Carolyn Cray

Avian aspergillosis, caused by Aspergillus fumigatus ( Af ), lacks sensitive antemortem diagnostics. Existing microbial cell-free DNA (cfDNA) tests are prone to contamination and require a high pathogen load. We hypothesized that infection-induced tissue damage in chickens creates differentially methylated regions (DMRs) in host cfDNA, enabling machine learning (ML) diagnostics. Serum cfDNA samples ( n = 124) were obtained from broiler chickens ( n = 76) with Af and non- Af infections ( Escherichia coli or Gallibacterium anatis ) and controls. Oxford Nanopore sequencing enabled DMR detection and ML training. Performance was evaluated using an independent set ( n = 49) and 10-repeat Monte Carlo cross-validation (CV) ( n = 490 evaluations per test) as quality control. A High Accuracy test (93 DMRs, neural network) achieved 98.0% accuracy (sensitivity 95%, specificity 100%, AUC 0.974, PR-AUC 0.928) in the independent set, with CV accuracy 92.0% [95% CI: 89.7%–94.4%]. A Fast test (35 DMRs, SVM) achieved 81.6% accuracy and CV accuracy 79.6% [74.9%–84.3%]. An In Situ test (5 DMRs, random forest) designed for field deployment achieved 71.4% accuracy and CV accuracy 62.9% [58.7%–67.0%]. Stratified CV accuracy showed 84.6% [65.1%–95.6%] correct classifications for E. coli and 100% [80.5%–100%] for G. anatis . Markers showed high bootstrap stability and predominantly overlapped EMARs and enhancers. In conclusion, we present MethylSense ( https://github.com/markusdrag/MethylSense ), an automated open-source software. The High Accuracy test achieved 92.0% [89.7%–94.4%] CV accuracy (CV sensitivity 94.5% [91.4%–97.6%], CV specificity 90.3% [87.8%–92.9%]). While validated in chickens, MethylSense is adaptable to other species and pathogens, offering scalable, contamination-resilient diagnostics for veterinary and conservation applications. IMPORTANCE: MethylSense is an automated software for training machine learning diagnostics using differentially methylated regions (DMRs) in cell-free DNA from Oxford Nanopore sequencing. We applied MethylSense to develop three Aspergillus fumigatus tests for chickens, each optimized for different clinical scenarios. The High Accuracy test (93 DMRs, neural network) demonstrated 98.0% accuracy, in a blinded test set ( n = 49) with sensitivity 95%, specificity 100%, ROC-AUC 0.974, and PR-AUC 0.928. Stratified 10-repeat Monte Carlo cross-validation ( n = 490) showed correct classifications of 84.6% [CI: 65.1%–95.6%] Escherichia coli and 100% [80.5%–100%] Gallibacterium anatis infected specificity samples. A Fast test for rapid In Situ test (5 DMRs, random forest) for field deployment via methylation-specific PCR achieved 71.4% accuracy (sensitivity 45%, specificity 89.7%). Bootstrap analysis demonstrated exceptional marker stability (80.6%–100%) with minimal batch effects, confirming robust host-based diagnostics.

Acute-phase proteins (APPs) are used in veterinary science for diagnosing disease, identifying acute and subclinical inflammatory processes, monitoring disease progression, assessing patient response to treatment, and as a general health screening tool. In turtles, the utility of APPs, such as albumin, haptoglobin, fibrinogen, and myeloid-related protein (MRP)-126, as inflammatory markers has been investigated in a small number of studies. Serum or plasma protein electrophoresis, immunoassays, and biochemistry assays are most commonly employed to study such APPs, with mass spectrometry–based proteomics emerging as an important tool. Protein electrophoresis RIs have been established for a few turtle species. However, very few commercial assays are available for measuring specific APPs in turtles, with even fewer reported validated assays. Turtles have significant inter- and intraspecies biological variation; consequently, RIs of protein electrophoresis and specific APP assays for individual species should be established according to guidelines and referenced to determine if sex, age, reproductive status, and health status influence the results. For small populations of animals, particularly those of high conservation value, subject-based RIs are recommended if population-based RIs are not feasible. Further studies, especially biomarker identification, assay development, and validation, are required to increase the tools for disease diagnosis and monitoring of this taxon and contribute to the healthcare and conservation of many endangered turtle species.

Serum protein electrophoresis is a tool widely used in veterinary medicine to aid in the detection of inflammation as well as monoclonal and polyclonal gammopathies. The use of agarose gel electrophoresis (AGE) has previously been described in some non-domesticated mammals, including the bongo and pronghorn antelope as well as in cattle. Capillary zone electrophoresis (CZE) represents a new method that provides a higher resolution and lower coefficient of variation. In the present study, banked samples (n = 24–30) of adult addax (Addax nasomaculatus), gemsbok (Oryx gazella), sable antelope (Hippotragus niger), scimitar horned oryx (Oryx dammah), waterbuck (Kobus elllipsiprymnus), and common wildebeest (Connochaetes taurinus) were examined to describe the CZE electrophoretogram and determine preliminary reference intervals. Interestingly, interspecies differences were observed with a lower A/G ratio in the addax, gemsbok, and sable antelope versus the scimitar horned oryx, waterbuck, and wildebeest. This was mostly related to the overall higher alpha and beta globulins in the former group. In addition, albumin quantitated by CZE was also compared to albumin levels determined by traditional bromocresol green methodology. While the methods were found to be significantly correlated (p < 0.01), r values ranged from 0.5 to 0.9 and BCG overestimated albumin by 0.16 to 0.52 g/dL. CZE may be a useful diagnostic tool in health assessments and reveal species differences, which may be driven by genetics, subclinical infection, and/or environment.

Serum protein electrophoresis and acute phase protein quantitation have been demonstrated as important tools in clinical pathology testing of nondomesticated mammals. The goal of this study was to validate a commercially available reagent for serum amyloid A (SAA) and obtain preliminary data for SAA and protein electrophoresis in lions and tigers. Serum samples from 32 tigers (Panthera tigris), including 16 clinically normal animals, and 29 lions (Panthera leo), including 22 clinically normal animals, were obtained from private facilities in south and central Florida between 2014 and 2025. SAA was defined as a major acute phase protein (APP) in these species, with 10–100-fold increases in clinically abnormal animals. The SAA reagent was validated for use based on linearity under dilution and coefficient of variation analyses. Given the size of the sample sets, reference intervals were generated for lions and descriptive statistics were performed for tigers. Significant increases in SAA as well as alpha-2 and beta globulins in clinically abnormal vs normal lions and tigers were observed (P < 0.05). This preliminary assessment provides valuable information for the use of SAA and protein electrophoresis and lays the groundwork to establish robust reference intervals as well as a clinical validation of the use of these assays for these two species.

Members of the microsporidial genus Encephalitozoon have the capacity to infect both mammals and birds, and E. cuniculi is most commonly found in rabbits. With a seroprevalence ranging up to 85%, E. cuniculi can be a problem in pet rabbits as well as in food production and laboratory animal science. While most infections are likely subclinical, there are three main clinical presentations: neurological, renal, and ocular. Typical clinical signs including vestibular disease and phacoclastic uveitis may develop with initial or relapsing infection, while renal infection is usually progressive and associated with non-specific clinical signs. High-sensitivity/specificity ante mortem diagnostic options are lacking, and serological testing most often provides adjunct rather than definitive information such that physical examination and other diagnostics are used more so for ruling out other differentials and comorbidities, rather than confirming infection. In the veterinary community, treatment regimens are variable given the lack of thorough studies and a consensus. The aim of this document is to present the available literature to give a concise review of this organism and its infection of rabbits as well as to propose guidelines and protocols for diagnostics and treatment regimens. In addition, the current challenges and recommendations for further studies are discussed.

The anatomical and physiological similarities between human and canine eyes suggest that dogs may serve as a valuable model for studying retinopathies and developing future gene therapies. This study aims to evaluate the similarities and differences between humans with GUCY2D gene variants causing Leber’s congenital amaurosis (LCA) and a group of German Spitz dogs with hereditary retinopathy due to variants in the same gene, to assess their potential as an animal model for gene therapy research. A review of medical records, genetic testing, and ophthalmological examinations was conducted, including data such as age, genotyping, fundus photography, visual acuity (VA), fundus autofluorescence, optical coherence tomography (OCT), and electroretinography (ERG). Both groups presented subtle fundus abnormalities and severely reduced or absent ERG responses. In humans, OCT scans revealed decreased retinal thickness and structural alterations in the outer retinal layers. Similarly, the affected dogs exhibited focal neurosensory retinal detachments. The German Spitz model with GUCY2D variants shows significant parallels in retinal structure and functional impairment and may represent a promising candidate for preclinical gene therapy studies for LCA.

Penguins under managed care are widely considered to have high susceptibility to infection by Aspergillus spp. Antemortem laboratory diagnostic options vary in sensitivity and specificity, and antibody detection has been problematic in penguin species given elevated levels of reactivity observed in clinically normal patients using traditional whole antigen enzyme-linked immunosorbent assays (ELISA). In the present study, an alternative assay was implemented to detect reactivity to Afmp1p, an Aspergillus cell wall antigen, in samples obtained from several different penguin species. With confirmed infection, abnormal protein electrophoretograms were consistently observed, and gliotoxin was detected in the majority of cases. An increase in reactivity to Afmp1p was observed in penguins with confirmed (n = 18, p < 0.0001) and probable (n = 13, p = 0.08) aspergillosis versus normal adult penguins (n = 33). Interestingly, increased reactivity to Afmp1p (p < 0.0001) was noted in normal adult penguins (n = 33) versus juvenile penguins (n = 22, p < 0.0001). Overall, the area under the curve for this assay was 0.890, with a sensitivity of 94.4% and a specificity of 57.6%, with an antibody assay cutoff of 1.0. Increasing reactivity resulted in an increase in specificity. These data support the use of Afmp1p antibody quantitation as part of a diagnostic workup in penguins with suspected aspergillosis.

Background: The Atlantic sturgeon has faced severe population declines due to overfishing and environmental changes, resulting in its critical conservation status. There is concern today for the survival of this species, leading to the initiation of studies focused on its conservation.Objectives: The aims of this study were to establish reference intervals (RIs) for plasma protein fractions in Atlantic sturgeon using agarose gel electrophoresis (AGE), to evaluate whether changes occur during the spawning season, and to compare results with those obtained using capillary zone electrophoresis (CZE).Methods: Between late May and June 2022, blood samples were collected from 81 spawning Atlantic sturgeon. Plasma from all individuals was analyzed using AGE, with a subset of 16 paired samples further evaluated using CZE for comparative analysis.Results: AGE revealed six distinct protein fractions, while CZE identified seven fractions. Significant differences in the proportions of fraction 3 (traditionally referred to as alpha‐1 globulins) were observed between early and late spawning seasons.Conclusion: Comparison of AGE and CZE results showed that while both methods provided useful data, CZE offered greater resolution and detection sensitivity. Our results provide important data for sturgeon health assessment; however, discrepancies between the two methods underscore the need for method‐specific RI.

The common loon (Gavia immer) is considered a sentinel of ecosystem health, and declines in this species have been linked to several different drivers. Loons are widely recognized as being very sensitive to fungal respiratory disease, and this has proven a major barrier to successful clinical care and rehabilitation. The goal of the present study was to assess the presence of antibody reactivity to Aspergillus with secondary testing including Aspergillus antigen and gliotoxin detection as well as plasma protein electrophoresis and complete blood count as sample volume permitted. Conducted over two years in collaboration with multiple centers in Maine, New Hampshire, and Florida, samples were collected in the Northeast from healthy wild adult and juvenile birds (n = 72) as well as clinically abnormal birds presented for rehabilitation (n = 29). In addition, a cohort of rehabilitation samples was obtained from clinically abnormal overwintering birds (n = 6). Necropsy results were available for those found moribund or euthanized (n = 29). Of these birds, eight were confirmed to have aspergillosis where the remainder were diagnosed with other complications. Only three of eight displayed antibody reactivity to Aspergillus using a recombinant antigen-based ELISA and four tested positive for the presence of gliotoxin. An abnormal electrophoretogram was present in samples from all eight birds. The presence of antibody reactivity was not observed in clinically normal loons and only in one of the necropsied loons without aspergillosis. Overall, aspergillosis appears uncommon in healthy, free-ranging birds but likely can be an opportunistic infection after a stress inducing event. Serological testing and protein electrophoresis may provide an opportunity to monitor the health of this species and may improve the ability to manage this species in captivity.

Protein electrophoresis is a tool used in the health assessments of non-mammalian vertebrates. In elasmobranchs, agarose gel electrophoresis (AGE) has been described in various species and a newer method called capillary zone electrophoresis (CZE) has been developed and implemented in the undulate skate (Raja undulata) and nursehound shark (Scyliorhinus stellaris). The study goals were to implement AGE and CZE methods on plasma samples from the sand tiger shark (Carcharias taurus) and examine differences in resolution as well as to calculate reference intervals (RI). Plasma was obtained from aquarium sharks (n = 23) and free-ranging sharks (n = 62) sampled during field research conducted from 2017 to 2023. As with previous reports, CZE was found to provide superior resolution with definition of two major globulin migrating fractions compared to AGE. Overall, the alpha and beta migrating fractions were well correlated between the methods (r = 0.92, 0.89, respectively, p < 0.0001). The correlation for the gamma fraction was weaker (r = 0.42, p = 0.002) as the CZE fraction was lower in concentration versus AGE. There were minor, but significant, differences between the concentration of some of the fractions in samples from sharks under managed care versus free-ranging animals which necessitated the production of two sets of RI. In total, this information may help in further studies to address the applicability of these tools in the management of this species under human care as well as in health assessments of free-ranging sharks.