José Luiz Catão-Dias

Worldwide Occurrence of Feline Hemoplasma Infections in Wild Felid Species

ABSTRACT While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroys cat, margay, and ocelot. “Candidatus Mycoplasma haemominutum” was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas “Candidatus Mycoplasma turicensis” was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.

Morphometry of canine cutaneous mast cell tumors.

Twenty-four canine cutaneous nodules, diagnosed as mast cell tumors by fine-needle aspiration biopsy and confirmed by histopathologic analysis by staining with hematoxylin and eosin (HE) and toluidine blue, were analyzed by computerized nuclear morphometry on panoptic- and HE-stained cytopathology slides. Two hundred nuclei per lesion were examined. The morphometric parameters investigated were nuclear area, mean diameter, perimeter, regularity factor, and ellipticity factor. Lesions were graded as I (well differentiated), II (intermediate differentiation), or III (poorly differentiated) according to the following morphologic features: invasiveness, cellularity and cellular morphology, mitotic index, and stromal reaction. Nuclear morphometric results were then compared with histopathologic grades. Values of nuclear area, mean diameter, and perimeter increased with increase in histopathologic grade, but statistical analysis revealed significant differences only between grades II and III and between grades I and III when HE was used (P, 0.01) and between grades I and III with panoptic stain (P, 0.05). The ellipticity factor and regularity factor did not reveal significant differences between histopathologic grades. The results indicate that nuclear morphometric analysis, in combination with the rapid and inexpensive cytopathology technique, can help in mast cell tumor grading, thus contributing to the establishment of a more precise prognosis and treatment.

First Evidence of Feline Herpesvirus, Calicivirus, Parvovirus, and Ehrlichia Exposure in Brazilian Free-ranging Felids

Serum samples from 18 pumas (Puma concolor), one ocelot (Leopardus pardalis), and two little spotted cats (Leopardus tigrinus) collected from free-ranging animals in Brazil between 1998 and 2004 were tested by indirect immunofluorescence (IFA) for antibodies to feline herpesvirus 1 (FHV 1), calicivirus (FCV), coronavirus (FCoV), parvovirus (FPV), Ehrlichia canis, Anaplasma phagocytophilum, and Bartonella henselae. Serum samples also were tested, by Western blot and ELISA, for feline leukemia virus (FeLV) specific antibodies and antigen, respectively, by Western blot for antibodies to feline immunodeficiency virus (FIV), and by indirect ELISA for antibodies to puma lentivirus (PLV). Antibodies to FHV 1, FCV, FCoV, FPV, FeLV, FIV, PLV or related viruses, and to B. henselae were detected. Furthermore, high-titered antibodies to E. canis or a closely related agent were detected in a puma for the first time.

Pathology of Toxoplasmosis in Captive New World Primates

Clinical information was available for 32 of 33 New World primates with fatal toxoplasmosis, all of which were subjected to a variable number of pathological observations. Death without apparent clinical signs occurred in 43.7% of cases. The most common clinical findings were malaise (40.6%), dyspnoea (18.7%), hypothermia (15.6%) and a sero-sanguinous or foamy nasal discharge (12.5%). Nutritional status was good in 71.8%, average in 18.7% and poor in 9.4%. The most common post-mortem findings were pulmonary congestion (78.8%), pulmonary oedema (75.8%), splenomegaly (57.6%) and mesenteric lymphadenitis (54.6%). The most common histopathological findings were multifocal necrotic hepatitis (97%), lymphadenitis (95.4%), interstitial pneumonia (90.3%) and necrotic splenitis (71.4%). The gross post-mortem changes in cebids were more variable than those observed in callitrichids, a fact that may complicate the diagnosis of toxoplasmosis in cebids.

Characterization of monkey enteropathogenic Escherichia coli (EPEC) and human typical and atypical EPEC serotype isolates from neotropical nonhuman primates.

ABSTRACT Enteropathogenic Escherichia coli (EPEC) has been associated with infantile diarrhea and mortality in humans in developing countries. While diarrhea is also a major problem among primates kept in captivity, the role of E. coli is unclear. This study was designed to characterize diarrheagenic E. coli recovered from the feces of 56 New World nonhuman primates, primarily marmosets (Callithrix spp.). Seventeen of the 56 primates had signs of diarrhea and/or enteritis. E. coli recovered from feces from these animals was tested by PCR for genes encoding virulence factors of diarrheagenic E. coli and for patterns of adherence to HeLa cells. In addition, isolates were characterized by the fluorescence actin staining test and by their ability to induce attaching and effacing lesions. PCR for the eae gene was positive in 10 of the 39 (27%) apparently healthy animals and in 8 of the 17 (47%) animals with diarrhea and/or enteritis. Colonies of eae+E. coli were serotyped and examined by PCR for genes encoding EPEC virulence markers. The eae+E. coli isolates recovered from both healthy and sick nonhuman primates demonstrated virulence-associated attributes similar to those of EPEC strains implicated in human disease and are designated monkey EPEC. The results presented here indicate that EPEC may be a significant pathogen for nonhuman primates, deserving further investigation. The similarities between the affected animals investigated in this study and human EPEC infections suggest that marmosets may represent an important model for EPEC in humans.

TOXOPLASMOSIS IN GOLDEN-HEADED LION TAMARINS (LEONTOPITHECUS CHRYSOMELAS) AND EMPEROR MARMOSETS (SAGUINUS IMPERATOR) IN CAPTIVITY

Abstract From 1991 to 1995, eight New World nonhuman primates of the family Callitrichidae belonging to the collection of Fundação Parque Zoológico de São Paulo died of toxoplasmosis. Of the eight affected nonhuman primates, four were Leontopithecus chrysomelas (one male, three females) and four were Saguinus imperator (two males, two females). The most commonly affected organs were the lungs, liver, and lymph nodes, with hemorrhagic and necrotic lesions. Histopathologic examination revealed protozoa that were morphologically consistent with Toxoplasma gondii. Immunohistochemical assays were strongly positive for T. gondii.

Outbreak of avian malaria associated to multiple species of Plasmodium in magellanic penguins undergoing rehabilitation in southern Brazil.

Avian malaria is a mosquito-borne disease caused by Plasmodium spp. Avian plasmodia are recognized conservation-threatening pathogens due to their potential to cause severe epizootics when introduced to bird populations with which they did not co-evolve. Penguins are considered particularly susceptible, as outbreaks in captive populations will often lead to high morbidity and rapid mortality. We used a multidisciplinary approach to investigate an outbreak of avian malaria in 28 Magellanic penguins (Spheniscus magellanicus) at a rehabilitation center during summer 2009 in Florianópolis, Brazil. Hemosporidian infections were identified by microscopic and molecular characterization in 64% (18/28) of the penguins, including Plasmodium (Haemamoeba) tejerai, Plasmodium (Huffia) elongatum, a Plasmodium (Haemamoeba) sp. lineage closely related to Plasmodium cathemerium, and a Haemoproteus (Parahaemoproteus) sp. lineage closely related to Haemoproteus syrnii. P. tejerai played a predominant role in the studied outbreak and was identified in 72% (13/18) of the hemosporidian-infected penguins, and in 89% (8/9) of the penguins that died, suggesting that this is a highly pathogenic parasite for penguins; a detailed description of tissue meronts and lesions is provided. Mixed infections were identified in three penguins, and involved P. elongatum and either P. tejerai or P. (Haemamoeba) sp. that were compatible with P. tejerai but could not be confirmed. In total, 32% (9/28) penguins died over the course of 16 days despite oral treatment with chloroquine followed by sulfadiazine-trimethoprim. Hemosporidian infections were considered likely to have occurred during rehabilitation, probably from mosquitoes infected while feeding on local native birds, whereas penguin-mosquito-penguin transmission may have played a role in later stages of the outbreak. Considering the seasonality of the infection, rehabilitation centers would benefit from narrowing their efforts to prevent avian malaria outbreaks to the penguins that are maintained throughout summer.

Surveillance using serological and molecular methods for the detection of infectious agents in captive Brazilian neotropic and exotic felids.

The aim of the current study was to investigate the exposure of captive wild felids to various infectious pathogens using serological and molecular methods. One hundred and fifty-nine neotropic felids and 51 exotic felids from 28 captive settings in Brazil were tested. While antibodies against Feline parvovirus and Feline coronavirus (FCoV), Feline calicivirus and Bartonella spp. were frequently detected by serologic tests, antibodies against Felid herpesvirus 1 or infection with hemotropic mycoplasmas were less prevalent. Serologic evidence of exposure to Ehrlichia spp., Feline immunodeficiency virus, and Feline leukemia virus (FeLV) was detected rarely, and infections with FeLV, Ehrlichia spp., and Cytauxzoon spp. were found infrequently. The detected Bartonella sequence was molecularly similar to B. koehlerae and B. henselae; for Cytauxzoon, the sequence resembled those from domestic cats. No Anaplasma phagocytophilum and Theileria spp. infections were detected. The positive test results varied significantly among different facilities and species. Additionally, FCoV seropositivity was more prevalent in captivity than in free-ranging populations. Results suggest that testing is appropriate prior to relocation of felids.

Nuclear Morphometry in Cytopathology: A Prognostic Indicator for Canine Cutaneous Mast Cell Tumors

Twenty-nine canine cutaneous mast cell tumors (MCTs) were morphometrically analyzed with regard to mean nuclear area (MNA) using cytopathology smears. The results showed a correlation between MNA and survival. When graded into 2 morphometrically different groups, there were statistically significant differences among high- and low-grade MCTs, regarding both Romanowsky-type stain and hematoxylin and eosin. Cytomorphometry could also separate histologic grade II tumors with better prognosis from the more aggressive MCTs. The results indicated that nuclear morphometry on cytopathology preparations can predict the biological behavior of cutaneous MCTs in dogs in an independent manner, yielding a rapid and reproducible diagnosis, which renders the method useful for veterinary oncology.

Parasitological and new molecular-phylogenetic characterization of the malaria parasite Plasmodium tejerai in South American penguins.

This study is the first report on mortality of Spheniscus magellanicus, penguin of South America, caused by Plasmodium tejerai, which was identified using morphological and molecular analyses. Blood stages (trophozoites, meronts and gametocytes) were reported and illustrated. The necropsy revealed marked splenomegaly and pulmonary edema, as well as moderate hepatomegaly and hydropericardium. The histopathology revealed the presence of tissue meronts in the macrophages and endothelial cells of multiple organs. The molecular analyses showed 5.6% of genetic divergence in cytochrome b gene between P. tejerai and Plasmodium relictum. Morphology of blood and tissue stages of P. tejerai is similar to P. relictum; the distinction between these two species requires experience in the identification of avian Plasmodium species. Molecular studies associated with reliably identified morphological species are useful for barcoding and comparisons with previous studies of wildlife malaria infections as well as for posterior phylogenetic and phylogeographic studies. S. magellanicus is a new host record of P. tejerai, which is the virulent parasite and worth more attention in avian conservation and veterinary medicine projects in South America.