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Clinical metrics of baseline health in sentinel seabird species can offer insight into marine ecosystem dynamics, individual and population health, and assist in wildlife rehabilitation and conservation efforts. Protein electrophoresis is useful for detecting changes in acute phase proteins and immunoglobulin levels that may indicate subtle inflammatory responses and/or infectious disease. Serum biochemistry can highlight nutritional status, metabolic derangements, and organ injury and function. However, baseline values for such health parameters are largely unknown for many seabird species. Therefore, the objective of this study is to establish baseline clinical health reference intervals for serum protein electrophoresis, acute phase proteins including serum amyloid A and haptoglobin, and biochemistry parameters in the rhinoceros auklet (Cerorhinca monocerata), a key sentinel species in the North Pacific. From 2013 to 2019, 178 wild, apparently healthy breeding adult rhinoceros auklets were captured across four breeding colonies in British Columbia, Canada (Lucy Island, Pine Island, Triangle Islands, and SGang Gwaay) and from one colony in Washington, United States (Protection Island). Reference intervals were calculated for protein electrophoresis fractions and acute phase proteins (n = 163), and serum biochemistry (n = 35) following established guidelines by the American Society of Veterinary Clinical Pathology. Animals were also assessed for the presence of antibodies to the influenza A virus. Approximately 48% (70/147) of sampled birds were seropositive for influenza A virus, with a prevalence of 50% (6/12) in 2013, 75% (47/63) in 2014, and 24% (17/72) in 2019. This work provides clinical baseline health metrics of a key North Pacific sentinel species to help inform marine ecosystem monitoring, recovery, and rehabilitation efforts in the Pacific Northwest.

Sarcoptic mange, caused by epidermal infection with Sarcoptes scabiei, negatively impacts the health, welfare, and local abundance of bare-nosed wombats (Vombatus ursinus) in Australia. Improved understanding of the host immune response to disease and its contribution to pathophysiology could be used to inform management actions for this species in and ex situ. To evaluate the immune response of bare-nosed wombats to sarcoptic mange, we validated three assays (haptoglobin, agarose gel electrophoresis, and micro-erythrocyte sedimentation rate) measuring non-specific markers of inflammation using serum samples from free-living wombats from Tasmania (n = 33). We then analysed correlations between the assay results for each non-specific marker of inflammation and wombat’s sarcoptic mange scores, and performed histopathological examinations to investigate association of the acute phase response with systemic amyloidosis. We present evidence that haptoglobin and erythrocyte sedimentation rate increased, and albumin decreased, in association with sarcoptic mange scores. This research demonstrates links between the acute phase response and sarcoptic mange severity in bare-nosed wombats, highlighting the utility of non-specific markers of inflammation for aiding assessment of the systemic effects of mange. Showing the value of agarose gel electrophoresis, we also identified specific acute phase proteins warranting future evaluation and found evidence of an immunoglobulin response in mange-affected wombats, revealed by increasing γ-globulins in association with apparent disease severity. Meanwhile, owing to its relatively low resource requirements and rapidity, the erythrocyte sedimentation rate assay may be useful as a point-of-care test to support therapeutic decisions in the field. Our methods and findings are likely to be applicable to a range of other clinical and population health scenarios in captive and free-living wombats, and species impacted by sarcoptic mange globally.

In 2022–2023, the first survey of clinicians regarding Encephalitozoon cuniculi infection of rabbits was undertaken. The purpose was to assess the current worldwide knowledge of clinicians regarding influence of breed and age, clinical presentations, use of various diagnostic tools, and treatment regimens and to explore how responses may differ by geographic location. Identifying mostly as exotic animal clinicians, 339 respondents participated, with nearly half from the United States and the remainder from 35 other countries. Neurological signs were most consistently reported as associated with Encephalitozoon cuniculi and the most recurrent clinical case presentation. Other responses showed differences by geographic location. For example, age predilection for disease varied, with 71.1% of international respondents indicating that infection was more common in young rabbits versus 28.9% who suggested it was more frequent in older rabbits. This was significantly different (p = 0.001) from the information from respondents from the United States, where 48.0% indicated that younger rabbits had an increased incidence versus 52.0% who indicated that older age increased occurrence. While some differences may be related to the practice composition and the training level of respondents, many responses were in alignment with the existing literature. Future inquiries should focus more on these discrepancies to aid in generating guidelines for the diagnosis and treatment of this infectious disease.

A recombinant vaccine for rabbit hemorrhagic disease virus 2, a highly pathogenic virus, was granted emergency use authorization in the United States after the detection and spread of the virus starting in 2018. The goal of the current study was to assess pet rabbits (n = 29) through physical examination and routine clinical pathology testing using repeated assessments post-vaccination. In addition, seroconversion was also monitored after the initial vaccination and booster vaccination. Neither owners nor clinicians detected any physical abnormalities in relationship to the vaccine protocol. Hematological and clinical biochemistry testing showed some changes although median values were within species specific reference intervals. A significant increase in antibody levels was observed at day 21 (post-initial vaccination) and day 49 (post-booster vaccination) versus that present at baseline (p < 0.0001). However, variability in study rabbits was noted with some individuals showing low antibody levels as well as a lower overall response in older rabbits (r = −0.56, p = 0.006). A second cohort of rabbits was assessed at 11–12 months post-initial vaccination. In this second group, antibody levels were not significantly different from baseline levels (p = 0.21). Additional studies should be conducted to further define the variability in seroconversion and the term of protection in pet rabbits as the industry moves forward in the optimization of RHDV2 vaccines.

Point-of-care (POC) glucose and β-hydroxybutyrate (β-HB) meters can potentially provide rapid insight into an elasmobranch’s metabolic state in clinical and field research settings. This study evaluated the diagnostic agreement of three commercial POC meters against reference laboratory methods for glucose and β-HB concentrations in stingrays. Blood was collected during anesthetized exams from 28 stingrays representing four species: cownose rays (Rhinoptera bonasus), Atlantic stingrays (Hypanus sabina), southern stingrays (Hypanus americanus), and yellow stingrays (Urobatis jamaicensis). Glucose and β-HB concentrations were measured with each POC meter using whole blood and plasma; in parallel, plasma glucose and β-HB concentrations were measured via reference laboratory methods. Agreement between POC meters and reference laboratory methods was assessed using Bland–Altman methods, Passing-Bablok regression, observed total error, percent relative error, and linear mixed effect models. Plasma glucose and β-HB concentrations determined by reference laboratory methods ranged from

Measurements of specific acute phase proteins (APP) and protein electrophoresis (EPH) fractions have been widely used to better assess the health of species under managed care across numerous taxa. To date, APP assays have not been validated in the hamadryas baboon (Papio hamadryas), and reference intervals have not yet been established. This information is critical for the interpretation of APP and EPH measurements used in the diagnosis of inflammatory diseases during routine veterinary care of this species. To obtain this information, banked serum samples from hamadryas baboons of various age, sex, and health status, under managed care at the North Carolina Zoo, were analyzed. A small pilot study found significantly higher serum amyloid A (SAA) and C-reactive protein (CRP) but not haptoglobin in baboons with acute inflammation compared to healthy counterparts, so these two APPs were investigated further. Reagents for serum amyloid A (SAA) and C-reactive protein (CRP) were validated, although differences in CRP reagents were observed. Based on the results of this study, SAA and CRP were defined as major APPs that were significantly increased in baboons with active inflammation or infection compared to healthy conspecifics. Baboons with acute inflammation additionally had significantly higher gamma globulins compared to healthy baboons. Although mean albumin concentrations were lower in baboons with acute inflammation, the difference from healthy baboons was not statistically significant. This study identifies SAA, CRP, and EPH as useful tools in the diagnosis of inflammatory disease in the hamadryas baboon and establishes reference intervals to aid in the future veterinary care of this species.

This retrospective study evaluated canine patients with presumptively diagnosed meningoencephalomyelitis (ME) based on neurological clinical signs, cerebrospinal fluid (CSF) analysis, cross-sectional imaging, and infectious disease testing with a limited neurological-focused polymerase chain reaction (PCR) panel performed on blood and CSF. The first goal was to determine the proportion of dogs where the condition was caused by an infectious agent versus a probable immune-mediated etiology (i.e., meningoencephalomyelitis of unknown origin; MUO) in our geographic region. The secondary goals of this study were to examine and define associations between abnormal CSF test results and cross-sectional neuroimaging findings, in addition to defining the age and most common neurological clinical signs in each group of ME. A total of 168 dogs matched the inclusion criteria with magnetic resonance imaging (MRI) performed in 130 dogs and computed tomography (CT) performed in 38 dogs. Presumptive MUO was observed in 152/168 (90.5%) of dogs and infectious ME was identified in 16/168 (9.5%) of dogs (p

Serum protein electrophoresis has been demonstrated to have utility in diagnostic workup, wellness exams, and prognosis. Agarose gel electrophoresis (AGE) has previously been described for use with serum from Asian elephants (Elephas maximus). As the newer method of capillary zone electrophoresis (CZE) is becoming more commonplace in veterinary diagnostic laboratories, serum samples from Asian elephants were examined using this method. CZE allowed for a reproducible definition of two beta fractions and, overall, showed a low coefficient of variation for fraction quantitation. Preliminary reference intervals were generated using samples primarily from an older population of 22 female elephants. Albumin levels determined by CZE were also compared with those determined by the bromocresol green method on a chemistry analyzer. It was found that the latter method overestimated the level of albumin with a mean positive bias of 11.6% or 0.38 g/dL, thus method-specific reference intervals should be used. Significant negative correlations were observed between A/G ratio determined by CZE and serum amyloid A levels (r = −0.47, p

IntroductionFungal diseases are a rising health problem globally, in humans, nonhuman animals, and plants. Emerging fungal diseases have been associated with mass mortality events. A recent example of fungal disease pathogenicity is sea turtle egg fusariosis (STEF). The pathogenicity of STEF has been linked to fungi within the Fusarium solani species complex (FSSC). This complex is composed of over 45 phylogenetically identifiable species commonly found in the environment. Species within the FSSC lineage have been isolated from the nests of multiple sea turtle species and are linked to decreased hatching success in all 7 of the extant sea turtle species. Fungi within this lineage are also known to cause cutaneous and subcutaneous infections. These fungi are not only a threat to sea turtles but also to other animals, including humans, that use coastal waters and beaches inhabited by Fusarium spp. The presence of Fusarium spp., in the context of sea turtle health, has not been investigated on southeastern Florida beaches which are fundamentally important for at least three sea turtle species that nest there in large numbers.MethodsWe performed a retrospective assessment of necropsy reports from dead captive leatherback neonates from 2010 to 2022 to assess the most common microscopic diagnoses and the presence of skin lesions associated with mycotic dermatitis. Additionally, live captive leatherbacks and dead-in-nest samples from the 2022 hatching season were used to assess the presence and effect of mycotic dermatitis in Juno Beach and Boca Raton, Florida, USA. This was accomplished by observing gross lesions, fungal cultures, and blood values.ResultsThe retrospective analysis of dead captive neonates revealed that the diagnosis of mycotic dermatitis on histopathology has significantly increased since 2010. The assessment of gross skin lesions associated with mycotic dermatitis in dead and live captive leatherback neonates also revealed a similar increase. Investigations in live captive leatherbacks revealed fungal cultures positive for Fusarium spp. and significant differences in blood values at emergence between healthy turtles and those that later developed mycotic dermatitis.DiscussionPositive dead-in-nest culture results suggest that Fusarium spp. are likely present in leatherback sea turtle nests in Boca Raton and Juno Beach, Florida, USA. Additionally, the occurrence of mycotic dermatitis in dead and live captive leatherback neonates suggests that the presence of Fusarium spp. in the nest likely affects leatherback neonates even after emergence.

Unique features of elephant hematology are known challenges in analytical methodology like two types of monocytes typical for members of the Order Afrotheria and platelet counts of the comparatively small elephant platelet. To investigate WBC differential and platelet data generated by an impedance-based hematology analyzer without availability of validated species-specific software for recognition of elephant WBCs and platelets, compared to manual blood film review. Blood samples preserved in ethylenediaminetetraacetic acid (EDTA) of 50 elephants (n = 35 Elephas maximus and n = 15 Loxodonta africana) were used. A Mann-Whitney test for independent samples was used to compare parameters between methods and agreement was tested using Bland-Altman bias plots. All hematological variables, including absolute numbers of heterophils, lymphocytes, monocytes, eosinophils, basophils, and platelets, were significantly different (p

IntroductionOne of the most important emerging infectious diseases of amphibians is caused by the fungal pathogen Batrachochytrium salamandrivorans (Bsal). Bsal was recently discovered and is of global concern due to its potential to cause high mortality in amphibians, especially salamander species. To date, little has been reported on the pathophysiological effects of Bsal; however, studies of a similar fungus, B. dendrobatidis (Bd), have shown that electrolyte losses and immunosuppression likely play a key role in morbidity and mortality associated with this disease. The goal of this study was to investigate pathophysiological effects and immune responses associated with Bsal chytridiomycosis using 49 rough-skinned newts (Taricha granulosa) as the model species.MethodsTaricha granulosa were exposed to a 1 × 107 per 10 mL dose of Bsal zoospores and allowed to reach various stages of disease progression before being humanely euthanized. At the time of euthanasia, blood was collected for biochemical and hematological analyses as well as protein electrophoresis. Ten standardized body sections were histologically examined, and Bsal-induced skin lesions were counted and graded on a scale of 1–5 based on severity.ResultsResults indicated that electrolyte imbalances and dehydration induced by damage to the epidermis likely play a major role in the pathogenesis of Bsal chytridiomycosis in this species. Additionally, Bsal-infected, clinically diseased T. granulosa exhibited a systemic inflammatory response identified through alterations in complete blood counts and protein electrophoretograms.DiscussionOverall, these results provide foundational information on the pathogenesis of this disease and highlight the differences and similarities between Bsal and Bd chytridiomycosis.

Aspergillosis remains difficult to diagnose in animals. Laboratory-based assays are far less developed than those for human medicine, and only few studies have been completed to validate their utility in routine veterinary diagnostics. To overcome the current limitations, veterinarians and researchers have to propose alternative methods including extrapolating from human diagnostic tools and using innovative technology. In the present overview, two specific examples were complementarily addressed in penguins and dolphins to illustrate how is challenging the diagnosis of aspergillosis in animals. Specific focus will be made on the novel application of simple testing in blood based on serological assays or protein electrophoresis and on the new information garnered from metabolomics/proteomics to discover potential new biomarkers. In conclusion, while the diagnostic approach of aspergillosis in veterinary medicine cannot be directly taken from options developed for human medicine, it can certainly serve as inspiration.

The black-eared opossum (Didelphis aurita) is a South American synanthropic marsupial. The presence of opossums in domestic spaces is relevant in the One-Health context since they are hosts of pathogens and ectoparasites that may affect the health of domestic animals and humans. In this study, we aim to determine the occurrence of hemoplasmas and selected tick-borne pathogens in free-ranging black-eared opossums, along with their molecular characterization, hematological and biochemical evaluation and factors associated with infection, in the municipality of Viçosa, State of Minas Gerais, southeastern Brazil. Thirty black-eared opossums were trapped between March 2021 and June 2022. Ectoparasites were collected. Hematological and biochemical analyses were performed. DNA from EDTA-blood samples were analyzed by PCR and qPCR assays. By molecular analyses, ‘Candidatus Mycoplasma haemoalbiventris’ was the most prevalent hemoparasite (73.3%), followed by Hepatozoon sp. (22.2%). Significant differences were observed in the number of platelets, and in the concentration of protein and globulins in the animals infected by ‘Ca. M. haemoalbiventris’ when compared with the negative group. This is the first report of ‘Ca. M. haemoalbiventris’ infection in D. aurita.

Serum samples collected from 37 clinically normal bongo (Tragelaphus eurycerus) and 13 abnormal bongo were tested using assays for acute-phase proteins (APPs) and by protein electrophoresis. Abnormal bongo samples (n = 27) had significantly higher levels of fibrinogen (FIB) (p < 0.001) and trending but not significantly increased haptoglobin (HP) (p = 0.07) vs. samples from normal bongo (n = 37). There were no significant differences in values for total white blood cell counts or for any of the fractions determined by protein electrophoresis. Clinically normal female bongo (n = 19) had significantly lower levels of FIB than normal males (n = 18) (p = 0.014), and this observation was also made with samples from the clinically abnormal group (p = 0.004). Many weak to moderate significant correlations were observed with increasing age, including increased globulins, FIB, and HP and decreased albumin-to-globulin (A/G) ratio and albumin. In clinical cases reviewed in this study, mild HP changes categorized this reactant as a minor APP, which contrasts with the major APP classification of HP in the related species of the cow. The preliminary data indicate that the quantitation of these APPs may offer value in assessing inflammation in this species, but additional studies are needed.

Baseline plasma electrophoresis profiles (EPH) are important components of overall health and may aid in the conservation and captive management of species. The aim of this study was to establish plasma protein fractions for free-ranging Blanding's turtles (Emydoidea blandingii) and evaluate differences due to age class (adult vs. sub-adult vs. juvenile), sex (male, female, or unknown), year (2018 vs. 2019), month (May vs. June vs. July), health status, and geographical location (managed vs. unmanaged sites). Blood samples were obtained from 156 Blanding's turtles in the summer of 2018 and 129 in 2019 at two adjacent sites in Illinois. Results of the multivariate analysis demonstrated that age class, sex, year, month, health status, and geographical location all contributed to the variation observed in free-ranging populations. Adult females had the highest concentration of many protein fractions, likely associated with reproductive activity. Juveniles had lower protein concentrations. Temperature and rainfall differences between years impacted concentrations between 2018 and 2019, while May and June of both years saw higher levels in some protein fractions likely due to peak breeding and nesting season. Individuals with evidence of trauma or disease also showed increased plasma protein fractions when compared to those that were considered healthy. The two sites showed a wide/large variation over the two years. All of these factors emphasize the importance of considering multiple demographic or environmental factors when interpreting the EPH fractions. Establishing ranges for these analytes will allow investigation into disease prevalence and other environmental factors impacting this endangered species.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Corona Virus-2), cause of COVID-19 (Coronavirus Disease of 2019), represents a significant risk to people living with pre-existing conditions associated with exacerbated inflammatory responses and consequent dysfunctional immunity. In this paper, we have evaluated the influence of obesity, a condition associated with chronic systemic inflammation, on the secretion of SARS-CoV-2-specific IgG antibodies in the blood of COVID-19 patients. Our hypothesis is that obesity is associated with reduced amounts of specific IgG antibodies. Results have confirmed our hypothesis and have shown that SARS-CoV-2 IgG antibodies are negatively associated with Body Mass Index (BMI) in COVID-19 obese patients, as expected based on the known influence of obesity on humoral immunity. Antibodies in COVID-19 obese patients are also negatively associated with serum levels of pro-inflammatory and metabolic markers of inflammaging and pulmonary inflammation, such as SAA (serum amyloid A protein), CRP (C-reactive protein), and ferritin, but positively associated with NEFA (nonesterified fatty acids). These results altogether could help to identify an inflammatory signature with strong predictive value for immune dysfunction. Inflammatory markers identified may subsequently be targeted to improve humoral immunity in individuals with obesity and in individuals with other chronic inflammatory conditions.

The Atlantic Goliath Grouper (Epinephelus itajara) population has rebounded from near extinction to an international status as vulnerable due in part to regional species recovery efforts. The southeastern US population has been recovering with the main spawning locations off the coasts of Florida. Despite their economic importance to the catch-and-release fishery and the dive industry, and their ecological importance as ecosystem engineers resulting in positive impacts on reefs and species richness, baseline health assessment information is very limited in this species to date. The objectives of this study were to: (1) establish reference intervals for hematological and plasma biochemical analytes, and report immune function, oxidative stress, and vitellogenin in mature males and females; (2) evaluate total length, age, and sex in relation to blood analytes in juvenile and mature fish; (3) assess analytes across sampled months in mature male and female fish; and (4) describe the typical light microscopy findings in liver and gill biopsies, including quantitative assessment of pigmented macrophage aggregates. Health indices are reported as reference intervals when applicable, or otherwise descriptively. Blood analyte correlations with length and age, sex differences, and comparisons across months provided relevant physiological considerations, including differences in protein/energy metabolism, tissue growth, sexual maturation, active reproduction, and antigenic stimulation. Liver histology identified changes associated with life stage, active reproduction, or of subclinically to clinically insignificant infectious and/or inflammatory processes. Hepatocellular vacuolation and pigmented macrophage aggregates were prominent. Pigmented macrophage aggregates correlated with total length, presumably from continuous antigenic stimulation and/or metabolic changes as fish grow. Gill histological findings were subtle. The data presented herein provide an essential baseline assessment of a suite of health variables in an iconic marine teleost species, serves as a springboard for future studies relevant to conservation physiology, and allows for population-level applications for conservation management and policy.

When South Florida became a hot spot for COVID-19 disease in March 2020, we faced an urgent need to develop test capability to detect SARS-CoV-2 infection. We assembled a transdisciplinary team of knowledgeable and dedicated physicians, scientists, technologists, and administrators who rapidly built a multiplatform, polymerase chain reaction- and serology-based detection program, established drive-through facilities, and drafted and implemented guidelines that enabled efficient testing of our patients and employees. This process was extremely complex, due to the limited availability of needed reagents, but outreach to our research scientists and multiple diagnostic laboratory companies, and government officials enabled us to implement both Food and Drug Administration authorized and laboratory-developed testing–based testing protocols. We analyzed our workforce needs and created teams of appropriately skilled and certified workers to safely process patient samples and conduct SARS-CoV-2 testing and contact tracing. We initiated smart test ordering, interfaced all testing platforms with our electronic medical record, and went from zero testing capacity to testing hundreds of health care workers and patients daily, within 3 weeks. We believe our experience can inform the efforts of others when faced with a crisis situation.

Acute phase reactants (APRs) have not been investigated in white rhinoceros (Ceratotherium simum). This study aimed to identify clinically useful APRs in this species. Reference intervals (RIs) were generated for albumin, fibrinogen, haptoglobin, iron and serum amyloid A (SAA) from 48 free-ranging animals, except for SAA (n = 23). APR concentrations between healthy animals and those with tissue injury (inflammation) (n = 30) were compared. Diagnostic performance was evaluated using receiver-operator characteristic (ROC) curve and logistic regression analyses. RIs were: albumin 18–31 g/L, fibrinogen 1.7–2.9 g/L, haptoglobin 1.0–4.3 g/L, iron 9.7–35.0 μmol/L, SAA 4.7 g/L were significant predictors of inflammatory status and together correctly predicted the clinical status of 91% of cases. SAA > 20 mg/L had a specificity of 100%. In conclusion, albumin and iron are negative and fibrinogen, haptoglobin and SAA positive APRs in the white rhinoceros. The combination of iron and haptoglobin had an excellent diagnostic accuracy for detecting inflammation.

Plasma chemistry is widely used in diagnostic and research settings in sea turtles. However, plasma discolorations such as hemolysis are often not considered in data interpretation. The objectives of this study were to (1) evaluate the effects of moderate hemolysis on plasma electrolytes, minerals, and proteins using dry chemistry analysis (DCA) and protein electrophoresis from nesting leatherback sea turtles (Dermochelys coriacea) from Florida and to (2) establish blood analyte reference intervals. Twenty-six plasma samples with absence of hemolysis were selected and sub-divided into one non-hemolytic aliquot and an aliquot that was experimentally manipulated to mimic moderate hemolysis. Plasma samples were analyzed for hemoglobin using a handheld photometer; sodium, potassium, chloride, magnesium, calcium, phosphorus, and total protein using DCA; and protein electrophoresis. Packed cell volume and hemoglobin were measured in corresponding whole blood samples. Reference intervals were established. All analytes except calcium and pre-albumin were significantly higher and the calcium:phosphorus and albumin:globulin ratios were significantly lower in hemolytic plasma compared to non-hemolytic plasma. Alpha2-globulins and potassium were the analytes most impacted by hemolysis, averaging 3.3- and 2.0-fold higher in hemolyzed samples, respectively, indicating that (1) hemoglobin migrates into the alpha2-globulin region in this species and (2) notable intracellular potassium is released into plasma with hemolysis. Attempted conversion factors for compensation of hemolysis were considered inaccurate for 4 of 16 analytes due to non-significant regression lines. We also report that PCV provides an estimate of hemoglobin (g/L) using the formula: (2.59 × PCV) + 24.59. Given the spurious effects of hemolysis, the degree of this artifact should be reported with biochemistry data, and samples with moderate to severe hemolysis should be excluded from datasets when interpreting electrolyte, mineral, and protein results. This will ensure accurate data interpretation for individual turtles in rehabilitation or research settings and population-level data relevant to conservation-focused projects.

Aspergillosis is a fungal disease due to Aspergillus molds that can affect both humans and animals. As routine diagnosis remains difficult, improvement of basic knowledge with respect to its pathophysiology is critical to search for new biomarkers of infection and new therapeutic targets. Large-scale proteomics allows assessment of protein changes during various disease processes. In the present study, mass spectrometry iTRAQ® (isobaric tags for relative and absolute quantitation) protocol was used for direct identification and relative quantitation of host proteins in diseased fluids and tissues collected from an experimental rat model challenged with Aspergillus, as well as in blood obtained from naturally-infected penguins. In all, mass spectrometry analysis revealed that proteome during aspergillosis was mostly represented by proteins that usually express role in metabolic processes and biological process regulation. Ten and 17 proteins were significantly ≥4.0-fold overrepresented in blood of Aspergillus-diseased rats and penguins, respectively, while five and 39 were negatively ≥4.0-fold depleted within the same samples. In rat lungs, 33 proteins were identified with positive or negative relative changes versus controls and were quite different from those identified in the blood. Except for some zinc finger proteins, kinases, and histone transferases, and while three pathways were common (Wnt, cadherin and FGF), great inter-species variabilities were observed regarding the identity of the differentially-represented proteins. Thus, this finding confirmed how difficult it is to define a unique biomarker of infection. iTRAQ® protocol appears as a convenient proteomic tool that is greatly suited to ex vivo exploratory studies and should be considered as preliminary step before validation of new diagnostic markers and new therapeutic targets in humans.

Chronically debilitated loggerhead sea turtles (Caretta caretta) (DT) are characterized by emaciation, lethargy, and heavy barnacle coverage. Although histopathological findings associated with this condition have been reported, only limited data is available on health variables with clinical application. The objectives of this study were to 1) to compare morphometrics, clinicopathological variables, and immune functions of DTs to a group of apparently healthy loggerhead turtles to better understand the pathophysiology of the condition and 2) to assess health parameters in live debilitated turtles as they recovered during rehabilitation in order to identify potential prognostic indicators. We examined and sampled 43 DTs stranded from North Carolina to Florida for 47 health variables using standardized protocols to further characterize the condition. DTs were grouped into categories of severity of the condition, and those that survived were sampled at four time points through rehabilitation. All groups and time points were compared among DTs and to clinically healthy loggerhead turtles. Compared to healthy turtles, DTs had significantly lower body condition index, packed cell volume (PCV), total white blood cell (WBC) count, lymphocytes, glucose (Glc), total protein, all protein fractions as determined by electrophoresis, calcium (Ca), phosphorus (P), Ca:P ratio, potassium (K), lymphocyte proliferation, and greater heterophil toxicity and left-shifting, uric acid (UA), aspartate aminotransferase, creatine kinase, lysozyme, and respiratory burst. From admission to recovery, hematology and plasma chemistry data improved as expected. The most informative prognostic indicators, as determined by correlations with a novel severity indicator (based on survival times), were plastron concavity, P, albumin, total solids, UA, lymphocyte proliferation, WBC, K, Glc, Ca:P, and PCV. The results of this study document the wide range and extent of morphometric and metabolic derangements in chronically debilitated turtles. Monitoring morphometrics and clinicopathological variables of these animals is essential for diagnosis, treatment, and prognosis during rehabilitation.

Investigation of globulin fractions by serum protein electrophoresis (SPE) is the first step towards evaluation of the proteome in the southern white rhinoceros (Ceratotherium simum simum). Furthermore, identification of changes in globulins in animals with poaching and other injuries can guide discovery of potentially useful biomarkers of inflammation. The aim of this study was to develop reference intervals for agarose gel SPE in healthy white rhinoceros and to compare these serum protein electrophoresis results to those from animals with tissue trauma. Reference intervals for total serum protein and agarose gel electrophoretic albumin and globulin fractions were generated using serum samples from 49 healthy free-ranging adult white rhinoceros. A standardised gating system together with identification of specific proteins by mass spectrometry aided in fraction identification. Six globulin fractions were identified: α1a, α1b, α2, β1, β2 and γ. Reference intervals were generated for total serum protein (76-111 g/L), albumin (10-27 g/L) and globulin fractions (α1a: 1.6-3.2 g/L; α1b: 1.7-3.6 g/L; α2: 16.1-26.6 g/L; β1: 6.6-18.2 g/L; β2: 11.8-30.4 g/L; γ: 10.4-23.1 g/L; albumin: globulin ratio: 0.12-0.39). Results were compared to those from 30 animals with various degrees and chronicities of tissue trauma. Wounded animals had lower concentrations of total serum protein, albumin, total globulin, α and β1 globulins, lower percentages of α2 and β1 globulins, and higher percentages of β2 and γ globulins. These protein changes are similar to those seen in human patients with wounds rather than classic acute phase or chronic inflammatory responses.

Invasive aspergillosis has been studied in laboratory by the means of plethora of distinct animal models. They were developed to address pathophysiology, therapy, diagnosis, or miscellaneous other concerns associated. However, there are great discrepancies regarding all the experimental variables of animal models, and a thorough focus on them is needed. This systematic review completed a comprehensive bibliographic analysis specifically-based on the technical features of rodent models infected with Aspergillus fumigatus. Out the 800 articles reviewed, it was shown that mice remained the preferred model (85.8% of the referenced reports), above rats (10.8%), and guinea pigs (3.8%). Three quarters of the models involved immunocompromised status, mainly by steroids (44.4%) and/or alkylating drugs (42.9%), but only 27.7% were reported to receive antibiotic prophylaxis to prevent from bacterial infection. Injection of spores (30.0%) and inhalation/deposition into respiratory airways (66.9%) were the most used routes for experimental inoculation. Overall, more than 230 distinct A. fumigatus strains were used in models. Of all the published studies, 18.4% did not mention usage of any diagnostic tool, like histopathology or mycological culture, to control correct implementation of the disease and to measure outcome. In light of these findings, a consensus discussion should be engaged to establish a minimum standardization, although this may not be consistently suitable for addressing all the specific aspects of invasive aspergillosis.

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, polysaccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflammatory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in rabbits.