Ultrastructure of the spermatozoa from a Florida manatee (Trichechus manatus latirostris)

Semen was opportunistically collected from a free-ranging, 10-year-old, 275 cm (total length) Florida manatee (Trichechus manatus latirostris) during rehabilitation treatments. Ultrastructure of the spermatozoa was examined by scanning and transmission electron microscopy and differed slightly from that described for other mammals. Comparisons to the manatee's closest phylogenetic relatives, the elephant and hyrax, were made. The manatee spermatozoa had a similar acrosome but a distinct annulus and lacked the dense bodies observed in the neck of the elephant spermatozoa. Additionally, manatee spermatozoa lacked the lateral vacuoles observed in the nuclear chromatin from of the hyrax spermatozoa. These data add to our understanding of manatees and allow for comparative studies with other species that may be useful in phylogenetic and reproductive studies.     

Mycobacteriosis in two captive Florida manatees (Trichechus manatus latirostris).

Two male Florida manatees (Trichechus manatus latirostris) died at the marine aquarium in Inagi City, Tokyo, Japan. Acid-fast bacteria were demonstrated in tuberculoid nodules in the lungs from both manatees. Mycobacterium marinum and M. fortuitum were isolated from one manatee; M. marinum and M. kansasii were cultured from the second animal. This report confirms the pathogenicity and potentially fatal outcome of mycobacterial infection in manatees. In addition, the pathologic response to infection with these mycobacteria in manatees is similar to that associated with Mycobacterium spp. in other animals.     

Corneal vascularization in the Florida manatee (Trichechus manatus latirostris) and three-dimensional reconstruction of vessels

The cornea of the Florida manatee is unique and unusual in its anatomy in that blood vessels have been found throughout. In all other animal species, this is considered a pathological condition impeding vision, and is usually caused by injury or trauma. The purpose of this study was to more clearly describe corneal vascularization by examining the architecture through three-dimensional reconstruction in order to find possible patterns in size, distribution, and location of blood vessels relative to gender, age, location, and season. Twenty-six eyes from 22 individuals were prepared for histologic examination and subsequent three-dimensional reconstruction. Every specimen examined had blood vessels in the cornea, comprising an average of 0.3% of total surface density (volume) of the cornea. No differences were found between individuals based on gender, age, and season. Environmental influences were not a significant factor either, which was not originally anticipated. The presence of vessels at the level of the anterior epithelium was surprising and it appeared that the vascularization was directed more anteriorly than was originally thought. The presence of blood vessels in a prenatal eye was also found. In all the eyes examined, no signs of injury or trauma could be observed. The presence of blood vessels appears to minimally impair vision based on their low density, size, and location. The association of vessels with the anterior epithelium and development of vessels within the fetus point to an evolutionary adaptation possibly due to the manatee's unique ability to move between water bodies.     

Electrocardiography in two subspecies of manatee (Trichechus manatus latirostris and T. m. manatus).

Electrocardiographic (ECG) measurements were recorded in two subspecies of awake, apparently healthy, wild manatees (Trichechus manatus latirostris and T. m. manatus) undergoing routine field examinations in Florida and Belize. Six unsedated juveniles (dependent and independent calves) and 6 adults were restrained in ventral recumbency for ECG measurements. Six lead ECGs were recorded for all manatees and the following parameters were determined: heart rate and rhythm; P, QRS, and T wave morphology, amplitude, and duration; and mean electrical axis (MEA). Statistical differences using a t-test for equality of means were determined. No statistical difference was seen based on sex or subspecies of manatees in the above measured criteria. Statistical differences existed in heart rate (P = 0.047), P wave duration (P = 0.019), PR interval (P = 0.025), and MEA (P = 0.021) between adult manatees and calves. Our findings revealed normal sinus rhythms, no detectable arrhythmias, prolonged PR and QT intervals, prolonged P wave duration, and small R wave amplitude as compared with cetacea and other marine mammals. This paper documents the techniques for and baseline recordings of ECGs in juvenile and adult free-living manatees. It also demonstrates that continual assessment of cardiac electrical activity in the awake manatee can be completed and can be used to aid veterinarians and biologists in routine health assessment, during procedures, and in detecting the presence of cardiac disease or dysfunction.     

Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris

Florida manatees (Trichechus manatus latirostris) are exposed to many conditions in their habitat that may adversely impact health and impair immune function in this endangered species. In an effort to increase the current knowledge base regarding the manatee immune system, the production of an important reactive nitrogen intermediate, nitric oxide (NO), by manatee peripheral blood mononuclear cells (PBMC) was investigated. PBMC from healthy captive manatees were stimulated with LPS, IFN-gamma, or TNF-alpha, either alone or in various combinations, with NO production assessed after 24, 48, 72, and 96 h of culture. NO production in response to LPS stimulation was significantly greater after 48, 72, or 96 h of culture compared to NO production after 24h of culture. A specific inhibitor of inducible nitric oxide synthase (iNOS), L-NIL (L-N(6)-(1-iminoethyl)lysine), significantly decreased NO production by LPS-stimulated manatee PBMC. Manatee specific oligonucleotide primers for iNOS were designed to measure expression of relative amounts of mRNA in LPS-stimulated manatee PBMC from captive manatees. NO production by PBMC from manatees exposed to red tide toxins was analyzed, with significantly greater NO production by both unstimulated and LPS stimulated PBMC from red tide exposed compared with healthy captive or cold-stress manatees. Free-ranging manatees produced significantly lower amounts of nitric oxide compared to either captive or red tide rescued manatees. Results presented in this paper contribute to the current understanding of manatee immune function and represent the first report of nitric oxide production in the immune system of a marine mammal.     

Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris).

Manatees (Trichechus manatus latirostris) are afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as "cold stress syndrome" and pneumonia. White-blood-cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase-response proteins, such as haptoglobin and serum amyloid A, have proven to be sensitive measures of inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including total white-blood-cell count, albumin: globulin ratio, gel electrophoresis analysis, C-reactive protein, alpha, acid glycoprotein, haptoglobin, fibrinogen, and serum amyloid A. Samples were collected from 71 apparently healthy and 27 diseased animals during diagnostic medical examination. Serum amyloid A, measured by ELISA, followed by albumin:globulin ratio, measured by plasma gel electrophoresis, were most sensitive in diagnosing inflammatory disease, with diagnostic sensitivity and specificity of approximately 90%. The reference interval for serum amyloid A is <10-50 microg/ml with an equivocal interval of 51-70 microg/ml. The reference interval for albumin:globulin ratio by plasma gel electrophoresis is 0.7-1.1. Albumin: globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology. Albumin:globulin ratio, measured by serum gel electrophoresis, has a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4-2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. The haptoglobin assay is significantly affected by hemolysis. Fibrinogen, measured by heat precipitation, has a reference interval of 100-400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.     

Clinical biochemistry in healthy manatees (Trichechus manatus latirostris).

Florida manatees (Trichechus manatus latirostris) are endangered aquatic mammals living in coastal and riverine waterways of Florida and adjacent states. Serum or plasma biochemical analyses are important tools in evaluating the health of free-ranging and captive manatees. The purpose of this study was to measure diagnostically important analytes in the plasma of healthy manatees and to determine whether there was significant variation with respect to location (free-ranging versus captive), age class (small calves, large calves, subadults, adults), and gender. No significant differences in plasma sodium, potassium, bilirubin, glucose, alanine aminotransferase, or creatine kinase were found among these classes of animals. Compared to free-ranging manatees, captive animals had significantly lower mean concentrations of plasma chloride, phosphate, magnesium, triglycerides, anion gap, and lactate. Captive manatees had significantly higher mean values of total CO2, calcium, urea, creatinine, alkaline phosphatase, gamma-glutamyltransferase, total protein, albumin, and albumin/globulin ratio than did free-ranging animals. Differences in the environments of these two groups, including diet, temperature, salinity, and stress, might account for some of these results. The higher plasma lactate and anion gap concentrations and lower total CO2 concentrations of free-ranging manatees were probably due to greater exertion during capture, but the lack of elevated plasma creatine kinase activity relative to captive animals indicates that there was no serious muscle injury associated with capture. Plasma phosphate decreased and total globulins increased with age. Plasma cholesterol and triglyceride concentrations were highest in small calves. Plasma aspartate aminotransferase was higher in large calves than in adults and subadults, and the albumin/ globulin ratio was higher in subadults than in adults. Plasma total CO2 was higher and chloride was slightly lower in females than in males.     

Gross and microscopic observations on the lingual structure of the Florida Manatee Trichechus manatus latirostris

The tongue of the Florida manatee (Trichechus manatus latirostris) was studied macroscopically, light and electron microscopically. The tongue was slender, muscular and firmly fixed in the oral cavity; only the cranial tip was free and mobile. Numerous filiform papillae were distributed over the dorsal surface of the rostral tongue. Multiple raised, round fungiform-like papillae were distributed over most of the dorsum. Typical fungiform papillae were restricted to the lateral margins of the tongue. Foliate papillae, presenting as multi-fossulate openings, were noted on the caudolateral margins. Open pits were located on the dorsocaudal surface and lateral walls. Microscopic examination showed that most of the lingual dorsum was covered with a thick stratified squamous epithelium. Open pits led to well-developed mucous salivary glands. Glands within the foliate papillae were mostly mucous, although some seromucous glands were present. Taste buds were restricted to the epithelium of the foliate papillae. Throughout the tongue, striated muscle was abundant below the epithelium. Blood vessels, lymph channels and nerve fibres were freely distributed throughout the intermuscular stroma. Nerve fibres reacted positively with neurone specific enolase (NSE) antibody throughout the tongue, including nerve bundles, glands and taste buds. Clear to translucent vacuoles were found juxtaposed to nuclei in the stratum spinosum in the foliate papillae epithelium.     

Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris

The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected each year by exposure to cold weather or harmful algal blooms (red tide; Karenia brevis). Exposures can be sublethal, resulting in stressed animals that are rescued and taken to authorized facilities for rehabilitation, or lethal if exposures are prolonged or unusually severe. To investigate whether sublethal environmental exposures can impair immune function in manatees, rendering animals vulnerable to disease or death, mitogen-induced proliferation was assessed in lymphocytes from manatees exposed to cold temperatures (N=20) or red tide (N=19) in the wild, and compared to lymphocyte responses from healthy free-ranging manatees (N=32). All animals sampled for this study were adults. Lymphocytes were stimulated in vitro with either concanavalin A (ConA) or phytohemagglutinin (PHA) and proliferation was assessed after 96 h using incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into newly synthesized DNA. Proliferation of lymphocytes from manatees rescued from exposure to red tide or cold-stress was approximately one-third that of lymphocytes from healthy free-ranging manatees. To examine the direct effects of red tide toxins on lymphocyte function, mitogen-induced proliferation was assessed following co-culture of lymphocytes with K. brevis toxin extracts. Stimulation indices decreased with increasing toxin concentration, with a significant decrease in proliferation occurring in the presence of 400 ng red tide toxins/ml. When lymphocytes from cold-stressed manatees were co-cultured with red tide toxin extracts, proliferative responses were reduced even further, suggesting multiple stressors may have synergistic effects on immune function in manatees.     

Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris)

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70 degrees C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma.     

Three-dimensional reconstruction of the angioarchitecture of the ciliary body of the West Indian manatee (Trichechus manatus)

OBJECTIVE: To examine the angioarchitecture of the ciliary body in the West Indian manatee (Trichechus manatus), through the use of three-dimensional reconstruction. PROCEDURE: Specimens from West Indian manatee were preserved in 10% buffered formalin, embedded in paraffin, serial sectioned and stained by Masson trichrome for light microscopic three-dimensional reconstruction and evaluation. RESULTS: The network of blood vessels in the ciliary processes of the West Indian manatee is fed by the major arterial circle that lies mostly near the base of the iris. The branching arterioles give rise to a capillary-sinusoidal bed that extends internally along each process, emptying into two sets of veins, one being elevated. The elevated and nonelevated veins join posteriorly before emptying into the choroidal venous system. CONCLUSIONS: The angioarchitecture of the ciliary body of the West Indian manatee is clearly unique when compared to those previously examined in land mammals. Three-dimensional reconstruction of paraffin sections is an effective means to evaluate vascular patterns in ocular specimens, especially those unavailable for corrosion casting.     

Comparative anatomy of the ciliary body of the West Indian manatee (Trichechus manatus) and selected species

OBJECTIVE: To examine the anatomy of the ciliary body in the West Indian manatee (Trichechus manatus), paying close attention to its vascularization and to compare to those of its distant relative, the African elephant (Loxodonta africana), the amphibious hippopotamus (Hippopotamus amphibius) and the aquatic short-finned pilot whale (Globicephala macrorhynchus). PROCEDURE: Specimens from each species were preserved in 10% buffered formalin, and observed stereomicroscopically before being embedded in paraffin, sectioned and stained by Masson trichrome, hematoxylin and eosin, and periodic acid-Schiff for light microscopic evaluation. RESULTS: The network of blood vessels in the ciliary processes of the West Indian manatee appear to have an intricate pattern, especially with regard to venous outflow. Those of the elephant are slightly less complex, while those of the hippopotamus and whale have different vascular patterns within the ciliary body. Musculature within the ciliary body is absent within the manatee and pilot whale. CONCLUSIONS: In general, there appears to be a direct relationship between the increased development of vasculature and the loss of musculature within the ciliary bodies of the aquatic and amphibious mammals presently studied. Specifically, the ciliary body of the West Indian manatee has a comparatively unique construction, especially with regard to its vasculature.     

A contribution for the definition of serum chemistry values in captive adults Antillean manatees (Trichechus manatus manatus Linnaeus, 1758)

Serum chemistry analyses represents a fundamental tool for the diagnosis and understanding of diseases in marine mammals. Although several studies are being conducted within the field of clinical pathology, haematological and serum chemistry data for Antillean manatees are deficient. The purpose of this study was to determine serum chemistry values for captive Antillean manatees within the CMA/Ibama facility in Brazil. Serum samples were obtained from five captive adult Antillean manatees fed with seagrass and analysed for aspartate aminotransferase, alanine aminotransferase, bilirubin, alkaline phosphatase, urea, creatinine, glucose, triglycerides, cholesterol, total protein, albumin, globulin, phosphate, chloride, calcium and uric acid. Blood chemistry parameters were determined using a semi-automatic analyzer. Maximum, minimum, mean and standard deviations were calculated for each serum chemistry parameter. Differences on the values of males and females were verified using an unpaired Student's t-test. All the parameters analysed were similar between sexes, with exception of AP, which was higher in females (191.43 +/- 31.86 U/l). Alanine aminotransferase and uric acid values for Trichechus manatus manatus are reported for the first time in this paper. This study is the first to report serum chemistry parameter values for long-term captive male and female Antillean manatees. Therefore, the lower values of albumin, phosphate, chloride, cholesterol and triglycerides obtained here highlight the importance of clinical pathology during health monitoring of captive marine mammals.     

Structure and presumptive function of the iridocorneal angle of the West Indian manatee (Trichechus manatus), short-finned pilot whale (Globicephala macrorhynchus), hippopotamus (Hippopotamus amphibius), and African elephant (Loxodonta africana)

The iridocorneal angles of prepared eyes from the West Indian manatee, short-finned pilot whale, hippopotamus and African elephant were examined and compared using light microscopy. The manatee and pilot whale demonstrated capacity for a large amount of aqueous outflow, probably as part of a system compensating for lack of ciliary musculature, and possibly also related to environmental changes associated with life at varying depths. The elephant angle displayed many characteristics of large herbivores, but was found to have relatively low capacity for aqueous outflow via both primary and secondary routes. The hippopotamus shared characteristics with both land- and water-dwelling mammals; uveoscleral aqueous outflow may be substantial as in the marine mammals, but the angular aqueous plexus was less extensive and a robust pectinate ligament was present. The angles varied greatly in size and composition among the four species, and most structures were found to be uniquely suited to the habitat of each animal.     

Methods used in veterinary practice to maintain the temperature of intravenous fluids

To determine the methods used in veterinary practice to maintain the temperature of intravenous fluids, and the users' impression of their efficacy, data were collected from a survey of 150 veterinary practices. Of the 99 per cent of the practices that warmed intravenous fluids, the use of a heat retention bag cover was most popular and was considered most effective. In a laboratory experiment, four methods of maintaining the temperature of intravenous fluids were compared. The results showed that there was a significant loss of heat through the giving set. A heat retention cover was an effective device for maintaining the temperature of the prewarmed fluid in its bag. The use of ;hot hands' (a sealed surgical glove containing warmed water) was most effective in reducing heat loss from the delivered fluid, and was aided by prewarming the giving set.     

Importance of amino-acid and electrolyte balance in experimental diets used to determine the apparent metabolisable energy (AME) value of wheat

1. An experiment was done with male, Ross broiler chicks to study the effect of diet composition and electrolyte balance on the measurement of diet AME and performance parameters in diets containing high levels of wheat inclusion. 2. The control diet (G) was based on wheat (667 g/kg) with full-fat soya, soyabean meal, Extrupro and herring meal providing the protein supplements. The experimental treatments were based on 2 diets, one based on wheat (790 g/kg) with casein as the protein supplement and the other containing 667 g/kg wheat plus casein, cellulose and starch. Each of these was formulated to contain one of three concentrations of sodium and potassium bicarbonate giving final values for dietary electrolyte balance of approximately 90, 180 and 280 meq/kg diet. 3. DM intake was numerically lower with the lower level of wheat inclusion and LWG (P < 0.05) and gain:food (P < 0.001) were reduced. ME:GE was similar for the two levels of wheat/casein but higher (P < 0.001) than for the control diet. 4. The two higher electrolyte balances improved DM intake (P < 0.01), LWG and gain:food (P < 0.01) compared with the diets containing no added bicarbonate. ME:GE was unaffected by electrolyte inclusion. 5. Viscosity of ileal digesta supernatant fell (P < 0.01) with increasing bicarbonate inclusion but the value for the highest electrolyte level was still higher than for the control diet. 6. It is concluded that addition of both arginine and electrolyte is needed with a wheat/casein diet to improve performance to levels seen with a more commercial diet but that the determination of diet AME, with the type of fat addition used in this study, is unaffected by electrolyte balance. The interaction between electrolyte balance and in vivo viscosity requires further study.     

A morphological study of the gross and light microscopic lesions of infectious anaemia in Atlantic salmon (Salmo salar).

Infectious anaemia in Atlantic salmon (Salmo salar) was studied by recording gross and light microscopic changes, and the development of lesions was studied in relation to haematocrit values. Gross lesions were characterised by ascites formation, congestion and enlargement of liver and spleen, congestion of the foregut and petechiae in the peritoneum. Histologically, lesions were demonstrated in the liver, being characterised by congestion in early stages (that is, haematocrit values around 25), dilatation of the sinusoids, and in later stages (haematocrit values 25 to 15) formation of blood-filled spaces bearing morphological resemblance to peliosis hepatis. At low haematocrit values (around 10), these changes comprised large areas of the liver parenchyma, that is, blood-filled areas coalesced, presenting islets of degenerate and necrotic hepatocytes. At this stage, haemorrhagic necroses were found. Spleen and kidney lesions were characterised by congestion. In the foregut, congestion and bleeding in lamina propria were observed. Liver lesions became more disseminated and severe with decreasing haematocrit values. Hypoxia due to anaemia alone cannot fully explain the development of the liver lesions.     

A comparison of gross pathology,histopathology, and mycobacterial culture for the diagnosis of tuberculosis in elk (Cervus elaphus).

Using the isolation of Mycobacterium bovis as the reference standard, this study evaluated the sensitivity, specificity and kappa statistic of gross pathology (abattoir postmortem inspection), histopathology, and parallel or series combinations of the two for the diagnosis of tuberculosis in 430 elk and red deer. Two histopathology interpretations were evaluated: histopathology I, where the presence of lesions compatible with tuberculosis was considered positive, and histopathology II, where lesions compatible with tuberculosis or a select group of additional possible diagnoses were considered positive. In the 73 animals from which M. bovis was isolated, gross lesions of tuberculosis were most often in the lung (48), the retropharyngeal lymph nodes (36), the mesenteric lymph node (35), and the mediastinal lymph nodes (16). Other mycobacterial isolates included: 11 M. paratuberculosis, 11 M. avium, and 28 rapidly growing species or M. terrae complex. The sensitivity estimates of gross pathology and histopathology I were 93% (95% confidence limits [CL] 84.97%) and 88% [CL 77.94%], respectively, and the specificity of both was 89% [CL 85.92%]). The sensitivity and specificity of histopathology II were 89% (CL 79.95%) and 77% (CL 72.81%), respectively. The highest sensitivity estimates (93-95% [CL 84.98%]) were obtained by interpreting gross pathology and histopathology in parallel (where an animal had to be positive on at least one of the two, to be classified as combination positive). The highest specificity estimates (94-95% [CL 91-97%] were generated when the two tests were interpreted in series (an animal had to be positive on both tests to be classified as combination positive). The presence of gross or microscopic lesions showed moderate to good agreement with the isolation of M. bovis (Kappa = 65-69%). The results showed that post-mortem inspection, histopathology and culture do not necessarily recognize the same infected animals and that the spectra of animals identified by the tests overlaps.     

Occurrence of antibodies to Toxoplasma gondii and Lepstospira spp. in manatees (Trichechus inunguis) of the Brazilian Amazon

The presence of Toxoplasma gondii and Leptospira spp. antibodies was investigated in 74 manatees (Trichechus inunguis [Mammalia: Sirenia]) kept in captivity in two rescue units in the northern region of Brazil. Antibodies to T. gondii were detected in 29 (39.2%) of 74 animals by using the modified agglutination test (titer, 1:25). For antibodies against Leptospira spp., sera were diluted 1:50 and tested against 24 strains ofleptospires by microscopic agglutination microtechnique, and positive samples were end titrated. Twenty-three (31.1%) of 74 animals were reactive to four serovars (Patoc 21/23, Castellonis 2/23, Icterohaemorrhagiae 1/23, and Butembo 1/ 23), with titers ranging from 100 to 1,600. This is the first report of antibodies against T. gondii and Leptospira spp. in T. inunguis from the Brazilian Amazon.