CRISPR-Cas9-mediated host signal reduction for 18S metabarcoding of host-associated eukaryotes.

Metabarcoding-based methods for identification of host-associated eukaryotes have the potential to revolutionize parasitology and microbial ecology, yet significant technical challenges remain. In particular, highly abundant host reads can mask the presence of less-abundant target organisms, especially for sample types rich in host DNA (e.g., blood and tissues). Here, we present a new CRISPR-Cas9-mediated approach designed to reduce host signal by selective amplicon digestion, thus enriching clinical samples for eukaryotic endosymbiont sequences during metabarcoding. Our method achieves a nearly 76% increased efficiency in host signal reduction compared with no treatment and a nearly 60% increased efficiency in host signal reduction compared with the most commonly used published method. Furthermore, the application of our method to clinical samples allows for the detection of parasite infections that would otherwise have been missed.

SERUM FENTANYL CONCENTRATIONS AND BEHAVIOR ASSOCIATED WITH TRANSDERMAL FENTANYL APPLICATION ON HEALTHY CORN SNAKES (PANTHEROPHIS GUTTATUS).

Snakes are common household pets and frequently managed in zoos. Geriatric snakes commonly develop osteoarthritis, leading to a declining quality of life that often results in euthanasia. Anecdotally, the application of transdermal fentanyl patches (TFP) appears to contribute to clinical improvement, including increased activity level, in osteoarthritic snakes presumed to be in pain. This study evaluated serum fentanyl concentrations over time and the effects of TFP on the normal behavior of healthy, captive, adult corn snakes (Pantherophis guttatus) using constant video monitoring. Serum fentanyl concentrations were evaluated over 4 wk during 12.5 µg/h TFP application, and the results demonstrated long-lasting (>4 wk) serum concentrations that were consistent with analgesic efficacy in mammalian species during TFP application. At 4 wk of TFP application, mean serum fentanyl concentrations were 11.5 ± 5.5 ng/ml. Snakes were videotaped for 1 wk prior to and 2 wk after 12.5 µg/h TFP application, and behavior was evaluated by an ethogram. Behavioral changes associated with TFP application included decreased mean time spent active, decreased mean number of climbs, and decreased mean number of water visits; feeding behavior was unchanged. Overall, these results suggest that TFP application may provide safe, clinically effective analgesia in healthy corn snakes for at least 4 wk without inducing deleterious side effects, and may therefore be appropriate analgesia for management of osteoarthritic snakes.

Maxillary Ameloblastoma in an Asiatic Black Bear (Ursus thibetanus).

An approximately 30-year-old intact female Asiatic black bear (Ursus thibetanus) was presented for annual examination and a history of ptyalism. A large 9.5 cm × 5 cm × 5 cm, firm, round mass was identified attached to the hard palate on physical examination. A computed tomography scan was performed, and the heterogeneous, mineral-attenuating mass was seen arising from the right aspect of the palatine bone and extending rostrally to the level of the last maxillary molars, caudally into the oropharynx, and dorsally into the nasal choana. Surgical debulking was performed to remove the portion of the mass within the oral cavity. Histopathologic analysis was consistent with a keratinizing ameloblastoma. Nine months postoperatively, the patient was asymptomatic for the tumor. The patient was euthanized 23-months postoperatively, and severe diffuse pustular dermatitis, growth of the ameloblastoma on the hard palate, and various degenerative and aging changes were noted on necropsy at that time. This is the first report of an ameloblastoma in a member of the Ursidae family.

FACTORS IMPACTING SUCCESSFUL REHABILITATION OF BIG BROWN BATS (EPTESICUS FUSCUS) IN A WISCONSIN WILDLIFE REHABILITATION CENTER: A 5-YEAR RETROSPECTIVE.

The big brown bat (Eptesicus fuscus; EPFU) is widely distributed throughout the Americas and plays critical roles in sustaining cave ecosystems and abating agricultural pests. In Wisconsin, EPFU is a threatened species with declining populations due to hibernacula disturbances, wind turbines, and habitat destruction. Due to their ecological and economic value, it is important to be able to release EPFU that enter wildlife rehabilitation centers back to the wild. This study evaluated the medical records of 454 EPFU (275 male, 179 female) admitted to a wildlife rehabilitation center in Wisconsin from 2015 to 2020. For each bat, the season at intake, examination findings, length of time in rehabilitation, and final outcome (released or not released) were recorded. Using a multiple variable logistic regression model, there was a statistically significant positive association between length of time in the rehabilitation center and likelihood of release (odds ratio [OR] 1.08; 95% CI 1.06-1.12); this association can be explained by the need to overwinter some otherwise healthy bats in rehabilitation during hibernation. The following examination findings were associated with a significantly lower likelihood of release: wing injury (OR 0.32; 95% CI 0.10-0.89) and decreased body condition (OR 0.29; 95% CI 0.12-0.64). When corrected for time spent in rehabilitation (potentially artificially lengthened due to hibernation), patients admitted in the summer and fall were less likely to be released than those admitted in the winter (OR 0.93; 95% CI 0.90-0.96 and OR 0.95; 95% CI 0.92-0.97, respectively). The results of this study can be used to help veterinarians and licensed rehabilitators better triage EPFU during admission to wildlife rehabilitation centers in order to improve management and promote successful release back to the wild.

MANAGEMENT OF MEDICAL COMPLICATIONS ASSOCIATED WITH A PRESUMED TETANUS INFECTION IN A NORTHWEST BORNEAN ORANGUTAN (PONGO PYGMAEUS PYGMAEUS).

An 18-yr-old female orangutan (Pongo pygmaeus pygmaeus) developed opisthotonus after sustaining conspecific bite wounds 3 wk earlier. The orangutan developed progressive tetraparesis and dysphagia, despite normal mentation, suggestive of tetanus. A tetanus vaccine had been administered at 2 yr of age, but none since. Brain magnetic resonance imaging, computed tomography, cerebral spinal fluid tap, and bloodwork were unremarkable. Viral, Baylisascaris, and tetanus toxin testing were negative. A femoral central venous catheter (CVC) was placed to provide medications, fluids, and parenteral nutrition. The orangutan received human tetanus immunoglobulin, tetanus toxoid, penicillin, methocarbamol, and analgesia. After 1 wk, the catheterized limb became edematous; a deep vein thrombosis (DVT) was diagnosed ultrasonographically. A cephalic CVC was placed, the limb casted, intravenous therapy reinitiated, and enoxaparin started. The orangutan became mobile days later, and progressively improved. Despite no compliance with enoxaparin, the DVT resolved without residual signs. This is the first reported case of presumptive tetanus and DVT in a great ape.

REPEATED USE OF A THIAFENTANIL-BASED ANESTHESIA PROTOCOL IN AN OKAPI (OKAPIA JOHNSTONI).

Seven anesthesia events were performed over 6 wk on a 1.5-yr-old female okapi (Okapia johnstoni) being managed for a fetlock injury. A combination of butorphanol (B) (median; range) (0.045; 0.031-0.046 mg/kg), medetomidine (M) (0.037; 0.031-0.037 mg/kg), ketamine (K) (0.553; 0.536-1.071 mg/kg), and thiafentanil (T) (0.0045; 0.0040-0.0046 mg/kg) was administered in a padded stall. One dart containing all drugs was used for the first two anesthesias. Subsequently, BM was administered 10 min prior to KT using two darts. Time (median; range) from initial injection to first effects (6; 3-7 min) and recumbency (14; 4-20 min) were recorded. Induction quality with the one-dart protocol was poor or fair and was good or excellent with the two-dart protocol. Following recumbency, the okapi was intubated and ventilated, and physiological parameters were recorded. Anesthesia was consistently achieved with BMKT, but induction was smoother with the staged two-dart approach. Neither resedation nor renarcotization was observed post-reversal.

REVIEW OF VETERINARY INTERVENTION IN REPRODUCTION OF CAPTIVE WESTERN LOWLAND GORILLAS (GORILLA GORILLA GORILLA) FROM 1996 TO 2016.

In order to gain an understanding of the frequency of veterinary intervention during the periparturient period of western lowland gorillas (Gorilla gorilla gorilla) in captivity between 1996 and 2016, a survey was sent to institutions associated with the Association of Zoos and Aquariums' Gorilla Species Survival Plan®. A total of 193 births were reported during the survey period, from 51/53 institutions. There were six incidences of prolonged labor (longer than 6 hr; 3%), six cases of cesarean section (3%), and three incidences of veterinary intervention that did not involve a cesarean section (2%). Fifty-one gorilla neonates (26%) were assist-reared (required intervention lasting longer than 24 hr). Out of 51 attempts to reintroduce neonates to dams or surrogates, 42 were accepted (82%), while nine attempts (18%) were deemed failures. The age group with the highest rate of maternal or surrogate acceptance after reintroduction was between 6 and 12 mo of age. Compared to data from a 1981 survey, the percentage of assist-reared gorillas decreased from 64% to 26%. Results show that veterinary intervention in the periparturient period is low, and there is a high rate of acceptance of neonates by either the dam or a surrogate after intervention. Advances in captive husbandry and veterinary knowledge have led to a reduced risk of veterinary intervention during gorilla parturition; however, the long-term effects on behavioral development of the neonate should be considered before removal of the infant from the dam for any period of time.

REFERENCE INTERVALS FOR ACUTE PHASE PROTEINS FOR KOALAS (PHASCOLARCTOS CINEREUS) AT THE SAN DIEGO ZOO.

The synthesis and circulating concentrations of acute phase proteins (APPs) are regulated in response to inflammation, infection, trauma, and neoplasia in many domestic and nondomestic species. The APP response is species specific; thus, assays must be validated, and reference intervals must be determined for each species. Koalas (Phascolarctos cinereus) are a vulnerable species, threatened by infectious and inflammatory diseases both under human care and in the wild. The ability to diagnose, treat, and provide prognosis for common koala health problems is challenged by the paucity of sensitive diagnostic tests. Assays for C-reactive protein, serum amyloid A, and haptoglobin were validated for use in koalas. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology based on serum samples from 26 healthy koalas at the San Diego Zoo. The reference intervals are as follows: C-reactive protein, 3.2-24.1 mg/L; serum amyloid A, 0.10-0.45 mg/L; haptoglobin, 0.10-0.64 mg/ml.

Management of respiratory complications in a western lowland gorilla (Gorilla gorilla gorilla) born by cesarean section.

A female western lowland gorilla (Gorilla gorilla gorilla) infant was delivered by cesarean section (C-section) to an 18-year-old primiparous dam after prolonged labor. The infant required resuscitation at birth and was hospitalized for management of pneumonia and associated respiratory distress secondary to the aspiration of meconium-stained amniotic fluid. The infant received nine days of intensive care with respiratory support, antibiotics, intravenous fluid therapy, anti-inflammatory drugs, bronchodilators, and chest physiotherapy. Respiratory support included nasal continuous positive airway pressure (CPAP), and supplemental oxygen. Complications included right lung atelectasis secondary to bronchial obstruction and pneumonia. Following the resolution of pneumonia, visual reintroductions with the troop were initiated. The dam was given a medical lactation supplement (metoclopramide) and her mammary glands were stimulated to increase milk production. After several days of visual and olfactory introductions, the infant was successfully physically reintroduced to her dam at 12 days of age; the dam showed appropriate maternal response and nursing of the infant. The infant had appropriate growth and development, and 4 years later, has no evidence of negative side effects of neonatal respiratory disease. This is the first reported case of management of neonatal gorilla respiratory disease associated with a C-section, with successful reintroduction to the family group after hospitalization.

CRYPTOCOCCUS NEOFORMANS VAR. GRUBII-ASSOCIATED RENAL AMYLOIDOSIS CAUSING PROTEIN-LOSING NEPHROPATHY IN A RED KANGAROO (MACROPUS RUFUS).

A 10-year-old male castrated red kangaroo (Macropus rufus) presented with mandibular swelling. Examination findings included pitting edema with no dental disease evident on examination or radiographs. The results of blood work were moderate azotemia, hypoalbuminemia, and severely elevated urine protein:creatinine ratio (9.9). Radiographs showed an interstitial pattern of the caudal right lung, and an abdominal ultrasound demonstrated scant effusion. Symptomatic and empirical therapy with antibiotics, anti-inflammatory drugs, and an angiotensin-converting enzyme (ACE) inhibitor did not resolve clinical signs. Due to poor prognosis and declining quality of life, euthanasia was elected. Necropsy revealed chronic granulomatous pneumonia of the caudal right lung lobe with intralesional Cryptococcus, identified as C. neoformans var. grubii by DNA sequencing. Severe bilateral glomerular and tubulointerstitial amyloidosis induced protein-losing nephropathy, leading to tri-cavitary effusion, subcutaneous edema, and cachexia. The authors speculate that renal amyloidosis was associated with chronic cryptococcal pneumonia in this red kangaroo.

LOW PREVALENCE OF WEST NILE VIRUS ANTIBODIES IN SELECT NORTHERN CALIFORNIA OWL SPECIES (2007-2014).

The objective of this study was to determine evidence of previous West Nile virus (WNV) infection in northern California owls. Owl serum samples were collected from birds presenting to a veterinary medical teaching hospital between 2007 and 2014 and were screened for the presence of WNV antibodies by an indirect enzyme immunoassay (EIA). Only one of 71 samples (1.41%) tested was positive by EIA and confirmed by a plaque reduction neutralization test; it was the most recent sample collected. The reason for the low prevalence of WNV in these California owls despite a high prevalence in sympatric avian species in the same region is unknown and should be a topic for further research.

Multi-method assessment of patients with febrile illness reveals over-diagnosis of malaria in rural Uganda.

BACKGROUND: Health clinics in rural Africa are typically resource-limited. As a result, many patients presenting with fever are treated with anti-malarial drugs based only on clinical presentation. This is a considerable issue in Uganda, where malaria is routinely over-diagnosed and over-treated, constituting a wastage of resources and an elevated risk of mortality in wrongly diagnosed patients. However, rapid diagnostic tests (RDTs) for malaria are increasingly being used in health facilities. Being fast, easy and inexpensive, RDTs offer the opportunity for feasible diagnostic capacity in resource-limited areas. This study evaluated the rate of malaria misdiagnosis and the accuracy of RDTs in rural Uganda, where presumptive diagnosis still predominates. Specifically, the diagnostic accuracy of "gold standard" methods, microscopy and PCR, were compared to the most feasible method, RDTs. METHODS: Patients presenting with fever at one of two health clinics in the Kabarole District of Uganda were enrolled in this study. Blood was collected by finger prick and used to administer RDTs, make blood smears for microscopy, and blot Whatman FTA cards for DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing. The accuracy of RDTs and microscopy were assessed relative to PCR, considered the new standard of malaria diagnosis. RESULTS: A total of 78 patients were enrolled, and 31 were diagnosed with Plasmodium infection by at least one method. Comparing diagnostic pairs determined that RDTs and microscopy performed similarly, being 92.6 and 92.0 % sensitive and 95.5 and 94.4 % specific, respectively. Combining both methods resulted in a sensitivity of 96.0 % and specificity of 100 %. However, both RDTs and microscopy missed one case of non-falciparum malaria (Plasmodium malariae) that was identified and characterized by PCR and sequencing. In total, based on PCR, 62.0 % of patients would have been misdiagnosed with malaria if symptomatic diagnosis was used. CONCLUSIONS: Results suggest that diagnosis of malaria based on symptoms alone appears to be highly inaccurate in this setting. Furthermore, RDTs were very effective at diagnosing malaria, performing as well or better than microscopy. However, only PCR and DNA sequencing detected non-P. falciparum species, which highlights an important limitation of this test and a treatment concern for non-falciparum malaria patients. Nevertheless, RDTs appear the only feasible method in rural or resource-limited areas, and therefore offer the best way forward in malaria management in endemic countries.

COMPARISON OF MEDETOMIDINE-KETAMINE AND BUTORPHANOL-AZAPERONE-MEDETOMIDINE IN CAPTIVE BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS).

The objective of this study was to compare a traditional partially reversible medetomidine-ketamine sedation with a more reversible butorphanol-azaperone-medetomidine combination in Bennett's wallabies ( Macropus rufogriseus ) maintained in a zoological collection. Fourteen animals were divided into two treatment groups. Individuals in group 1 received an intramuscular (i.m.) injection of butorphanol (0.54 ± 0.05 mg/kg), azaperone (0.22 ± 0.02 mg/kg), and medetomidine (0.16 ± 0.02 mg/kg). Individuals in group 2 received an i.m. injection of ketamine (5.43 ±1.16 mg/kg) with medetomidine (0.05 ± 0.014 mg/kg). For group 1, sedation was reversed with atipamezole (0.81 ± 0.069 mg/kg i.m.) and naltrexone (1.08 ± 0.09 mg/kg i.m.). For group 2, sedation was reversed with atipamezole (0.27 ± 0.056 mg/kg i.m.). There were no significant differences between the groups in mean time to induction, time spent on gas anesthesia, or time to standing after reversal was administered. Animals in both groups required supplemental gas anesthesia to facilitate intubation. No adverse reactions or effects were noted with either protocol; however, the BAM protocol did not provide sufficient sedation for handling in all animals and may not be suitable for use in this species.

Antemortem diagnosis of hydrocephalus in two Congo African grey parrots (Psittacus erithacus erithacus) by means of computed tomography.

CASE DESCRIPTION: A 7-year-old and a 10-year-old Congo African grey parrot (Psittacus erithacus erithacus; parrots 1 and 2, respectively) were evaluated because of neurologic deficits. CLINICAL FINDINGS: Parrot 1 had an 8- to 9-month history of lethargy and anorexia, with a recent history of a suspected seizure. Parrot 2 had a 6-month history of decreased activity and vocalizing, with an extended history of excessive water intake; a water deprivation test ruled out diabetes insipidus, and psychogenic polydipsia was suspected. Both birds had ophthalmologic asymmetry, with anisocoria detected in parrot 1 and unilateral blindness in parrot 2. Metal gastrointestinal foreign bodies were observed on whole-body radiographs of both birds, but blood lead concentrations were below the range indicated for lead toxicosis. Findings on CT of the head were consistent with hydrocephalus in both cases. TREATMENT AND OUTCOME: Parrot 1 received supportive care and died 3 months after the diagnosis of hydrocephalus. Parrot 2 was treated with omeprazole and prednisolone for 10 days without any improvement in neurologic deficits; euthanasia was elected, and hydrocephalus was confirmed on necropsy. No underlying or concurrent disease was identified. CLINICAL RELEVANCE: Hydrocephalus should be considered a differential diagnosis for parrots evaluated because of CNS signs. Computed tomography was an excellent screening tool to diagnose hydrocephalus in these patients. Compared with MRI, CT is more frequently available and offers reduced scanning times, reduced cost, and less concern for interference from metallic foreign bodies.

Molecular detection of Plasmodium in free-ranging birds and captive flamingos (Phoenicopterus chilensis) in Chicago.

Frozen blood samples from 13 species of free-ranging birds (n = 65) and captive Chilean flamingos (Phoenicopterus chilensis) (n = 46) housed outdoors in the Chicago area were screened for Plasmodium. With the use of a modified polymerase chain reaction, 20/65 (30.8%) of free-ranging birds and 26/46 (56.5%) of flamingos were classified as positive for this parasite genus. DNA sequencing of the parasite cytochrome b gene in positive samples demonstrated that eight species of free-ranging birds were infected with five different Plasmodium spp. cytochrome b lineages, and all positive Chilean flamingos were infected with Plasmodium spp. cytochrome b lineages most closely related to organisms in the Novyella subgenus. These results show that Chilean flamingos may harbor subclinical malaria infections more frequently than previously estimated, and that they may have increased susceptibility to some Plasmodium species.

Co-infection and cross-species transmission of divergent Hepatocystis lineages in a wild African primate community.

Hemoparasites of the apicomplexan family Plasmodiidae include the etiological agents of malaria, as well as a suite of non-human primate parasites from which the human malaria agents evolved. Despite the significance of these parasites for global health, little information is available about their ecology in multi-host communities. Primates were investigated in Kibale National Park, Uganda, where ecological relationships among host species are well characterized. Blood samples were examined for parasites of the genera Plasmodium and Hepatocystis using microscopy and PCR targeting the parasite mitochondrial cytochrome b gene, followed by Sanger sequencing. To assess co-infection, "deep sequencing" of a variable region within cytochrome b was performed. Out of nine black-and-white colobus (Colobus guereza), one blue guenon (Cercopithecus mitis), five grey-cheeked mangabeys (Lophocebus albigena), 23 olive baboons (Papio anubis), 52 red colobus (Procolobus rufomitratus) and 12 red-tailed guenons (Cercopithecus ascanius), 79 infections (77.5%) were found, all of which were Hepatocystis spp. Sanger sequencing revealed 25 different parasite haplotypes that sorted phylogenetically into six species-specific but morphologically similar lineages. "Deep sequencing" revealed mixed-lineage co-infections in baboons and red colobus (41.7% and 64.7% of individuals, respectively) but not in other host species. One lineage infecting red colobus also infected baboons, but always as the minor variant, suggesting directional cross-species transmission. Hepatocystis parasites in this primate community are a diverse assemblage of cryptic lineages, some of which co-infect hosts and at least one of which can cross primate species barriers.