Canine hepatozoonosis in Brazil: description of eight naturally occurring cases

Eight cases of canine hepatozoonosis were diagnosed at the Veterinary Hospital (Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu), between October 1993 and April 1994. Clinical signs included anorexia, pale mucous membranes, weight loss, pain, diarrhoea, vomit, gait abnormalities, fever, polyuria and polydipsia. Haematologic findings revealed anaemia in seven cases, leucocytosis with neutrophilia in three cases, lymphopenia in three cases and monocytosis in four cases. Serum biochemistries included alterations in many parameters. Thr micrometry of Hepatozoon canis gametocytes ranged from 6.8 x 4.0 mu m to 7.5 x 4.5 mu m. Parasitaemia ranged from less than 0.5% to 2%. In all the cases reported other concurrent diseases were present. Diagnosis of canine hepatozoonosis was made by identifying H. canis gametocytes within leucocytes in stained blood smears. (C) 1998 Elsevier B.V. B.V.

SPECIES OF COCCIDIA OCCURRING IN LAMBS IN SAO-PAULO STATE, BRAZIL

The purpose of this experiment was to characterize the species of Eimeria affecting lambs and their infection pattern. Faecal samples were collected from each animal at 14-day intervals, beginning when lambs were 2 weeks old and ending when they were 32 weeks old. The oocysts were counted and identified as E. intricata, E. parva, E. pallida, E. crandallis, E. bakuensis, E. weybridgensis, E. ahsata and E. ovinoidalis. The highest oocyst counts were observed when the lambs were 4-8 weeks old.

Molecular characterization of feline Hepatozoon species from Brazil

Feline Hepatozoon species from Brazil was molecular identified and characterized for the first time in São Paulo state, Brazil. Partial sequences of the 18S rRNA gene from the Hepatozoon from three naturally infected cats were analyzed. Sequences revealed that feline Hepatozoon was closely related to the canine Hepatozoon canis from Brazil. (C) 2005 Elsevier B.V All rights reserved.

Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara (Hydrochoerus hydrochaeris) from Brazil

A total of 145 capybara (Hydrochoerus hydrochaeris) fecal samples from the state of Sdo Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife. (c) 2007 Elsevier B.V. All rights reserved.

DISTRIBUTION OF THE NUMBER OF EURYTREMA SP EGGS PER GRAM OF FECES IN NATURALLY INFECTED CATTLE

Eurytrema sp. egg counts (epg) in the feces of naturally infected cattle were performed and the technique employed showed 94.2% probability of detecting positive cases of the infection with a single examination independently of the host parasite burden. It was also demonstrated that the epg of Eurytrema sp. follows a negative binomial distribution model and is characterized by its small magnitude.

CELL-MEDIATED AND HUMORAL IMMUNE-RESPONSES IN IMMUNIZED AND OR DERMATOBIA-HOMINIS INFESTED RABBITS

The cell-mediated and humoral immune response of rabbits to antigens from larvae of Dermatobia hominis were analyzed by leucocyte migration inhibition factor assay (MIF), immunodiffusion (ID) and passive hemagglutination (PH) test in rabbits immunized with D. hominis extract, in rabbits immunized and infested with the parasite and rabbits infested with D. hominis. Twenty rabbits were divided into five groups: Group 1, rabbits immunized with a crude antigen extract, evaluated for 40 weeks at 4 week intervals; Group 2, rabbits immunized and infested with newly hatched larvae at 14 weeks post immunization (PI) and evaluated as Group 1; Group 3, rabbits immunized, evaluated for 28 weeks at 2 week intervals; Group 4, rabbits immunized and infested at 4 weeks PI and evaluated as Group 3; Group 5, rabbits infested and evaluated for 24 weeks at 2 week intervals. Different patterns of reactivity were observed in the infested and immunized animals: immunized rabbits developed antibodies and cellular immune responses earlier and at higher levels during immunization than the infested rabbits; the infestation at 14 weeks PI, when the cell-mediated and humoral immune response began to decrease, or at 4 weeks PI when these parameters were at higher levels, elicited an anamnestic response. After the spontaneous elimination of larvae by the host, from the 4th week PI onwards, high titers of antibodies and migration inhibition indices were maintained for a long period. These results suggest that the onset of cellular and humoral immune responses after immunization may be important as a biological control of myiasis and contribute to better understanding of the immune defense mechanism of the host against D. hominis.

Acquired resistance of horses to Amblyomma cajennense (Fabricius, 1787) ticks

Acquired immunity of horses to larvae, nymphs and adults of the Amblyomma cajennense tick was evaluated through three consecutive experimental infestations of tick-bite naive hosts. Data from these infestations were compared to those from field-sensitized horses and donkeys. It was observed that tick-bite naive horses developed a low level of resistance after two infestations as shown by a significant decrease in larval yield and a tendency for lower engorged weight of nymphs during third infestation. Ticks fed on field-sensitized horses had a similar biological performance to that observed on the third infestation of tick-bite naive horses but the mean engorged nymph weight was significantly lower than that of the first infestation from tick-bite naive horses. Donkeys presented the strongest resistance with significantly lower engorged weights of all instars and of the egg mass compared to the first infestation of tick-bite naive horses. Donkeys also displayed a significantly higher resistance than field-sensitized horses as demonstrated by significantly lower egg mass weights. Overall these results indicate that donkeys but not horses maintain a strong resistance to A. cajennense ticks. The importance of these findings in relation to vectoring of tick-borne diseases is discussed. (C) 2003 Elsevier B.V. All rights reserved.

Biological and DNA evidence of two dissimilar populations of the Rhipicephalus sanguineus tick group (Acari : Ixodidae) in South America

In this work, the biology, mitochondrial DNA and fertility of hybrids from two strains of Rhipicephalus sanguineus, from Brazil and Argentina, were compared. Engorged larvae, nymphs and adults from Argentina weighed more and the engorgement period of adult females was significantly longer than those of their Brazilian counterparts, whereas adult female tick yield rate was higher for the Brazilian strain. High intraspecific divergence of mitochondrial DNA was detected between R. sanguineus from Brazil and Argentina. on the other hand, a strong genetic relationship was detected between European and Argentinean R. sanguineus populations while the Brazilian population appeared to be related to the African Rhipicephalus turanicus. Adult hybrid females laid eggs, which were mostly unviable, whereas a mean of more than 1400 larvae hatched per egg mass from pure Brazilian and Argentinean strains. These results showed that differences between these strains are greater than previously assumed and that the biosysternatic status of R. sanguineus ticks from South America should be re-evaluated. Wide variations, such as these might account for the reported worldwide differences in biology and vector capacity of this species. (c) 2005 Elsevier B.V. All rights reserved.

Skin hypersensitivity tests in buffaloes parasitized with Toxocara vitulorum.

Skin tests were done using larval extract and excretory-secretory (ES) antigens injected intradermally in the neck area of 30, 11- to 200-day-old buffalo calves and nine 27- to 100-day postparturition buffalo cows, the skin of the buffaloes infected with Toxocara vitulorum, mainly calves, demonstrated a hypersensitive response to antigens, especially to the larval extract antigens. Skin hypersensitivity responses were characterized by the presence of dermal nodules with progressive induration and an increase of up to four times the size of the original area at 30 min (immediate type) and at 72 h (delayed type) after injection, Histological preparations of skin reactions at 72 h showed a typical mononuclear cell infiltration, with eosinophils and perivascular cuffing in most of the animals, Fecal examination of 75 animals showed that 65 (86.7%) buffalo calves (9-115 days old) were parasitized with T. vitulorum. The peak of egg output from these animals occurred when they were approximately 45 days old.

Evaluation of transformation growth factor beta(1), interleukin-10, and interferon-gamma in male symptomatic and asymptomatic dogs naturally infected by Leishmania (Leishmania) chagasi

The aims of this study were to evaluate the immunomodulatory role of TGF-beta(1), 1L-10, and INF-gamma in spleen and liver extracts and supernatant cultures of white spleen cells from male symptomatic and asymptomatic dogs, naturally infected by Leishmania (Leishmania) chagasi. Thirty dogs from Aracatuba, São Paulo, Brazil, an endemic leishmaniosis area, were selected by positive ELISA serological reaction for Leishmania sp. and divided into two groups: asymptomatic (n=15) and symptomatic (n=15) consisting of animals with at least three characteristic signs (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachex a, locomotion problems, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy). After euthanasia, spleen and liver fragments were collected for ex vivo quantification of TGF-beta(1), IL-10, and INF-gamma. Naturally active in vitro produced TGF-beta(1) was also evaluated in spleen cell culture supernatant. Spleen and liver extract of asymptomatic dogs had higher mean TGF-beta(1) levels than symptomatic dogs. High concentrations of IL-10 were found in spleen, and mainly in liver extract of both groups. Higher INF-gamma concentrations were found in spleen extracts of symptomatic dogs, and in liver extracts of asymptomatic dogs. Extract of this cytokire was lower in spleen extract. Although INF-gamma is being produced in canine infection, mean levels of TGF-beta(1) and IL-10 from spleen and liver extracts were quantitatively much higher; suggesting that immune response in both asymptomatic and symptomatic dogs A as predominantly type Th2. (c) 2006 Elsevier B.V. All rights reserved.

Experimental toxoplasmosis in broiler chicks

To evaluate chicken toxoplasmosis both as an economic and a public health subject, 84 broiler chicks of a commercial strain, 30 days old, were distributed into seven groups of 12 birds (three replications of four chicks) experimentally infected with three developing T. gondii stages of the P strain as follows: tachyzoites. intravenous (two groups: 5.0 x 10(5) and 5.0 x 10(6)), cysts, per os (two groups: 1.0 x 10(2) and 1.0 x 10(3)) and oocysts, per os (three groups: 5.0 x 10(2), 5.0 x 10(3) and 5.0 x 10(4)). Twelve chicks received only a placebo (control group). During the next 30 days the following parameters were estimated: productivity (weight gain and feed conversion), clinical signs, including rectal temperature and parasitemia (bioassay). No clinical signs suggesting toxoplasmosis were seen and no statistical differences on productivity standards were found in comparison between inoculated and control chicks. However, fowls inoculated with tachyzoites and oocysts occasionally showed hyperthermia. Some haematological changes were detected in fowls inoculated with T. gondii. Anatomo-histopathological changes were not observed. From 14 parasitemias detected, 35.7% appeared on the 5th day after inoculation and 57.1% of them resulted from oocysts inoculation. After 30-35 days all birds were slaughtered: fragments from 12 organs or tissues from each of them were subjected to artificial peptic digestion and after that injected into T. gondii antibody-free mice (IIFR). T. gondii was detected in brain (12), pancreas (five), spleen (five), retina (five), kidney (two), heart (four), proventriculus (three), liver (two), intestine (two), lung (one), and skeletal muscle (one). Similar to observations with parasitemia, from 42 T. gondii isolations, 59.5% came from chicks which had received oocysts. It can thus be inferred that the developing form, expelled by cats, is the most important for T. gondii chicken infection and that brain is the most infected organ in birds. Attention must be paid to the potential importance of chicken meat in public health, since T. gondii was isolated from skeletal and heart muscles. (C) 1997 Elsevier B.V. B.V.

Species diversity and seasonality of free-living ticks (Acari : Ixodidae) in the natural habitat of wild Marsh deer (Blastocerus dichotomus) in Southeastern Brazil

This study evaluated the presence and seasonal activity of free-living ticks in remaining marsh areas by the Parana river, in Brazil. Eight field trips (once per season) for collection of ticks were performed during 2 years. Using CO2 traps, dragging, and visual inspection of vegetation, five free-living tick species were collected, in the following order of abundance: Amblyomma cajennense, Amblyom\ma dubitatum, Amblyomma triste, Amblyomma coelebs, and Amblyomma nodosum. The seasonal pattern of A. cajennense was characterized by the highest peaks for adult ticks in the summer/spring months, for nymphs in the winter and for larvae in the autumn and winter. A. dubitatum and A. triste presented similar seasonal patterns characterized by peaks of adult ticks in the autumn. Nymphs of A. dubitatum peaked in the winter of the first year and in the winter/spring of the second year. A. triste was the only species to be collected in significantly higher numbers in the marsh than in surrounding drier areas such as forest patches. Among domestic animals living close the marsh areas, horses were infested by Anocentor nitens, A. cajennense, and Boophilus microplus, bovines were infested solely by B. microplus, and dogs were infested by Rhipicephalus sanguineus. Adults of A. triste showed to be well adapted to the marsh environment. This result, at least partially, explains local previous observations on the association of A. triste with marsh deer, as this vertebrate host inhabits mainly the marsh area. (c) 2006 Published by Elsevier B.V.

Hepatozoon canis infection of wild carnivores in Brazil

Hepatozoon canis was diagnosed in a crab-eating fox (Cerdocyon thous) found on a highway in the region of Botucatu, São Paulo, Brazil, after being hit by a car. The fox had bilateral fractures of the olecranon, which was corrected by osteosynthesis. Hematologic findings included a neutrophilia, eosinophilia, monocytosis and mild anemia. In the Leishman-stained blood film, gametocytes of Hepatozoon canis in neutrophils were identified measuring 9.1+/-0.54x5.3+/-0.46 mu m. (C) 1997 Elsevier B.V. B.V.

Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: Optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiveroperating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P-neg=0.83), but a poor positive concordance (P-pos=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninuin was significantly associated with T gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis. (c) 2006 Elsevier B.V. All rights reserved.

Comparison of two immunochromatographic assays and the indirect immunofluorscence antibody test for diagnosis of Trypanosoma cruzi infection in dogs in south central Louisiana

Two rapid tests evaluated in dogs considered to be of high risk of Infection with the Chagas parasite Trypanosoma cruzi using two immunochromatographic assays. Trypanosoma Detect (TM) for canine, InBios, Seattle, WA and CHAGAS STAT-PAK (TM) assay, Chembio Diagnostic Systems, Medford, NY, in south central Louisiana. For this purpose a serological survey was carried out in a total of 122 dogs and a serum bank was created. These 122 animals were first tested by IFAT that was used as the standard test From the serum bank 50 samples were tested using the two rapid Chagas assays and results compared to the standard test IFAT The serological survey using IFAT showed it prevalence of T cruzi infection in 22.1% of the tested dogs. In the immunochromatographic assays. 13 and 11 animals were positive on rapid assay Trypanosoma Detect (TM) for canine, InBios and CHAGAS STAT-PAK (TM), Chembio Diagnostic Systems, respectively compared to 11 positive by IFAT. These two immunochromatographic tests have shown high susceptibility and specificity compared to our standard method IFAT. The rapid, easy and accurate screening assays used in conjunction with confirmatory tests, would be an excellent tool for veterinarians to diagnose T cruzi infection. Early detection of T cruzi infection may prevent complications through an effective treatment. Greater awareness by veterinarians of the risk. clinical findings, history along with diagnostic methods will contribute greatly to an understanding of the true prevalence of Chagas disease in dogs in Louisiana. (c) 2009 Elsevier B.V. All rights reserved.