Detection of a novel spotted fever group Rickettsia in the gophertortoise tick.

The gophertortoise tick, Amblyomma tuberculatum (Marx), is distributed throughout the southeastern United States, and its immature life stages have been reported to occasionally bite humans. Here we report detection of a novel spotted fever group (SFG) Rickettsia in A. tuberculatum ticks collected in the southern United States. Among questing ticks collected in Georgia, 10 pools of larvae were identified as gophertortoise ticks, A. tuberculatum. Each of these samples was positive for SFG Rickettsiae. The restriction fragment-length polymorphism profiles were identical to each other, but distinct from those of other rickettsiae previously found in Amblyomma spp. ticks. Partial genetic characterization of the novel agent was achieved by sequencing the 17 kDa, gltA, ompB, ompA, rpoB, and sca4 genes. Analysis of a concatenated tree of four genes (gltA, ompB, ompA, and sca4) demonstrates close relatedness of the detected Rickettsia to several SFG Rickettsia spp. The identical rickettsial DNA was detected in 50 and 70% of adult A. tuberculatum ticks from Mississippi and Florida, respectively. The results indicate wide distribution of a novel Rickettsia, capability for transovarial transmission, and high prevalence in tested tick populations.

Molecular characterization of "Candidatus Anaplasma testudinis": An emerging pathogen in the threatened Florida gopher tortoise (Gopherus polyphemus).

Members of the family Anaplasmataceae are obligate intracellular bacteria that replicate within membrane bound vacuoles in the cytoplasm of cells in vertebrate and invertebrate hosts. This study reports a putative new Anaplasma species in gopher tortoises in Florida. Two Florida gopher tortoises (Gopherus polyphemus) presented at the University of Florida Veterinary Hospital with anemia and intracytoplasmic vacuoles filled with bacteria within erythrocytes. The bacteria within these parasitophorous vacuoles were morphologically similar to Anaplasma marginale. We inoculated ISE6 cells with blood from one tortoise and isolated bacterial colonies consistent with A. marginale. Molecular characterization targeting Anaplasmataceae 16S rRNA sequences indicated that the clinical isolate, named here provisionally as "Candidatus Anaplasma testudinis", grouped within the genus Anaplasma on a separate clade, most closely related to the A. marginale, Anaplasma ovis and Anaplasma centrale group. We next screened archived red blood cells from 38 wild gopher tortoises with documented clinical anemia. Fourteen of the 38 wild tortoises, representing 5 of 11 geographical locations were PCR-positive for Anaplasmataceae spp. Sequencing analysis revealed 16S rRNA sequence identical to "Ca. A. testudinis". The clinical presentation of significant anemia associated with "Ca. A. testudinis" in a threatened species could have conservation implications. Importantly, the availability of a clinical isolate will aid further studies to develop diagnostic tests and to investigate potential tick vectors and infectivity for other wildlife and domestic animal species.

Social behavior drives the dynamics of respiratory disease in threatened tortoises.

Since the early 1990s, morbidity and mortality in tortoise populations have been associated with a transmissible, mycoplasmal upper respiratory tract disease (URTD). Although the etiology, transmission, and diagnosis of URTD have been extensively studied, little is known about the dynamics of disease transmission in free-ranging tortoise populations. To understand the transmission dynamics of Mycoplasma agassizii, the primary etiological agent of URTD in wild tortoise populations, we studied 11 populations of free-ranging gopher tortoises (Gopherus polyphemus; n = 1667 individuals) over five years and determined their exposure to the pathogen by serology, by clinical signs, and by detection of the pathogen in nasal lavages. Adults tortoises (n = 759) were 11 times more likely to be seropositive than immature animals (n = 242) (odds ratio = 10.6, 95% CI = 5.7-20, P < 0.0001). Nasal discharge was observed in only 1.4% (4/296) of immature tortoises as compared with 8.6% (120/1399) of adult tortoises. Nasal lavages from all juvenile tortoises (n = 283) were negative by PCR for mycoplasmal pathogens associated with URTD. We tested for spatial segregation among tortoise burrows by size class and found no consistent evidence of clustering of either juveniles or adults. We suggest that the social behavior of tortoises plays a critical role in the spread of URTD in wild populations, with immature tortoises having minimal interactions with adult tortoises, thereby limiting their exposure to the pathogen. These findings may have broader implications for modeling horizontally transmitted diseases in other species with limited parental care and emphasize the importance of incorporating animal behavior parameters into disease transmission studies to better characterize the host-pathogen dynamics.

Evanescent-wave biosensor for field serodiagnosis of tortoise mycoplasmosis.

Disease has become an increasingly important issue for wildlife management over the past two decades. Adequate surveillance is fundamental for disease prevention and control, thus there is an increasing need for diagnostic assays for wildlife management. The objective of this study was to evaluate the performance of a field-portable biosensor adapted for rapid detection of specific antibodies in tortoise plasma that reflect a history of exposure to Mycoplasma agassizii, which is an agent of tortoise upper respiratory tract disease. Banked plasma samples were tested in two blinded trials, and the parameters that define the reliability of a diagnostic test were estimated based on externally validated tortoise plasma controls. The mean sensitivity of the biosensor (ability to identify exposed tortoises in the group of all exposed individuals) was 78%; the mean specificity (unexposed individuals with negative test result, out of all unexposed individuals tested) was 73%; the mean positive predictive value (exposed individuals with positive test, out of all individuals with positive test) was 82%; the mean negative predictive value (unexposed individuals with negative test, out of all individuals with negative test) was 68%. In a 15-min field-portable format, the biosensor was able to discriminate between true seropositive (n=34) and true seronegative (n=23) tortoise plasma with overall accuracy of 84%. The goals established for the tortoise population can help managers decide whether potential diagnostic errors should impact management decision-making, and whether the benefits of the field-portable format of the biosensor assay outweigh any potential disadvantages.

Mycoplasma insons sp. nov., a twisted mycoplasma from green iguanas (Iguana iguana).

Mycoplasma insons sp. nov., first cultured from the choanae and tracheae of healthy green iguanas (Iguana iguana) from El Salvador, was readily distinguished from all previously described mollicutes and assigned to the Mycoplasma fastidiosum phylogenetic cluster by 16S rRNA gene sequence comparisons. Growth inhibition assays distinguished the isolates serologically from the other two members of that cluster. Many M. insons cells exhibit a remarkable twisted rod morphology despite lacking a cell wall. The organism is nonmotile, produces acid from glucose, but does not hydrolyze arginine, esculin, or urea. Mycoplasma insons 16S rRNA gene was also detected by PCR in packed blood cells from culture-negative iguanas. The type strain I17P1(T) has been deposited with the Mollicutes Collection at Purdue University and with the American Type Culture Collection (ATCC BAA-1435) in the USA. A limited number of cultures generated by the authors have also been deposited with the Culture Collection, University of Göteborg, in Sweden (CCUG 53461).

Mycoplasmosis in green iguanas (Iguana iguana).

Mycoplasma iguanae was the suspected etiology of spinal disease in feral iguanas (Iguana iguana) from Florida. In an experimental infection study, juvenile iguanas were inoculated with M. iguanae intravenously or by instillation into the nares. Blood samples obtained at intervals postinoculation were all culture negative for mycoplasma. Gross anatomic and histologic findings at necropsy 12 wk postinoculation were unremarkable. Mycoplasmas were cultured in high numbers from the posterior choanae and upper trachea of some inoculated and control iguanas at necropsy. The 16S rDNA gene sequence of these isolates revealed they were all a previously undescribed strain, Mycoplasma insons proposed species nova. M. iguanae. Mycoplasma iguanae was not recovered from the conjunctivae, choanae, trachea, lung, coelomic cavity, blood, heart, liver, spleen, limb joints, brain, or spinal cord of inoculated iguanas, and the iguanas did not seroconvert. We conclude that M. iguanae is unlikely to be an agent of acute disease in iguanas and that M. insons can be considered as normal flora in the respiratory tract of iguanas.

Lesions associated with a novel Mycoplasma sp. in California sea lions (Zalophus californianus) undergoing rehabilitation.

From July 1999 to November 2001, Mycoplasma sp. was cultured from lesions in 16 California sea lions (Zalophus californianus) undergoing rehabilitation. The Mycoplasma sp. was the likely cause of death of four animals in which it was associated with either pneumonia or polyarthritis. The most common lesion associated with this bacterium was subdermal abscessation, found in 12 animals. Other lesions included intramuscular abscesses, septic arthritis, and lymphadenopathy. Infection was associated with a leukocytosis and left shift in 12 animals. Animals with abscesses improved clinically after surgical lancing, irrigation, and systemic antibiotic therapy. The mycoplasma isolates had a consistent 16S rRNA sequence dissimilar from other Mycoplasma spp. and represent a novel species, Mycoplasma zalophi proposed sp. nov.

Mycoplasmosis and upper respiratory tract disease of tortoises: a review and update.

Tortoise mycoplasmosis is one of the most extensively characterized infectious diseases of chelonians. A 1989 outbreak of upper respiratory tract disease (URTD) in free-ranging Agassiz's desert tortoises (Gopherus agassizii) brought together an investigative team of researchers, diagnosticians, pathologists, immunologists and clinicians from multiple institutions and agencies. Electron microscopic studies of affected tortoises revealed a microorganism in close association with the nasal mucosa that subsequently was identified as a new species, Mycoplasma agassizii. Over the next 24 years, a second causative agent, Mycoplasma testudineum, was discovered, the geographic distribution and host range of tortoise mycoplasmosis were expanded, diagnostic tests were developed and refined for antibody and pathogen detection, transmission studies confirmed the pathogenicity of the original M. agassizii isolate, clinical (and subclinical) disease and laboratory abnormalities were characterized, many extrinsic and predisposing factors were found to play a role in morbidity and mortality associated with mycoplasmal infection, and social behavior was implicated in disease transmission. The translation of scientific research into management decisions has sometimes led to undesirable outcomes, such as euthanasia of clinically healthy tortoises. In this article, we review and assess current research on tortoise mycoplasmosis, arguably the most important chronic infectious disease of wild and captive North American and European tortoises, and update the implications for management and conservation of tortoises in the wild.

Mycoplasma agassizii strain variation and distinct host antibody responses explain differences between enzyme-linked immunosorbent assays and Western blot assays.

The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A405 values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations.

Effects of mycoplasmal upper respiratory tract disease on morbidity and mortality of gopher tortoises in northern and central Florida.

Gopher tortoise (Gopherus polyphemus) populations on four tracts of public lands in northern and central Florida were studied from 1998 to 2001 to assess the effects of mycoplasmal upper respiratory tract disease (URTD). Adult gopher tortoises (n=205) were marked for identification, serum and nasal flush samples were obtained for mycoplasmal diagnostic assays, and clinical signs of URTD (nasal discharge, ocular discharge, palpebral edema, and conjunctivitis) were evaluated. A subset of tortoises (n=68) was radio-instrumented to facilitate repeated sampling and document potential mortality. Presence of serum antibody to Mycoplasma agassizii was determined by enzyme-linked immunosorbent assay (ELISA), and mollicutes species were detected in nasal flushes by polymerase chain reaction (PCR). Antibody prevalence varied among sites and years but was highest in 1998, exceeding 70% at two sites. Only 11 tortoises (5%) were positive by PCR, and three species (M. agassizii, M. testudineum, and a nonpathogenic Acholeplasma) were identified in nasal flush specimens. Nasal discharge, though rare (6% of tortoises), was significantly correlated with higher ELISA ratios, study site, and positive PCR status. Mortality events (n=11) occurred on two of the three M. agassizii-positive sites; no mortality was observed on the M. agassizii-negative control site. However, none of the tested variables (ELISA result, study site, year, sex, presence of clinical signs, or carapace length) showed significant ability to predict the odds of death. Mycoplasmal URTD is believed to be a chronic disease with high morbidity but low mortality, and follow-up studies are needed to detect long-term effects.

Development and use of an indirect enzyme-linked immunosorbent assay for detection of iridovirus exposure in gopher tortoises (Gopherus polyphemus) and eastern box turtles (Terrapene carolina carolina).

Iridoviruses, pathogens typically associated with fish and amphibians, have recently been shown to cause acute respiratory disease in chelonians including box turtles, red-eared sliders, gopher tortoises, and Burmese star tortoises. Case reports of natural infections in several chelonian species in the United States have been reported, however the prevalence remains unknown in susceptible populations of free-ranging chelonians. To determine the prevalence of iridovirus exposure in free-ranging gopher tortoises (Gopherus polyphemus) in the southeast United States, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate plasma samples from wild gopher tortoises (G. polyphemus) from: Alabama (n=9); Florida (n=658); Georgia (n=225); Louisiana (n=12); Mississippi (n=28); and unknown locations (68) collected between 2001 and 2006. Eight (1.2%) seropositive tortoises were identified from Florida and seven (3.1%) from Georgia for an overall prevalence of 1.5%. Additionally, a population of eastern box turtles was sampled from a private nature sanctuary in Pennsylvania that experienced an outbreak of iridovirus the previous year, which killed 16 turtles. Only 1 turtle out of 55 survivors tested positive (1.8%). Results suggest a low exposure rate in chelonians to this pathogen; however, it is suspected that this is an underestimate of the true prevalence. Since experimental transmission studies and past outbreaks have shown a high rate of mortality in infected turtles, turtles may die before they develop an antibody response. Further, the duration of the antibody response is unknown and may also cause an underestimate of the true prevalence.

Improved enzyme-linked immunosorbent assay to reveal Mycoplasma agassizii exposure: a valuable tool in the management of environmentally sensitive tortoise populations.

The precarious status of desert (Gopherus agassizii) and gopher (Gopherus polyphemus) tortoises has resulted in research and conservation efforts that include health assessments as a substantial component of management decision-making. Therefore, it is critical that available diagnostic tests for diseases impacting these species undergo rigorous standardization and validation. Since 1992, analysis of exposure of tortoises to Mycoplasma agassizii, an etiological agent of upper respiratory tract disease, has relied on the detection of specific M. agassizii antibody by enzyme-linked immunosorbent assay (ELISA). We report here substantive refinements in the diagnostic assay and discuss the implications of its use in wildlife conservation and management. The ELISA has been refined to include more stringent quality control measures and has been converted to a clinically more meaningful titer reporting system, consistent with other diagnostic serologic tests. The ELISA results for 5,954 desert and gopher tortoises were plotted, and a subset of these serum samples (n = 90) was used to determine end-point titers, to establish an optimum serum dilution for analyzing samples, and to construct a standard curve. The relationship between titer and A405 was validated using 77 serum samples from known positive (n = 48) and negative (n = 29) control tortoises from prior transmission studies. The Youden index, J, and the optimal cut point, c, were estimated using ELISA results from the 77 control sera. Based on this evaluation, the refinement has substantially improved the performance of the assay (sensitivity of 0.98, specificity of 0.99, and J of 0.98), thus providing a clinically more reliable diagnostic test for this important infection of tortoises.