Quantification of chlamydiae in the endangered Houston toad (Anaxyrus houstonensis).

Two primer set/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as Chlamydia pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brain swabs, and ultimately specifically identify and quantify chlamydiae, specifically C. pneumoniae in these samples.

Quantification of members of the Mycobacterium chelonae-abscessus complex in lesions of the endangered houston toad (Anaxyrus houstonensis).

Illumina-based 16S rRNA V3 amplicon sequencing of total DNA obtained from soft tissue lesions (joint granulomas) of the endangered Houston toad (Anaxyrus houstonensis) demonstrated that many reads represented members of the actinobacterial Mycobacterium chelonae-abscessus complex. In order to quantify members of this complex in those lesions, we designed three complex-specific primer set/probe combinations (sets I, II and III) targeting variable regions on the 23S rRNA gene for SybrGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Both SybrGreen- and Taqman-based analyses specifically detected members of the M. chelonae-abscessus complex in lesion samples, with numbers between 104 and 107 cells per 100-mg sample. Values within individual samples were generally comparable between SybrGreen- and Taqman-based detection methods and between all primer set/probe combinations, except for SybrGreen-based analyses of a few samples analyzed with primer set I that used a less specific forward primer. The development of highly specific detection and quantification methods for members of the M. chelonae-abscessus complex in lesion samples can enable group specific tracking of these organisms, particularly in captive or stewardship settings where source and transmission monitoring are valuable tools to husbandry and species conservation.

DETECTION OF GLIOTOXIN BUT NOT BIS(METHYL)GLIOTOXIN IN PLASMA FROM BIRDS WITH CONFIRMED AND PROBABLE ASPERGILLOSIS.

Aspergillosis remains a difficult disease to diagnose antemortem in many species, especially avian species. In the present study, banked plasma samples from various avian species were examined for gliotoxin (GT), which is a recognized key virulence factor produced during the replication of Aspergillus species hyphae and a secondary metabolite bis(methyl)gliotoxin (bmGT). Initially, liquid chromatography-tandem mass spectrometry methods for detecting GT and bmGT were validated in a controlled model using sera obtained from rats experimentally infected with Aspergillus fumigatus. The minimum detection level for both measurements was determined to be 3 ng/ml, and the assay was found to be accurate and reliable. As proof of concept, GT was detected in 85.7% (30/35) of the samples obtained from birds with confirmed aspergillosis and in 60.7% (17/28) of samples from birds with probable infection but only in one of those from clinically normal birds (1/119). None of the birds were positive for bmGT. Repeated measures from birds under treatment suggests results may have prognostic value. Further studies are needed to implement quantitative methods and to determine the utility of this test in surveillance screening in addition to its use as a diagnostic test in birds with suspected aspergillosis.

Retrospective Survey of Amphibian Pathology Cases at Texas A&M University System (2016-2020).

Dramatic declines in amphibians worldwide highlight the need for a better understanding of diseases affecting these species. To delineate the health issues of amphibians submitted to Texas A&M University System (2016-2020), the databases were queried on the basis of defined selection criteria. A total of 502 anurans (157 frogs [44 species] and 345 toads [10 species]) and 30 caudatans (23 salamanders [6 species] and 7 newts [4 species]) were reviewed. A most likely cause of death or major pathological finding (CD-MPF) leading to euthanasia was identified in 295 (55%) Anura cases and 15 (50%) Caudata cases. Of the 532 records reviewed, anurans included 492 captive, seven free-ranging and three undetermined specimens. All caudatans were captive. The most common CD-MPF in anurans was infectious/inflammatory (228/295; 77%), involving mycobacteriosis (73/228; 32%), chlamydiosis (44/228; 19%) and mycosis (32/228; 14%). Neoplasia was less common (28/295; 9%). Infectious/inflammatory lesions (14/15; 93%) were the main CD-MPFs in caudatans. Infectious diseases are a significant threat to captive amphibians in Texas and these results may aid personnel involved in amphibian conservation programmes, veterinarians and diagnosticians.

MYCOBACTERIA IN SKIN LESIONS AND THE HABITAT OF THE ENDANGERED HOUSTON TOAD (ANAXYRUS HOUSTONENSIS).

Head-starting of the federally endangered Houston toad (Anaxyrus houstonensis), that is, the release of egg strands, tadpoles, and metamorphic juveniles produced in captivity into the original breeding ponds, requires assessment of potential threats for the transmission of pathogens from captive to free-ranging toads. We used Illumina-based 16S rRNA V3 amplicon sequencing to investigate the community structure of bacteria from skin lesions of captive Houston toad and habitat (pond) samples. Proteobacteria, alone or together with Actinobacteria and, in some samples, Cyanobacteria represented virtually all reads in tissue lesion samples, whereas pond samples were much more diverse, with Acidobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, and Verrucomicrobia present with little variation between samples. If present in lesions, Actinobacteria were largely represented by Mycobacteriaceae, and here mainly by one sequence identical to sequences of members of the Mycobacterium chelonae-abscessus complex. In pond samples, mycobacteria represented only a small portion of the actinobacteria, although at higher diversity with six distinct reads. Sequences for reads obtained from pond samples were identical to those representing the M. chelonae-abscessus complex, a group with Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium avium, a group with Mycobacterium vaccae, Mycobacterium fortuitum, Mycobacterium poriferae, and a group with Mycobacterium elephantis and Mycobacterium celeriflavum, whereas sequences of high similarity were detected for reads related to those of Mycobacterium holsaticum, Mycobacterium pallens, and Mycobacterium obuense, and Mycobacterium goodii. Our results indicated that lesions observed on the Houston toad in captivity are not the result of mycobacteria in every case, and that the presence of mycobacteria in the captive colony does not represent a novel pathogen threat to the wild populations because such bacteria are also seen in the natural pond habitats for the Houston toad.

Primary Hepatic Vascular Neoplasm in a Houston Toad (Anaxyrus houstonensis).

An 11-year-old captive, female Houston toad (Anaxyrus houstonensis) became lethargic and reluctant to move. This toad had been administered human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone (LHRH) annually as part of a hormone-assisted captive propagation programme for 8 years. Due to poor prognosis, euthanasia was elected. At necropsy, the liver was moderately enlarged, diffusely dark red and had a nodular superficial appearance. Microscopically, the hepatic nodules consisted of poorly demarcated, non-encapsulated, paucicellular infiltrative neoplastic growths. These consisted of spindle cells, similar to sinusoidal endothelial cells, that formed irregular blood-filled vascular channels resembling sinusoids separated by thin bands of collagen and reticulin fibres. Neoplastic cells dissected through and replaced hepatic cords and bile ducts. Based on the cytohistomorphological features and analogy with vascular neoplasms in other vertebrates, a diagnosis of low-grade angiosarcoma was made. To the best of our knowledge, this is the first record of primary hepatic vascular neoplasia in an amphibian. It is not known if the long-term administration of HCG and LHRH had any oncogenic role in this animal. Vascular neoplasia should be included in the differential diagnosis of hepatomegaly and non-specific lethargy and health decline in amphibian species.

CONGENITAL HYPOTHYROIDISM IN A BORNEAN ORANGUTAN (PONGO PYGMAEUS) AND A SUMATRAN ORANGUTAN (PONGO ABELII).

Congenital hypothyroidism (CH) in humans is most commonly caused by disruption of thyroid gland development (dysgenesis) or an inherited defect in thyroid hormone biosynthesis (dyshormonogenesis). CH has not been previously documented in great apes. This report describes the clinical presentation, diagnosis, and treatment of CH in a 9-mo-old male Bornean orangutan (Pongo pygmaeus) and a 6-wk-old female Sumatran orangutan (Pongo abelii). Primary CH due to thyroid dysgenesis was confirmed in the Bornean orangutan using sonography and radioisotope scintigraphy. Although commercial thyroid immunoassays are not validated for use in orangutans, in comparison to age-matched controls, thyroid-stimulating hormone level was markedly elevated, and serum thyroxine (T4) and free T4 levels were markedly decreased in both cases. Oral supplementation with levothyroxine sodium resulted in noticeable clinical improvement in both orangutans within 30 days of initiating treatment.

Chlamydia pneumoniae Polioencephalomyelitis and Ganglionitis in Captive Houston Toads (Anaxyrus houstonensis).

Chlamydia pneumoniae is a ubiquitous pathogen causing disease in humans, mammals, birds, reptiles, and amphibians. Since 2012, C. pneumoniae infection has caused neurologic disease and mortality in a breeding colony of endangered Houston toads (Anaxyrus houstonensis) at the Houston Zoo. The purpose of this report is to present the histopathologic and ultrastructural characteristics of C. pneumoniae infection in Houston toads. Fourteen cases were evaluated by histopathology and 1 case was evaluated by electron microscopy. The major histopathologic finding was necrotizing and histiocytic polioencephalomyelitis and ganglionitis. Bacteria formed intracytoplasmic inclusions within neurons but frequently extended into the surrounding tissue from necrotic cells. Ultrastructural evaluation showed the bacteria formed reticulate and elementary bodies characteristic of Chlamydia spp.

REVIEW OF MORTALITY AND EFFECTIVENESS OF NEONATAL TREATMENT IN CAPTIVE ATTWATER'S PRAIRIE CHICKENS ( TYMPANUCHUS CUPIDO ATTWATERI).

A retrospective study of mortality and neonatal treatment for 975 Attwater's prairie chickens ( Tympanuchus cupido attwateri) was conducted from 2009-2015. Gross necropsy, histopathology, and medical records for chicks less than 8 wk of age at the Houston Zoo were reviewed and summarized. The total mortality rate was 36% ( n = 352). Yolk sac infection was the most common cause of death (21%; n = 73) followed by maladaptation (19%; n = 68), musculoskeletal abnormalities (19%; n = 66), necrotic enteritis (13%; n = 44), and mucoid enteritis (11%; n = 39). Gavage feeding of a highly digestible formula was associated with a higher proportion of necrotic enteritis and a reduced occurrence of both mucoid enteritis and maladaptation. Meloxicam administration had a significantly reduced incidence of mucoid enteritis. Survival past 10 days of age, prevention of neonatal bacterial infection, and supportive treatment with meloxicam are important factors to increase chick survival and improve captive breeding success for Attwater's prairie chickens. Gavage feeding of a diet low in digestible carbohydrates has the potential for reducing disease due to necrotic enteritis in this species.

CLINICAL INFECTION OF CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS) WITH ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 4.

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants. A number of EEHV types and subtypes exist, where most deaths have been caused by EEHV1A and EEHV1B. EEHV4 has been attributed to two deaths, but as both diagnoses were made postmortem, EEHV4 disease has not yet been observed and recorded clinically. In this brief communication, two cases of EEHV4 infection in juvenile elephants at the Houston Zoo are described, where both cases were resolved following intensive treatment and administration of famciclovir. A quantitative real-time polymerase chain reaction detected EEHV4 viremia that correlated with clinical signs. High levels of EEHV4 shedding from trunk wash secretions of the first viremic elephant correlated with subsequent infection of the second elephant with EEHV4. It is hoped that the observations made in these cases--and the successful treatment regimen used--will help other institutions identify and treat EEHV4 infection in the future.

CLINICAL INFECTION OF TWO CAPTIVE ASIAN ELEPHANTS (ELEPHAS MAXIMUS) WITH ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1B.

The ability of prior infection from one elephant endotheliotropic herpesvirus (EEHV) type to protect against clinical or lethal infection from others remains an important question. This report describes viremia and subsequent shedding of EEHV1B in two juvenile 4-yr-old Asian elephants within 3 wk or 2 mo following significant infections caused by the rarely seen EEHV4. High levels of EEHV1B shedding were detected in the first elephant prior to emergence of infection and viremia in the second animal. The EEHV1B virus associated with both infections was identical to the strain causing infection in two herd mates previously. High EEHV viremia correlated with leukopenia and thrombocytopenia, which was followed by leukocytosis and thrombocytosis when clinical signs started to resolve. The observations from these cases should be beneficial for helping other institutions monitor and treat elephants infected with EEHV1, the most common virus associated with lethal hemorrhagic disease.

Hematology and plasma biochemistry analytes in five age groups of immature, captive-reared loggerhead sea turtles (Caretta caretta).

Abstract: Blood samples of 85 immature, apparently healthy, captive-reared loggerhead sea turtles (Caretta caretta) were analyzed for 13 hematologic variables and total solids of 5 age groups (8, 20, 32, 44, and 56 mo old) and for 20 plasma biochemical analytes of 4 age groups (20 to 56 mo old). Each individual turtle was sampled under similar conditions during a blood collection period of 3 days. Hematologic analytes included packed cell volume, white blood cell (WBC) counts, WBC estimates, and leukocyte differentials. Biochemical analysis included albumin, alanine aminotransferase, alkaline phosphatase, amylase, aspartate aminotransferase, blood urea nitrogen, calcium, chloride, cholesterol, creatine kinase, creatinine, gamma glutamyltransferase, globulins, glucose, phosphorous, potassium, sodium, total bilirubin, total protein, total solids, and uric acid. In due consideration of small sample size in all five age groups, the results of hematologic and biochemical analysis were used to determine ranges for these analytes and to compare values among consecutive age groups. Several significant differences in some hematologic and biochemical variables were identified and need to be considered in the interpretation of blood work of immature, growing sea turtles in human care.

Acute visceral toxoplasmosis in captive dik-dik (Madoqua guentheri smithi).

Acute toxoplasmosis was diagnosed in 2 captive dik-dik (Madoqua guentheri smithi) in the Houston Zoo. Both animals became ill suddenly and died in spite of supportive therapy. Toxoplasma gondii was identified in tissues of both animals immunohistochemically, and antibodies to T. gondii were found in titers of 1:800 or more in both animals upon examination by the modified agglutination rest. The cause of death was considered to be toxoplasmic pneumonia. This is the first report of toxoplasmosis in M. g. smithi.