Reliability, clinical performance and trending ability of a pulse oximeter and pulse co-oximeter, in monitoring blood oxygenation, at two measurement sites, in immobilised white rhinoceros (Ceratotherium simum).

BACKGROUND: Monitoring blood oxygenation is essential in immobilised rhinoceros, which are susceptible to opioid-induced hypoxaemia. This study assessed the reliability, clinical performance and trending ability of the Nonin PalmSAT 2500 A pulse oximeter's and the Masimo Radical-7 pulse co-oximeter's dual-wavelength technology, with their probes placed at two measurement sites, the inner surface of the third-eyelid and the scarified ear pinna of immobilised white rhinoceroses. Eight white rhinoceros were immobilised with etorphine-based drug combinations and given butorphanol after 12 min, and oxygen after 40 min, of recumbency. The Nonin and Masimo devices, with dual-wavelength probes attached to the third-eyelid and ear recorded arterial peripheral oxygen-haemoglobin saturation (SpO2) at pre-determined time points, concurrently with measurements of arterial oxygen-haemoglobin saturation (SaO2), from drawn blood samples, by a benchtop AVOXimeter 4000 co-oximeter (reference method). Reliability of the Nonin and Masimo devices was evaluated using the Bland-Altman and the area root mean squares (ARMS) methods. Clinical performance of the devices was evaluated for their ability to accurately detect clinical hypoxemia using receiver operating characteristic (ROC) curves and measures of sensitivity, specificity, and positive and negative predictive values. Trending ability of the devices was assessed by calculating concordance rates from four-quadrant plots. RESULTS: Only the Nonin device with transflectance probe attached to the third-eyelid provided reliable SpO2 measurements across the 70 to 100% saturation range (bias - 1%, precision 4%, ARMS 4%). Nonin and Masimo devices with transflectance probes attached to the third-eyelid both had high clinical performance at detecting clinical hypoxaemia [area under the ROC curves (AUC): 0.93 and 0.90, respectively]. However, the Nonin and Masimo devices with transmission probes attached to the ear were unreliable and provided only moderate clinical performance. Both Nonin and Masimo devices, at both measurement sites, had concordance rates lower than the recommended threshold of ≥ 90%, indicating poor trending ability. CONCLUSIONS: The overall assessment of reliability, clinical performance and trending ability indicate that the Nonin device with transflectance probe attached to the third-eyelid is best suited for monitoring of blood oxygenation in immobilised rhinoceros. The immobilisation procedure may have affected cardiovascular function to an extent that it limited the devices' performance.

Bovine tuberculosis disturbs parasite functional trait composition in African buffalo.

Novel parasites can have wide-ranging impacts, not only on host populations, but also on the resident parasite community. Historically, impacts of novel parasites have been assessed by examining pairwise interactions between parasite species. However, parasite communities are complex networks of interacting species. Here we used multivariate taxonomic and trait-based approaches to determine how parasite community composition changed when African buffalo (Syncerus caffer) acquired an emerging disease, bovine tuberculosis (BTB). Both taxonomic and functional parasite richness increased significantly in animals that acquired BTB than in those that did not. Thus, the presence of BTB seems to catalyze extraordinary shifts in community composition. There were no differences in overall parasite taxonomic composition between infected and uninfected individuals, however. The trait-based analysis revealed an increase in direct-transmitted, quickly replicating parasites following BTB infection. This study demonstrates that trait-based approaches provide insight into parasite community dynamics in the context of emerging infections.

Cardiopulmonary responses of free-ranging African elephant (Loxodonta africana) bulls immobilized with a thiafentanil-azaperone combination.

OBJECTIVE: To determine the time course and certain cardiopulmonary effects of trunk-breathing elephants immobilized with thiafentanil-azaperone. STUDY DESIGN: Prospective descriptive study. ANIMALS: A convenience sample of 10 free-ranging African elephant bulls (estimated weight range: 3000-6000 kg). METHODS: Elephants were immobilized using thiafentanil (15-18 mg) and azaperone (75-90 mg) administered by dart. Once recumbent, the respiratory rate, minute ventilation (V˙e), end-tidal carbon dioxide (Pe'CO2), arterial blood pressure and heart rate were recorded immediately after instrumentation and at 5 minute intervals until 20 minutes. Arterial blood gases were analysed at the time of initial instrumentation and at 20 minutes. On completion of data collection, thiafentanil was antagonized using naltrexone (10 mg mg-1 thiafentanil; administered intravenously). A stopwatch was used to record time to recumbency (dart placement to recumbency) and time to recovery (administration of antagonist to standing). Data were compared using a one-way anova. Data are presented as mean ± standard deviation. RESULTS: All elephants were successfully immobilized, and there were no significant changes in cardiopulmonary variables over the monitoring period. Average time to recumbency was 12.5 (± 3.9) minutes. The measured V˙e was 103 (± 30) L minute-1. The average heart and respiratory rates over the 20 minute immobilization were steady at 49 (± 6) beats minute-1 and 5 (± 1) breaths minute-1, respectively. The mean arterial blood pressure was 153 (± 31) mmHg. The elephants were acidaemic (pH: 7.18 ± 0.06), mildly hypoxaemic (PaO2: 68 ± 15 mmHg; 9.1 ± 2.0 kPa) and hypercapnic (PaCO2: 52 ± 7 mmHg; 6.9 ± 0.9 kPa). Average time to recovery was 2.2 ± 0.5 minutes. CONCLUSION AND CLINICAL RELEVANCE: African elephant bulls can be successfully immobilized using thiafentanil-azaperone. Recumbency was rapid, the cardiopulmonary variables were stable over time, and recovery was rapid and complete. Mild hypoxaemia and hypercapnia were evident.

Mycobacterium bovis Infection in Free-Ranging African Elephants.

Mycobacterium bovis infection in wildlife species occurs worldwide. However, few cases of M. bovis infection in captive elephants have been reported. We describe 2 incidental cases of bovine tuberculosis in free-ranging African elephants (Loxodonta africana) from a tuberculosis-endemic national park in South Africa and the epidemiologic implications of these infections.

Development of a cytokine gene expression assay for the relative quantification of the African elephant (Loxodonta africana) cell-mediated immune responses.

Immunological assays are the basis for many diagnostic tests for infectious diseases in animals and humans. Application in wildlife species, including the African elephant (Loxodonta africana), is limited however due to lack of information on immune responses. Since many immunoassays require both identified biomarkers of immune activation as well as species-specific reagents, it is crucial to have knowledge of basic immunological responses in the species of interest. Cytokine gene expression assays (GEAs) used to measure specific immune responses in wildlife have frequently shown that targeted biomarkers are often species-specific. Therefore, the aim of this study was to identify elephant-specific cytokine biomarkers to detect immune activation and to develop a GEA, using pokeweed mitogen stimulated whole blood from African elephants. This assay will provide the foundation for the development of future cytokine GEAs that can be used to detect antigen specific immune responses and potentially lead to various diagnostic tests for this species.

Immobilization of African buffaloes (Syncerus caffer) using etorphine-midazolam compared with etorphine-azaperone.

OBJECTIVE: To compare induction times and physiological effects of etorphine-azaperone with etorphine-midazolam immobilization in African buffaloes. STUDY DESIGN: Randomized crossover study. ANIMALS: A group of 10 adult buffalo bulls (mean body weight 353 kg). METHODS: Etorphine-azaperone (treatment EA; 0.015 and 0.15 mg kg-1, respectively) and etorphine-midazolam (treatment EM; 0.015 and 0.15 mg kg-1, respectively) were administered once to buffaloes, 1 week apart. Once in sternal recumbency, buffaloes were instrumented and physiological variables recorded at 5 minute intervals, from 5 minutes to 20 minutes. Naltrexone (20 mg mg-1 etorphine dose) was administered intravenously at 40 minutes. Induction (dart placement to recumbency) and recovery (naltrexone administration to standing) times were recorded. Arterial blood samples were analysed at 5 and 20 minutes. Physiological data were compared between treatments using a general linear mixed model and reported as mean ± standard deviation. Time data were compared using Mann-Whitney U test and reported as median (interquartile range) with p ≤ 0.05. RESULTS: Actual drug doses administered for etorphine, azaperone and midazolam were 0.015 ± 0.001, 0.15 ± 0.01 and 0.16 ± 0.02 mg kg-1, respectively. Induction time for treatment EA was 3.3 (3.6) minutes and not different from 3.2 (3.2) minutes for treatment EM. The overall mean arterial blood pressure was significantly lower for treatment EA (102 ± 25 mmHg) than that for treatment EM (163 ± 18 mmHg) (p < 0.001). The PaO2 for treatment EA (37 ± 12 mmHg; 5.0 ± 1.6 kPa) was not different from that for treatment EM (43 ± 8 mmHg; 5.8 ± 1.1 kPa). Recovery time was 0.8 (0.6) minutes for treatment EA and did not differ from 1.1 (0.6) minutes for treatment EM. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment EA was as effective as treatment EM for immobilization in this study. However, systemic arterial hypertension was a concern with treatment EM, and both combinations produced clinically relevant hypoxaemia. Supplemental oxygen administration is recommended with both drug combinations.

Shuni Virus in Wildlife and Nonequine Domestic Animals, South Africa.

We screened nonequine animals with unexplained neurologic signs or death in South Africa during 2010-2018 for Shuni virus (SHUV). SHUV was detected in 3.3% of wildlife, 1.1% of domestic, and 2.0% of avian species. Seropositivity was also demonstrated in wildlife. These results suggest a range of possible SHUV hosts in Africa.

Mycobacterium bovis Infection in African Wild Dogs, Kruger National Park, South Africa.

We screened African wild dogs (Lycaon pictus) in Kruger National Park, South Africa, for Mycobacterium bovis infection using an interferon-gamma release assay. We detected M. bovis sensitization in 20 of 21 packs; overall apparent infection prevalence was 83%. These animals experience high infection pressure, which may affect long-term survival and conservation strategies.

Mycobacterium bovis in a Free-Ranging Black Rhinoceros, Kruger National Park, South Africa, 2016.

In 2016, an emaciated black rhinoceros (Diceros bicornis) was found in Kruger National Park, South Africa. An interferon-γ response was detected against mycobacterial antigens, and lung tissue was positive for Mycobacterium bovis. This case highlights the risk that tuberculosis presents to rhinoceros in M. bovis-endemic areas.

Short-read full-length 16S rRNA amplicon sequencing for characterisation of the respiratory bacteriome of captive and free-ranging African elephants (Loxodonta africana).

Hypervariable region sequencing of the 16S ribosomal RNA (rRNA) gene plays a critical role in microbial ecology by offering insights into bacterial communities within specific niches. While providing valuable genus-level information, its reliance on data from targeted genetic regions limits its overall utility. Recent advances in sequencing technologies have enabled characterisation of the full-length 16S rRNA gene, enhancing species-level classification. Although current short-read platforms are cost-effective and precise, they lack full-length 16S rRNA amplicon sequencing capability. This study aimed to evaluate the feasibility of a modified 150 bp paired-end full-length 16S rRNA amplicon short-read sequencing technique on the Illumina iSeq 100 and 16S rRNA amplicon assembly workflow by utilising a standard mock microbial community and subsequently performing exploratory characterisation of captive (zoo) and free-ranging African elephant (Loxodonta africana) respiratory microbiota. Our findings demonstrate that, despite generating assembled amplicons averaging 869 bp in length, this sequencing technique provides taxonomic assignments consistent with the theoretical composition of the mock community and respiratory microbiota of other mammals. Tentative bacterial signatures, potentially representing distinct respiratory tract compartments (trunk and lower respiratory tract) were visually identified, necessitating further investigation to gain deeper insights into their implication for elephant physiology and health.

Pansteatitis of unknown etiology associated with large-scale Nile crocodile (Crocodylus niloticus) mortality in Kruger National Park, South Africa: pathologic findings.

Annual mortality events in Nile crocodiles (Crocodylus niloticus) in the Olifants River Gorge in Kruger National Park, South Africa, were experienced between 2008 and 2012, during which at least 216 crocodiles died. Live crocodiles were lethargic. Necropsy examination of 56 affected crocodiles showed dark yellow-brown firm nodules in both somatic fat and the abdominal fat body. In all of the 11 crocodiles submitted for histology, degenerative, necrotic, and inflammatory changes supported a diagnosis of steatitis in both fat types. Crocodiles are apex predators in this anthropogenically changed aquatic ecosystem that is used by humans upstream and downstream from the park for domestic, agricultural, fishing, and recreational purposes. This pathologic review of pansteatitis in crocodiles in the Olifants River system was part of a broad multidisciplinary research program. To date, no definitive causative agent has been identified. Epidemiologic evidence suggests that this event may have been a one-time event with long-standing repercussions on the health of the crocodiles. Pathologic findings are rarely documented in wild crocodilians. This study also reports on other conditions, including the presence of coccidian oocysts, capillarid and filaroid nematodes, digenetic trematodes, and pentastomes.

Reliability of the Enterprise Point-of-Care (EPOC) blood analyzer's calculated arterial oxygen-hemoglobin saturation in immobilized white rhinoceroses (Ceratotherium simum).

BACKGROUND: Enterprise Point-of-Care (EPOC) blood analysis is used routinely in wildlife veterinary practice to monitor blood oxygenation, but the reliability of the EPOC calculated arterial oxygen-hemoglobin saturation (cSaO2 ) has never been validated in the white rhinoceros (Ceratotherium simum), despite their susceptibility to hypoxemia during chemical immobilization. OBJECTIVES: We aimed to evaluate the reliability of the EPOC cSaO2 by comparing it against arterial oxygen-hemoglobin saturation (SaO2 ) measured by a co-oximeter reference method in immobilized white rhinoceroses. METHODS: Male white rhinoceroses in two studies (both n = 8) were immobilized by darting with different etorphine-based drug combinations, followed by butorphanol or saline (administered intravenously). Animals in both studies received oxygen via intranasal insufflation after 60 min. Blood samples were drawn, at predetermined time points, from a catheter inserted into the auricular artery and analyzed using the EPOC and a co-oximeter. Bland-Altman (to estimate bias and precision) and area root mean squares (ARMS) plots were used to determine the reliability of the EPOC cSaO2 compared with simultaneous co-oximeter SaO2 readings. RESULTS: The rhinoceros were acidotic (pH of 7.3 ± 0.1 [mean ± standard deviation]), hypercapnic (PaCO2 of 73.7 ± 10.5 mmHg), and normothermic (body temperature of 37.4 ± 1.8°C). In total, 389 paired cSaO2 -SaO2 measurements were recorded (the cSaO2 ranged between 13.2% and 99.0%, and the SaO2 ranged between 11.8% and 99.9%). The EPOC cSaO2 readings were unreliable (inaccurate, imprecise, and poor ARMS) across the entire saturation range (bias -6%, precision 5%, and ARMS 8%). CONCLUSIONS: The EPOC cSaO2 is unreliable and should not be used to monitor blood oxygenation in immobilized white rhinoceroses.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS (EEHV) IN FREE-RANGING AFRICAN ELEPHANTS (LOXODONTA AFRICANA) IN THE KRUGER NATIONAL PARK, SOUTH AFRICA.

Elephant endotheliotropic herpesvirus (EEHV) infection can cause acute, often fatal, EEHV hemorrhagic disease in free-ranging and human-managed Asian elephants (Elephas maximus) and human-managed African elephants (Loxodonta africana). However, significant knowledge gaps exist pertaining to the presence of EEHV in free-ranging African elephant populations. We retrospectively screened 142 opportunistically collected samples (blood, n=98; bronchoalveolar lavage (BAL) fluid, n=21; trunk wash (TW) fluid, n=23) obtained between 2010 and 2020 from 98 free-ranging African elephants in the Kruger National Park, South Africa, for the presence of different EEHVs, as well as determining the real-time quantitative PCR positivity rate in this population. With the use of validated, previously published DNA extraction and real-time quantitative PCR protocols provided by the National Elephant Herpesvirus Laboratory (Washington, DC, USA), EEHV was detected in nine male African elephants from samples collected in 2011 (n=1), 2013 (n=1), 2018 (n=2), 2019 (n=4), and 2020 (n=1). Viral detection was more common in respiratory compared with blood samples. Six elephants tested positive for EEHV2 subtype (blood, n=2; BAL, n=3; TW, n=2), including one individual that tested positive on matched respiratory samples (BAL and TW). Four elephants tested positive for EEHV3-4-7 (blood, n=1; BAL, n=2; TW, n=1), whereas EEHV6 was not detected in any of the study animals. One elephant tested positive for both EEHV2 and EEHV3-4-7 in the same BAL sample. Even though the levels of viremia varied between 158 and 1,292 viral genome equivalents/mL blood and viral shedding of EEHV2 and EEHV3-4-7 was detected in respiratory samples, no clinical signs were observed in these apparently healthy elephants. These findings are consistent with reports of asymptomatic EEHV infection in human-managed African elephants.

"Spotting" Mycobacterium bovis infection in leopards (Panthera pardus) - novel application of diagnostic tools.

Background: Mycobacterium bovis (M. bovis) is the causative agent of animal tuberculosis (TB) which poses a threat to many of South Africa's most iconic wildlife species, including leopards (Panthera pardus). Due to limited tests for wildlife, the development of accurate ante-mortem tests for TB diagnosis in African big cat populations is urgently required. The aim of this study was to evaluate currently available immunological assays for their ability to detect M. bovis infection in leopards. Methods: Leopard whole blood (n=19) was stimulated using the QuantiFERON Gold Plus In-Tube System (QFT) to evaluate cytokine gene expression and protein production, along with serological assays. The GeneXpert® MTB/RIF Ultra (GXU®) qPCR assay, mycobacterial culture, and speciation by genomic regions of difference PCR, was used to confirm M. bovis infection in leopards. Results: Mycobacterium bovis infection was confirmed in six leopards and individuals that were tuberculin skin test (TST) negative were used for comparison. The GXU® assay was positive using all available tissue homogenates (n=5) from M. bovis culture positive animals. Mycobacterium bovis culture-confirmed leopards had greater antigen-specific responses, in the QFT interferon gamma release assay, CXCL9 and CXCL10 gene expression assays, compared to TST-negative individuals. One M. bovis culture-confirmed leopard had detectable antibodies using the DPP® Vet TB assay. Conclusion: Preliminary results demonstrated that immunoassays and TST may be potential tools to identify M. bovis-infected leopards. The GXU® assay provided rapid direct detection of infected leopards. Further studies should aim to improve TB diagnosis in wild felids, which will facilitate disease surveillance and screening.

EFFECT OF BOMA CONFINEMENT ON HEMATOLOGIC AND BIOCHEMICAL VALUES IN FREE-RANGING WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN KRUGER NATIONAL PARK, SOUTH AFRICA.

Boma adaptation is an important component of rhinoceros translocations to allow transition to new diets, restricted space, and quarantine for disease screening. However, up to 20% of recently captured white rhinoceros (Ceratotherium simum) do not adjust to captivity, resulting in early release or even death. The causes and physiologic consequences of maladaptation to boma confinement are poorly understood. The aim of this investigation was to evaluate hematologic and serum biochemical changes in maladapted rhinoceros compared to animals that adapted under the same boma conditions. Ninety-six white rhinoceros were captured between 2009 and 2011 in Kruger National Park, South Africa and placed in bomas prior to translocation. Weight, complete blood count, and serum biochemical panel results were recorded when rhinoceros were placed in the boma and repeated on the day of release. In this study, the mean duration of boma confinement for maladapted white rhinoceros was 13 d (range 8-16 d) compared to 89.9 d (range 39-187 d) for adapted animals. Mean weight loss between capture and release was significantly greater in maladapted rhinoceros (224.0 versus 65.9 kgs; P<0.001). Although adapted rhinoceros had statistically significant changes in some hematologic and biochemical values, most were not considered clinically relevant. In contrast, the maladapted rhinoceros had significant changes at the time of early release from the boma, including evidence of leukocytosis with left shift, lymphopenia, eosinopenia, decreased red blood cell count and hematocrit, increased serum creatine kinase, and decreased serum calcium, phosphorus, and magnesium values. Along with loss of body condition, these findings were consistent with a stress-associated catabolic response. These changes occurred in the first 2 wk of confinement, and the results provide a foundation for evaluating adaptation in white rhinoceros. Future studies should focus on factors that improve adaptation and welfare of recently confined free-ranging white rhinoceros.

EFFECT OF CAPTURE AND IMMOBILIZATION ON BOMA ADAPTATION IN FREE-RANGING WHITE RHINOCEROS (CERATOTHERIUM SIMUM) IN KRUGER NATIONAL PARK, SOUTH AFRICA.

Ninety-six white rhinoceros (Ceratotherium simum) were captured between February and October 2009-2011 in Kruger National Park, South Africa and placed in boma confinement before translocation. Of these, 19 rhinoceros did not adapt to the bomas and required early release (n=18) or died (n=1). The available immobilization data and physiologic parameters, including blood gas analyses, were compared between adapted and maladapted rhinoceros to determine whether predisposing causes could be identified. There were no statistical differences in age category, sex, or body weight at capture between adaptation cohorts. The recorded immobilization data, physiologic values, blood gas analytes, hematologic, or serum chemistry values were not statistically different between adapted and maladapted rhinoceros at capture, except maladapted rhinoceros had lower median serum aspartate aminotransferase, blood urea nitrogen, and phosphorus values; however, these statistically different values were not clinically important. Therefore, observable demographic or capture-related factors did not appear to predispose white rhinoceros to maladaptation to boma confinement. Further investigations into factors affecting adaptation should be performed to minimize the effect on rhinoceros health and welfare.

Adaptation and Diagnostic Potential of a Commercial Cat Interferon Gamma Release Assay for the Detection of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis (M. bovis) infection in wildlife, including lions (Panthera leo), has implications for individual and population health. Tools for the detection of infected lions are needed for diagnosis and disease surveillance. This study aimed to evaluate the Mabtech Cat interferon gamma (IFN-γ) ELISABasic kit for detection of native lion IFN-γ in whole blood samples stimulated using the QuantiFERON® TB Gold Plus (QFT) platform as a potential diagnostic assay. The ELISA was able to detect lion IFN-γ in mitogen-stimulated samples, with good parallelism, linearity, and a working range of 15.6-500 pg/mL. Minimal matrix interference was observed in the recovery of domestic cat rIFN-γ in lion plasma. Both intra- and inter-assay reproducibility had a coefficient of variation less than 10%, while the limit of detection and quantification were 7.8 pg/mL and 31.2 pg/mL, respectively. The diagnostic performance of the QFT Mabtech Cat interferon gamma release assay (IGRA) was determined using mycobacterial antigen-stimulated samples from M. bovis culture-confirmed infected (n = 8) and uninfected (n = 4) lions. A lion-specific cut-off value (33 pg/mL) was calculated, and the sensitivity and specificity were determined to be 87.5% and 100%, respectively. Although additional samples should be tested, the QFT Mabtech Cat IGRA could identify M. bovis-infected African lions.

Diagnosis of Mycobacterium bovis infection in free-ranging common hippopotamus (Hippopotamus amphibius).

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection are crucial for effective surveillance and management, especially in wildlife populations. There are, however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection, and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB-endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevalence of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB-endemic ecosystems and evaluate serological and other diagnostic tools in this species.

Comparison of interferon gamma release assay and CXCL9 gene expression assay for the detection of Mycobacterium bovis infection in African lions (Panthera leo).

Mycobacterium bovis (M. bovis) infection has been identified in both domestic and wild animals and may threaten the conservation of vulnerable species including African lions (Panthera leo). There is a need to develop accurate ante-mortem tools for detection of M. bovis infection in African big cat populations for wildlife management and disease surveillance. The aim of this study was to compare the performances of two immunological assays, the QuantiFERON®-TB Gold Plus (QFT) Mabtech Cat interferon gamma release assay (IGRA) and QFT CXCL9 gene expression assay (GEA), which have both shown diagnostic potential for M. bovis detection in African lions. Lion whole blood (n=47), stimulated using the QFT platform, was used for measuring antigen-specific CXCL9 expression and IFN-γ production and to assign M. bovis infection status. A subset (n=12) of mycobacterial culture-confirmed M. bovis infected and uninfected African lions was used to compare the agreement between the immunological diagnostic assays. There was no statistical difference between the proportions of test positive African lions tested by the QFT Mabtech Cat IGRA compared to the QFT CXCL9 GEA. There was also a moderate association between immunological diagnostic assays when numerical results were compared. The majority of lions had the same diagnostic outcome using the paired assays. Although the QFT Mabtech Cat IGRA provides a more standardized, commercially available, and cost-effective test compared to QFT CXCL9 GEA, using both assays to categorize M. bovis infection status in lions will increase confidence in results.

Cytokine biomarker discovery in the white rhinoceros (Ceratotherium simum).

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, disrupts conservation programs of threatened species such as the white rhinoceros (Ceratotherium simum). Interferon gamma release assays have been developed for the diagnosis of M. bovis infection in rhinoceros, however, the discovery of additional diagnostic biomarkers might improve the accuracy of case detection. The aim of this pilot study was therefore to evaluate a novel unbiased approach to candidate biomarker discovery and preliminary validation. Whole blood samples from twelve white rhinoceros were incubated in Nil and TB antigen tubes of the QuantiFERON® TB Gold (In-Tube) system after which RNA was extracted and reverse transcribed. Using the equine RT2 profiler PCR array, relative gene expression analysis of samples from two immune sensitized rhinoceros identified CCL4, CCL8, IL23A, LTA, NODAL, TNF, CSF3, CXCL10 and GPI as upregulated in response to antigen stimulation. Novel gene expression assays (GEAs) were designed for selected candidates, i.e. CCL4, CXCL10 and IFNG, and analysis of QFT-processed samples showed the CXCL10 GEA could distinguish between five M. bovis-infected and five uninfected rhinoceros. These findings confirm the value of the equine RT2 profiler PCR array as a useful tool for screening biomarkers for the diagnosis of M. bovis infection in rhinoceros.