

When determining interpretive criteria for CLSI standards and guidelines, document developers rely on published studies or studies brought forth by sponsors. 41 It cannot be overemphasized that these studies must be conducted using standardized AST methods. For these studies to be most impactful and useful for CLSI or other standards setting organizations to consider for inclusion, consistent methods with appropriate QC, clear citations regarding which method was used for testing, atmospheric conditions, and appropriate test media must be used. 21 , 37 , 41 Importantly, as new CLSI and EUCAST clinical breakpoints are adopted or updated, it is imperative that interpretive criteria used in VDLs are updated; this is particularly critical when using automated AST instruments. It is imperative that result interpretations from automatic AST readers are also compared against a standard to ensure that correct interpretations were applied. Many users implicitly trust instrument software to provide accurate results; however, user review and validation should always be woven into software adoption and subsequent updates. 42 A recent survey 43 found that even in human laboratories the most updated CLSI breakpoints are not always used. New requirements from the College of American Pathologists will require updates to be made within 3 years of a new breakpoint development; however, these requirements are not in place for veterinary medicine. 44

Clinical Outcome Studies

Controlled clinical outcome studies regarding the efficacy of antimicrobial treatment protocols are scarce for veterinary medicine. Outcome studies would help define effective treatment regimens for different types of infections and thereby improve antimicrobial stewardship by providing evidence for treatment recommendations. In human medicine, large retrospective cohort studies 45 – 47 have been conducted to determine which CLSI breakpoints accurately predict response to selected antimicrobials, further validating developed breakpoints. In veterinary medicine, these studies would optimally occur in large teaching hospitals associated with a VDL, but VDLs could also partner with local clinics to establish, revise, or validate existing clinical breakpoints. Although more expensive to conduct, prospective studies using defined treatment regimens in a hospital setting would also be highly beneficial for the collection of phenotypic AST data.

Transparent AMU reporting should be encouraged and tools supporting collection and analysis of these data should be developed. 48 AMU reporting supports studies targeting prescribing practices and is valuable for the development of antimicrobial stewardship programs because evidence of inappropriate selection may help improve treatment choice. 48 – 50 Several studies 49 , 51 , 52 have found inappropriate selection of a third-generation cephalosporin over peer-reviewed first-line treatment options; interventions after targeted training were successful at minimizing inappropriate treatment selection.

Beyond treatment outcomes, studies demonstrating the clinical utility of rapid genotypic ART methods in conjunction with phenotypic AST are necessary to prove genotypic ART provides a faster response, better patient outcomes, reduced treatment costs, and reduction of AMR in veterinary medicine. 53 Commercial genotypic ART platforms to detect MDROs are available and have been used with success in human medicine. Because MDROs have One Health importance, these assays, such as those to detect CP-CREs, could be adapted for use with appropriate testing and infection control response based on positive test results. These test methods could help to minimize the spread of nosocomial infections in veterinary hospital settings. It has been suggested that de-escalation of antimicrobial therapies is a sufficient justification for use of such test systems; however, other considerations may include reduction in length of hospital stays, reduced patient mortality, and improved clinical decision-making. 53

Education and Training

The education of veterinarians and veterinary students on appropriate AMU is a critical need in veterinary medicine. Targeted education has the potential to improve antimicrobial prescribing practices. 49 , 50 , 54 Studies focused on development of training programs and behavior modification outcomes are necessary to further improve current strategies and expand training capacity will help shape the future of veterinary training.

Beyond research, there is a critical need for training veterinary microbiology specialists and microbiologists working in the laboratory. Due to retirements, changing laboratory test needs, and the small number of training programs, VDLs are experiencing a shortage of trained laboratorians and specialty-trained veterinary microbiologists. 55 Funding for veterinary microbiology training programs is necessary to ensure continued workforce development and expertise is available to VDLs and veterinarians.

Path Forward

Advances in test methodologies, phenotypic AST interpretive criteria, clinical outcomes, and surveillance efforts will ultimately improve patient care and preserve the efficacy of critically important antimicrobials. 3 , 4 Studies targeted at veterinary medicine must be prioritized for funding to ensure that antimicrobials remain effective for generations to come. Manufacturers of diagnostic tools must commit to partnership with VDLs to develop better test methods that capture the most current clinical breakpoints or recommended technologies while remaining affordable to clients. Education of veterinarians, veterinary students, and veterinary laboratory personnel is a critical need worthy of funding. Because these studies and training needs are necessarily interdisciplinary, effort should be made to include partnerships with diverse stakeholders across the One Health spectrum to ensure the most efficient and sustainable strategies are employed for use.

Acknowledgments

None reported.

Disclosures

Many of the authors are voluntary (unpaid) members of the CLSI-Veterinary AST subcommittee. The views expressed in this article do not necessarily reflect those of X-ZL’s affiliation, Health Canada.

No AI-assisted technologies were used in the generation of this manuscript.

The authors have nothing to disclose.

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Sarzynski SH , Lawandi A , Warner S , et al . Association between minimum inhibitory concentration values and mortality risk in patients with Stenotrophomonas maltophilia infections: a retrospective cohort study of electronic health records from 148 US hospitals . JAC Antimicrob Resist . 2023 ; 5 ( 2 ): dlad049 . doi: 10.1093/jacamr/dlad049

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Diagnostic lab & vet workforce development. United States Animal Health Association . Accessed December 20, 2023. https://www.usaha.org/diagnostic-lab-vet-workforce-development

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Editorial: Diagnostic Procedures in Veterinary Microbiology and Infectious Diseases

Valentina stefanetti.

1 Department of Veterinary Medicine, University of Perugia, Perugia, Italy

Doreene Hyatt

2 Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, United States

Fabrizio Passamonti

The authors are pleased to have been invited to serve as Guest Editors for the Research Topic entitled: “Diagnostic Procedures in Veterinary Microbiology and Infectious Diseases.” This Research Topic is a collection of 19 manuscripts (14 original research papers, four brief research reports, and one mini-review article) published from 130 authors, which, so far, have received thousands of views. The geographical areas represented include Europe ( n = 6), North America ( n = 5), South America ( n = 1), Australia ( n = 1), and Asia ( n = 6).

Infectious diseases are relevant to veterinary medicine, both in companion animals and in livestock, due to their ability to induce clinical illness and/or economic losses. Additionally, infectious diseases of animals are relevant to public health in that the causative organisms can be carried by animals and be spread to humans to cause zoonotic disease. Knowledge of the pathological mechanisms that microorganisms have developed is a critical issue for a correct and prompt diagnosis, which can be life-saving for animals. Inspired by the recent SARS-CoV-2 pandemic, we thought about the importance of working from the One-Health perspective and about how timely diagnosis of infectious diseases represents a significant challenge.

Diagnosticians use two primary methods to detect infectious diseases: confirming the presence of the microorganism or detecting antibodies against the organism. Despite the availability of an array of techniques, among the last-generation assays, no single test is usually considered the definitive golden standard, and that is the reason why there is still an urgent need for research on infectious disease diagnosis.

Therefore, out of the various research questions proposed by manuscripts submitted in this Research Topic section, the large majority of contributing studies describe new advances, i.e., novel assays or improvement of pre-existing tests for diagnostic purposes, in veterinary microbiology for several animal species.

Pig production is one of the fastest growing livestock sectors in the world. That is probably the reason why several contributions in our Research Topic are related to the main infectious diseases, which represent a significant threat for the swine industry worldwide. Of note, African swine fever (ASF) has caused great economic losses in the pork industry, and the current lack of commercially available vaccines makes the prevention and control of ASF worldwide even more challenging. Yu et al. , described the production and characterization of p30 monoclonal antibodies and the subsequent development of an ELISA kit for early detection of ASF virus (ASFV). This kit has high diagnostic specificity and sensitivity and is able to detect seroconversion in infected pigs as early as 10 days post-infection (dpi). Therefore, this assay is a very useful tool for early detection of ASFV. Jia et al. , described another method for ASFV DNA quantification, which utilizes a next-generation PCR platform, nanofluidic chip digital PCR. Due to its high sensitivity, this assay appears to be very useful, especially for the recognition of early ASFV infection. Velazques-Salinas et al. , in an attempt to find a potential vaccine to protect pigs against ASFV, developed attenuated vaccine candidates by deleting critical viral genes associated to virulence. In their paper, they describe how they developed three new quantitative PCR (qPCR) assays for differentiation between infected and vaccinated animals (DIVA). Collectively, the results of this study demonstrate the potential of qPCR assays as tests supporting ASFV vaccination.

Among other porcine viruses explored in our collection, Mai et al. , investigated the high coinfection status of novel porcine parvovirus 7 (PPV7). Sows with porcine circovirus 3 (PCV3) experience reproductive failure, suggesting that PPV7 may play a role in infection as cofactor by enhancing PCV3 replication. More analyses of the exact PPV7 pathogenesis mechanism are warranted, and there is an urgent need to further investigate coinfections in the porcine field. Liu et al. , studied porcine astroviruses (PAstVs), which are prevalent in pigs and of which five genotypes have been reported worldwide. Multiple PAstV genotypes and coinfection and genetic recombination events have often been reported. However, there is still no diagnostic method available for PAstV genotype detection. In this study, a multiplex reverse-transcription PCR (RT-PCR) method, which is efficient and convenient for the diagnosis of five known PAstV genotypes, was established. This method is a valuable tool for the differential diagnosis of PAstVs circulating in pig herds and will facilitate the surveillance of PAstV coinfection.

The emergence of new viruses or variants due to spillover or mutation or recombination events makes monitoring of porcine enteric coronavirus (CoV) of utmost importance in order to curtail their spread and allow for updated diagnostic tools. The emergence of new CoVs and the re-emergence of porcine epidemic diarrhea virus (PEDV) worldwide require studies characterizing these CoVs on pig farms in order to allow an accurate differential diagnosis of viral diarrhea. Although Puente et al. , demonstrated that PEDV is the only CoV currently circulating in Spain, they highlighted the need for constantly monitoring porcine enteric CoVs in order to prevent their spread and allow for updated diagnostic tools. So, gastrointestinal infectious diseases affect pigs worldwide and are one of the major concerns in many countries. The disease occurs usually at the post-weaning stage and impairs pig performance, weakens welfare, and causes economic losses to farmers due to medication costs as well as slow growth. One of the main challenges to investigate the health status of pigs is to find a less stressful and non-invasive sampling method. Sali et al. , explored the dynamics of salivary biomarkers such as adenosine deaminase (ADA), haptoglobin (Hp), and cortisol from saliva samples of growing pigs exposed to LPS challenge. Their study suggests that ADA and Hp can be used as inflammatory biomarkers in pigs.

Infectious diseases of livestock are a major threat to global animal health and welfare and their effective diagnosis is crucial also for human health. Okda et al. , developed diagnostic tools to differentiate between influenza D virus (IDV), a novel orthomyxovirus emerging in cattle worldwide, and the human influenza C virus (ICV), through an ELISA test. It is critical to develop diagnostic tools and assays to differentiate between ICV and IDV due to their similar genomic structures. These authors successfully developed a blocking ELISA assay able to differentiate between these two closely related viruses.

Bovine respiratory disease (BRD) is one of the major causes of losses for the cattle industry worldwide, and the efforts to improve diagnostic procedures to identify pathogens caused BRD are remarkably increasing, also to provide some indication for veterinarians. Klompmaker et al. , used high-throughput real-time PCR to detect some bovine respiratory pathogens and quantified the cut-off values for pathogens associated with BRD in an effort to analyze the association between these findings and clinical observations. The authors found that it is possible to suggest clinically relevant cut-off values with statistically significant associations with clinical scores indicating respiratory disease for P. multocida, M. bovis , and H. somni . This study thereby presented an interesting approach for objective and veterinary field-relevant diagnostic test interpretation. The same research group, in the wider project of Goecke et al. , aimed to design and develop a high-throughput real-time PCR system for the detection of significant respiratory and enteric viral and bacterial bovine pathogens. Nowadays, detection of these pathogens is costly and time consuming due to the methodology used and due to the fact that several different PCR assays are needed to cover the wide range of circulating pathogens. Their study shows that the high-throughput real-time PCR method allows simultaneous analysis of a large number of samples and contributes to more detailed diagnostics.

Another biomolecular assay was developed by Hole et al. , for the diagnosis of vesicular stomatitis virus (VSV). Vesicular stomatitis virus causes a disease in susceptible livestock that is clinically indistinguishable from foot-and-mouth disease (FMD). Rapid testing is therefore critical to identify VSV and rule out FMD. The authors improved a previously published multiplex real-time RT-PCR (mRRT-PCR) assay. In this paper they highlight the challenges that the large genetic variability of VSV poses for virus detection by mRRT-PCR and show the importance of frequent re-evaluation and validation of diagnostic assays for VSV to ensure high sensitivity and specificity. Similarly, Subbiah et al. , focused on the emerging field of next-generation sequencing and metagenomics to address the problem of detecting disease conditions in veterinary science with a metagenomic shotgun sequencing approach for unbiased detection of the microbiome. The authors used horses that were suspected to have tick-borne diseases and demonstrated the detection of A. phagocytophilum , suggesting that this approach can be used to detect blood-borne pathogens.

In recent years, the incidence of brucellosis has increased annually, causing economic losses in various countries. Therefore, the development of rapid, sensitive, and specific diagnostic techniques for brucellosis has become critical. Yin et al. , used “immunoinformatic” technology to predict the B cell epitopes in the major outer membrane proteins of Brucella , establishing an ELISA method for brucellosis diagnosis based on a multi-epitope fusion protein that can be used to assess the serum of bovine, goats, and other livestock.

One of the aims of our Research Topic section was to find new diagnostic approaches to replace the so-called golden standard tests. Dall'Ara et al. , demonstrated the utility of an in-clinic ELISA test in detecting protective antibodies against canine parvovirus (CPV) in adult dogs and compared it with the golden standard, the hemagglutination inhibition (HI) test, both after vaccination and/or infection. Moreover, in unvaccinated puppies, specific CPV maternally derived antibody titers can be measured more easily by an in-clinics ELISA test, allowing the prediction of the best time of vaccination, thus reducing the rate of vaccination failures.

Silvestri et al. , investigated the methicillin resistance of S. pseudintermedius ( SP ) through a new approach. The resistance is mediated by the mecA gene, which encodes the PBP2a protein and conveys resistance to β-lactams. The authors showed that immunofluorescence is a promising technique, with a good capability of correctly identifying resistant and sensitive SP . Immunofluorescence has the potential to be applied as a screening method, independent from the golden standard, bacterial culture. Therefore, it represents a new interesting tool for both research and diagnostics.

Guedes et al. , investigated the usefulness of the ranking technique to predict the most likely infecting serogroup of Leptospira . The microscopic agglutination test (MAT) used for the serological diagnosis of leptospirosis, as a robust and inexpensive method, is still the reality in many laboratories worldwide. However, both the performance and the interpretation of the MAT vary from region to region, making standardization difficult. Of course, MAT replacement may be difficult, but the authors proposed the ranking technique as another way of interpreting the results obtained by MAT, in order to refine the data and reduce the occurrence of cross-reactions between the serogroups, thus demonstrating that this technique can be useful in the MAT for predicting the most likely infecting serogroup of Leptospira and can be applied in epidemiological studies involving herds.

Hwang et al. , performed an interesting study on chronic wasting disease (CWD), which is a transmissible spongiform encephalopathy in free-ranging and captive cervid species. The disease process is ultimately fatal and occurs through the misfolding of a normally occurring protein. Detection of this misfolded protein is the only known means by which a prion disease can be diagnosed, and it is typically performed after death. The authors developed an approach for the detection of this misfolded protein using a technique known as real-time quaking-induced conversion (RT-QuIC) from secretions and excretions. Real-time quaking-induced conversion is a powerful tool to detect infectious fecal prions from CWD-infected white-tailed deer, well before the onset of clinical disease.

Interestingly, Peng et al. , developed an intelligent genotyping platform, PmGT, for P. multocida strains according to their whole genome sequences using web 2.0 technology. Using PmGT, the authors determined capsular genotypes, LPS genotypes, and MLST genotypes as well as the main virulence factor genes of P. multocida isolates from different host species based on their whole genome sequences published on NCBI. The results revealed a close association between the genotypes and pasteurellosis rather than between genotypes and host species. With the advent of high-quality, inexpensive DNA sequencing platforms, PmGT represents a more efficient tool for P. multocida diagnosis in both epidemiological studies and clinical settings.

Finally, a review article by Cameron et al. , was included in our Research Topic section, covering the current knowledge and limitations of the use of field-based nucleic acid amplification technology termed loop-mediated isothermal amplification (LAMP) for the diagnosis of honey bee pathogens and pests. Loop-mediated isothermal amplification assays have become an important tool in the last few years in the detection of both exotic and endemic pathogens in the livestock industry.

Taken together, all the published papers in this Research Topic contribute to the development and improvement of techniques and strategies to control animal infectious diseases, exploring novel diagnostic tests, and new technologies that have the potential both to advance our understanding of bacterial/viral animal infections and to improve current diagnostic and control strategies.

Author Contributions

All authors listed as Guest Editor for this Research Topic have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Diagnostic Procedures in Veterinary Microbiology and Infectious Diseases

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Infectious disease agents capable of inducing clinical illness are quite common in veterinary medicine and their prompt diagnosis is often life-saving for animals. Knowledge of the pathological mechanisms developed by is critical for correct diagnosis and possible therapy. Additionally, animals can sometimes ...

Keywords : Veterinary Microbiology, Veterinary Infectious Diseases, Diagnostic tests, Diagnostics

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Forging the future of veterinary virology

May 30, 2024

“This is what I’ve been missing my whole life.” That’s what Estefany L. Cotto-López thought during her first course in virology as a masters student—and it’s what led her to the Veterinary Sciences PhD program at the University of Minnesota College of Veterinary Medicine (CVM).

With a BS in microbiology and two masters degrees—one in environmental health from the University of Puerto Rico Medical Sciences Campus, and another in One Health from the University of Florida—Cotto-López is no stranger to academic life. In her first year at the College of Veterinary Medicine, both the academic challenges she’s faced and the successes she’s experienced have grown.

Plus, she survived her first Minnesota winter.

“I didn’t know how I was going to deal with snow—this is a completely different environment for me—but this winter wasn’t so bad!” Cotto-López reported.

She had plenty to keep her busy. Cotto-López joined the lab of  Dr. Yuying Liang and  Dr. Hinh Ly   to pursue her interest in virology and zoonotic diseases (diseases that can spread between animals and humans). Her interest in working with animals had developed during an internship she completed with the Veterinary Services program at the United States Department of Agriculture. “They thought I wouldn’t last long because I come from a public health background and went from working in offices to working on farms,” Cotto-López described. “But I was thrown in to work with cattle and I fell in love with it.”

Her current work focuses on a swine virus called Porcine Reproductive and Respiratory Syndrome (PRRS) virus, a serious disease that manifests in reproductive failure or death in pigs due to complications from fetal development or pneumonia. In addition to animal welfare concerns, the disease causes significant economic loss, costing U.S. pork producers over half a billion dollars each year.

With the Liang/Ly lab team, Cotto-López is exploring the use of Pichinde virus—a virus that’s not known to cause disease in animals or humans—to develop improved vaccines for PRRS and other diseases.

Her work on this cutting edge of virology has quickly won her accolades, including an invitation to participate in a highly competitive summer training program through the Kansas State University’s Center of Excellence for Emerging and Zoonotic Animal Diseases. The 2024 BSL-3 Training/Transboundary Animal Disease Summer Program is sponsored by the USDA Agricultural Research Service and will be conducted at the  Biosecurity Research Institute   of the  Kansas State University, adjacent to the  National Bio and Agro-Defense Facility (NBADF). The NBADF is a newly constructed biocontainment lab for large animal research that’s the first of its kind in the United States, and one of only a handful in the world.

“I want to get as much training as possible to learn how to work with pathogens at a high biosecurity level,” said Cotto-López. “My ultimate goal is to work in a BSL4 facility”—a facility with the highest level of biosecurity, such as the NBADF, where high-consequence animal pathogens can safely be studied.

Along with that honor, she received the Maheswaran Graduate Fellowship from the College of Veterinary Medicine, along with a  MNDrive Global Food Ventures award for her work toward the development of vaccines for swine diseases.

With her regular travels between the Liang/Ly lab on the U of M St. Paul campus and Worthington, MN where she works with pigs at the Vaxxinova company, the summer program at Kansas State, and a trip to Ohio to attend the annual conference of the American Society for Virology (“It’s like Diseyland for virologists”), Cotto-López’s summer is shaping up to be just as head-spinning as her first year at CVM.

“There’s a line in a song by the band Florence and the Machine that goes ‘Happiness hit her like a train on a track,’ and it’s the perfect description,” she said. “I’ve had doors open for everything at the same time. It’s been a lot to handle all at once. But I’m living my dream! I just need to remember to stop and breathe sometimes.” 

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Tags: Veterinary and Biomedical Sciences

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Research identifies possible new pathway to treatment of colorectal cancer

by Kevin Myatt, Virginia Tech

Research led by Irving Coy Allen in the Virginia-Maryland College of Veterinary Medicine has unlocked a pathway to possible future treatments for colorectal cancer in humans.

A paper published in May in Cellular and Molecular Gastroenterology and Hepatology focuses on NF-kB-inducing kinase (NIK) and its importance in triggering cellular responses that reduce the risk of the development of colorectal cancer .

"The gene itself is colloquially called NIK, and it encodes a protein that is a kinase, which means it basically turns on or turns off—mostly turns on—lots of different genes and pathways," said Allen, professor of inflammatory diseases in the Department of Biomedical Sciences and Pathology.

"It gives us a central spoke in a hub of biological mechanisms that can be targeted with possible therapeutics. We do know that there are companies working on developing drugs to target NIK. We're hoping that this can provide them with incentive to go after these drug candidates more aggressively."

Colorectal cancer is second deadliest form of cancer in the United States, killing over 52,000 people in 2023. Present treatment options are often based on chemotherapy and can be difficult for the patient to endure.

"By identifying new markers and new drug targets, it may provide us with better therapeutic approaches that can minimize side effects and improve overall patient outcomes," Allen said.

While much of the study was conducted examining mice, and that alone could have generated valuable research, Allen's team took the extra step of translating it directly to human patients.

"By modeling it in mice, we were able to identify things to look for in humans," Allen said. "Through collaboration with the Duke University Medical Center and colleagues here at the Virginia Tech Carilion School of Medicine, we were able to get human specimens to confirm that what we were observing in the mouse models was also true in in human colorectal cancer patients."

The medical application of the findings will be determined in years to come as other researchers seek to identify treatments that can target NIK and its interactions with other proteins.

"Our study identified changes in a significant signaling pathway in human patients," Allen said. "That presents a variety of possible targets that have not been previously evaluated in that pathway where you could potentially design therapeutics."

Publication of the paper represents the conclusion of a lengthy study for Allen. Graduate students Kristin Eden '06, DVM '10, Ph.D. '18; Holly Morrison Ph.D. '23; and Brie Trusiano Ph.D. '24 took turns helping Allen with the research and carrying it to the finish line.

"When I first came here 12 years ago, my postdoctorate work had identified some hints that this pathway might be important in the context of colorectal cancer and also in the context of inflammatory bowel disease," Allen said. "Completion of this work has been very satisfying, knowing that it helped to launch the careers of these three talented graduate students and several undergraduate researchers as well."

Allen said because his studies on the NIK pathway in colorectal cancer are concluded, he and his team will turn toward other types of cancer impacted by NIK and the signaling pathways it controls. But he hopes others can carry the findings forward for life-saving improvements in colorectal cancer treatment.

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Green microbe profile: rhizophagus intraradices —a review of benevolent fungi promoting plant health and sustainability.

1. Introduction

2. taxonomy and characteristics, 3. mycorrhizal symbiosis, 4. role of r. intraradices in promoting plant growth, 5. nutrient cycling and soil health, 6. environmental restoration and ecosystem resilience, 7. sustainable agriculture and organic farming, 8. agro-ecological relevance of glomeromycota and r. intraradices.

• (b) Mycorrhiza–plant interaction: yielding plant disease biocontrol
• (c) Mycorrhiza–microorganisms interaction
• (d) Mycorrhiza–soil interaction
• (e) Biogeochemical cycles and mycorrhiza

9. Genomic Research in Glomeromycota

10. negative effects of amf, 11. challenges and future perspectives, 12. conclusions, author contributions, institutional review board statement, data availability statement, conflicts of interest.

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Onyeaka, H.N.; Akinsemolu, A.A.; Siyanbola, K.F.; Adetunji, V.A. Green Microbe Profile: Rhizophagus intraradices —A Review of Benevolent Fungi Promoting Plant Health and Sustainability. Microbiol. Res. 2024 , 15 , 1028-1049. https://doi.org/10.3390/microbiolres15020068

Onyeaka HN, Akinsemolu AA, Siyanbola KF, Adetunji VA. Green Microbe Profile: Rhizophagus intraradices —A Review of Benevolent Fungi Promoting Plant Health and Sustainability. Microbiology Research . 2024; 15(2):1028-1049. https://doi.org/10.3390/microbiolres15020068

Onyeaka, Helen N., Adenike A. Akinsemolu, Kehinde Favour Siyanbola, and Victoria Ademide Adetunji. 2024. "Green Microbe Profile: Rhizophagus intraradices —A Review of Benevolent Fungi Promoting Plant Health and Sustainability" Microbiology Research 15, no. 2: 1028-1049. https://doi.org/10.3390/microbiolres15020068

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