Experimental inoculation of Neospora caninum tachyzoites in eared doves (Zenaida auriculata).

Neospora caninum is an apicomplexan parasite distributed worldwide. Although a positive association between the presence of birds and abortions in cattle associated to N. caninum has been reported, the role of the birds in the epidemiologic cycle of the parasite is unknown. To the best knowledge, no experimental studies have evaluated N. caninum in the eared dove, Zenaida auriculata. Therefore, we aimed to determine whether Z. auriculat can act as intermediate host for N. caninum. Eighteen birds were divided into four groups, G1, G2, G3, and G4 (control); G1, G2 and G3 received 2 × 106 tachyzoites of NC-1 strain via different routes: subcutaneous, intramuscular, and intraperitoneal, respectively. G4 composed of three birds. Serum samples were collected weekly, and one bird each from G1, G2 and G3 was euthanized on the 7th and 14th day post-inoculation (dpi). The remaining birds were euthanized after the 28th dpi. Tissues from the doves were evaluated using histopathological analysis, PCR and dog bioassay to detect the parasite. Dogs were fed with tissues from the birds and monitored for 30 days. Serum samples were collected weekly from the dogs for serological analysis, and feces samples were collected daily until the end of the experiment for coproparasitological examinations. No dove showed clinical signs of the infection; however, all of them seroconverted after the inoculation, with stronger immunological response in the G3 birds. The lung tissue of one G3 bird showed positive PCR results; it was euthanized on the 7th dpi, and an inflammatory infiltrate was observed in the lung and kidney from this dove. The dogs did not shed oocysts or seroconverted. Our results indicate that the intraperitoneal route induced infection in the doves; however, the parasite may have been eliminated by the host, and the doves may be resistant to chronic infection.

Genotyping of Toxoplasma gondii isolated from pigs for human consumption.

The present study aimed to isolate and genotype strains of T. gondii from pigs slaughtered for human consumption in South Brazil. Blood and tissues (heart, diaphragm, liver, tongue, and masseter) from 400 animals were collected at two slaughterhouses. Sera were obtained, and antibodies against T. gondii were detected by both indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT). The tissues of animals that tested positive in MAT, IFAT, or both (cut-off ≥ 64) were bioassayed. Twenty-six (6.5%) of the 400 animals were positive by serology. A total of 18 (69.2%) out of those 26 were positive in the mouse bioassay. The isolates were characterized by using 10 PCR-RFLP genetic markers. Fourteen isolates were fully genotyped, and four isolates were genotyped using nine of the 10 markers. All isolates belonged to ToxoDB PCR-RFLP genotype #206. The present study reports on genotype #206 in pigs for the first time, and it confirms the atypical nature of the Brazilian T. gondii isolates. Additionally, even with low levels of antibodies detected in pig herds, pork presents a T. gondii infection risk for humans.

IMMUNOHISTOCHEMICAL AND MOLECULAR EVIDENCE OF PUTATIVE NEORICKETTSIA INFECTION IN COATIS ( NASUA NASUA) FROM SOUTHERN BRAZIL.

The pathologic, molecular, and immunohistochemical findings associated with Neorickettsia helminthoeca are described in coatis ( Nasua nasua). Tissue sections (small intestine, lungs, kidney, liver, and spleen) of coatis ( n = 3) that died at the Bela Vista Biological Refuge, Foz do Iguaçu, Paraná, southern Brazil were routinely processed from histopathology. Selected formalin-fixed paraffin-embedded (FFPE) tissue sections of the small intestine, lungs, and spleen were used in an immunohistochemical (IHC) assay designed to identify the antigens of N. helminthoeca. Additionally, FFPE tissue sections of the small intestine were used to demonstrate antigens of canine parvovirus-2 (CPV-2) by IHC. Histopathology revealed chronic enteritis in all coatis. Parasitic enteritis was diagnosed in two coatis; one of these contained examples of a trematode within the lumen of the small intestine and the ovum of a trematode encysted in the intestinal mucosa. Other significant pathologic findings included interstitial pneumonia ( n = 2) and pyogranulomatous splenitis ( n = 1). Positive immunolabeling for N. helminthoeca was identified within macrophages of the small intestine and reticuloendothelial cells within the germinal centers of the spleen of all coatis; the intestinal trematode was N. helminthoeca IHC-positive. All pulmonary sections revealed negative immunolabeling for N. helminthoeca. Furthermore, the antigens of CPV-2 were not identified in the intestine of any coati. These findings indicate that these coatis were infected by N. helminthoeca, but since clinical and gross pathological findings were not recorded, it is uncertain if this pathogen produced clinical disease in this canid host; therefore, coatis may be asymptomatic or dead-end hosts for this organism.

First molecular detection of Haemoproteus spp. and Plasmodium spp. in eared doves (Zenaida auriculata) in Brazil.

The aim of this study was to verify the presence and identify the species of haemosporidian parasites in eared doves (Zenaida auriculata) in Brazil. Two hundred and eleven male and female eared doves were trap-captured in four different regions of Londrina city, in southern Brazil. Whole blood was collected in EDTA tubes through heart puncture after euthanasia in a CO2 chamber. A nested PCR targeting the mitochondrial cytochrome b gene (cyt b) of Haemoproteus spp./Plasmodium spp. was performed, followed by an enzymatic digestion to identify the genus. Phylogenetic trees were constructed to determine the closely related species. Out of 211 eared doves, 209 (99.05%) were positive for Haemoproteus spp. and/or Plasmodium spp. RFLP analysis showed that 72.72% (152/209) of eared doves were positive only for Haemoproteus spp., 6.22% (13/209) were positive only for Plasmodium spp., and 21.05% (44/209) of eared doves had mixed infections. Genetic analysis found four samples that were homologous with Haemoproteus multipigmentatus and one that was homologous with Plasmodium sp. This is the first molecular study of hemoparasites from eared doves in Brazil, and it is also the first description of H. multipigmentatus and Plasmodium spp. infection in eared doves in Brazil.

Hemotropic mycoplasmas (hemoplasmas) in free-ranging bats from Southern Brazil.

Hemotropic mycoplasmas (hemoplasmas) are bacteria distributed worldwide and affect domestic and wildlife animals and human beings. Hemoplasmas have been described infecting hematophagous and non-hematophagous bats; however, transmission risk and zoonotic potential in vampire bats remain to be fully established. This study aimed to evaluate the presence of hemotropic mycoplasma species in free-ranging bats from this area using a universal PCR protocol for hemoplasmas. Accordingly, ten blood samples were collected from six male common vampire bats (Desmodus rotundus), two male hairy-legged vampire bats (Diphylla ecaudata), and two female non-hematophagous Pallas's mastiff bats (Molossus sp.) from the Curitiba's region, Paraná State, Southern Brazil. A total of eight (8/10) blood samples were positive byconventional PCR; five (5/6) Desmodus rotundus, two (2/2) Diphylla ecaudata, and one (1/2) Molossus sp. bats. The analyses of the partial sequence of the 16S rDNA gene suggest that the hemoplasma detected in Desmodus rotundus in South Brazil has a high identity compared to the hemoplasma circulating in vampire bats from Central and South America.

Cloning, expression, and characterization of the MSP1a and MSP1b recombinant proteins from PR1 Anaplasma marginale strain, Brazil.

The Anaplasma marginale is a bacterium that has obligate intraerythrocytic multiplication in cattle causing important economic loss. The A. marginale major surface protein 1 (MSP1) complex, heterodimer composed of MSP1a and MSP1b, has been identified as adhesins for bovine erythrocytes. The objectives of this study were to sequences the msp1beta gene and produce and characterize recombinant MSP1a and MSP1b from a Brazilian strain of A. marginale, PR1. The msp1alpha and msp1beta genes from the PR1 strain were cloned and expressed in E. coli BL21 Star using the vectors pET102 and pET101/D-TOPO. Antibodies were produced against the recombinant proteins and were shown to react with rMSP1a and rMSP1b demonstrating a molecular mass of 70kDa to 105kDa and 100kDa, respectively for these proteins. Bovine erythrocytes were agglutinated by BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhibited by the presence of the IgY anti-rMSP1a, confirming the adhesion function of these proteins. Additionally, using the IgY anti-rMSP1a and rMSP1b in a IFI, the presence of rMSP1a and rMSP1b was confirmed on the outer membrane of the recombinant E. coli BL21. Our results show that the msp1beta gene from the PR1 strain has both the conserved region and contain the defined polymorphism regions previously described for other strains of A. marginale. The results from this study confirm adhesive functions for rMSP1a and rMSP1b from PR1 strain in bovine erythrocytes invasion.

First study of Cryptosporidium spp. occurrence in eared doves (Zenaida auriculata).

Cryptosporidium is a protozoan parasite with a wide range of hosts, including humans. However, only a few Cryptosporidium species have been described in birds (C. meleagridis, C. baileyi, C. galli and C. avium). The aim of this study was to investigate the occurrence of Cryptosporidium spp. in feces of eared doves (Zenaida auriculata), followed by molecular characterization of the parasite. A total of 196 animals of both sexes were trap-captured; the animals were culled and the intestinal contents were collected for DNA extraction. After extraction, a nested-PCR (nPCR), which amplifies a fragment of the 18S rRNA gene of Cryptosporidium spp., was performed. The amplicons obtained were purified and sequenced. PCR analysis revealed that 30 animals (15.3%) were positive for Cryptosporidium spp. There was no significant sex-dependent enrichment of Cryptosporidium occurrence (p > 0.05). Only 15 out of the 30 positive samples were successfully sequenced and their species determined, of which, 13 (86.7%) and 2 (13.3%) were C. meleagridis and C. galli, respectively. Herein, we present for the first time a molecular characterization of Cryptosporidium from feces of eared doves (Z. auriculata) and propose that these birds are a potential source of C. meleagridis infection in humans.

Detection of Hemotropic Mycoplasma sp. in white-eared opossums (Didelphis albiventris) from Southern Brazil.

Opossums are marsupials from the New World of the genus Didelphis and known as synanthropic animals due to their proximity with human beings. To date, 'Candidatus Mycoplasma haemodidelphis' has been solely found infecting the North American opossum (Didelphis virginiana). Accordingly, the aim of this study was to screen eight white-eared opossums (Didelphis albiventris) from a public park in Maringa city, Paraná State, southern Brazil, for hemoplasma infection. Blood samples were taken from caudal venipuncture, and DNA was extracted and further screened by a pan-hemoplasma PCR assay. Seven out of eight (87.50%; CI 95%: 47.35-99.68%) white-eared opossums were positive for Mycoplasma spp. Sequencing of the 16S rRNA fragment showed 98,97% identity with 'Ca. M. haemodidelphis' detected in the USA. Three out of eight (37.50%; CI 95%: 8.52-75.51%) white-eared opossums were infested by Amblyomma dubitatum ticks. This is the first report on detection of a potentially novel hemotropic Mycoplasma sp. infecting opossums from South America.

Tick-borne pathogens in carthorses from Foz do Iguaçu City, Paraná State, southern Brazil: A tri-border area of Brazil, Paraguay and Argentina.

Tick-borne diseases (TBD) constitute an important group of illness affecting animals and humans worldwide. In Brazil, carthorses are frequently exposed to ticks and tick-borne pathogens, leading to impairment of horse performance and imposing restrictions by the international veterinary authorities for the importation of horses. Accordingly, this study has aimed to i) determine the prevalence of the TBD agents Theileria equi, Babesia caballi, Ehrlichia spp., and hemotropic mycoplasmas in carthorses, ii) identify the tick species parasitizing the animals, and iii) determine factors associated with exposure/infection in Foz do Iguaçu City, Parana state, southern Brazil. A total of 103 carthorses were screened for anti-T. equi and anti-Ehrlichia spp. antibodies by indirect fluorescent antibody assays (IFA). Samples were also tested by PCR assays targeting the 18S rRNA gene of T. equi and B. caballi, and 16S rRNA gene of hemoplasmas. Additionally, PCR assays targeting the 16S rRNA, disulfide bond formation protein (dsb) and tandem repeat proteins 36 (trp36) genes of Ehrlichia spp. were also performed. Antibodies to T. equi and Ehrlichia spp. were detected in 43/103 (41.75%; 95% CI: 32.10-51.88%) and 5/103 (4.85%; 95% CI: 1.59-10.97%) horses by IFA, respectively. DNA of T. equi and B. caballi were found in 25/103 (24.27%; 95% CI: 16.36-33.71%) and 10/103 (9.71%; 95% CI: 4.75-17.13%) carthorses, respectively, and all tested negative for Ehrlichia spp. and hemoplasmas. All sequences showed ≥99% identity with multiple T. equi and B. caballi 18S rRNA gene sequences deposited in GenBank. Overall, 191 Dermacentor nitens ticks were collected from 25/103 (24.27%) animals. Carthorses older than 5 years were more likely to be positive for T. equi (p < 0.05). In conclusion, equine piroplasmosis agents are highly prevalent in carthorses from Foz do Iguaçu City. The low prevalence of Ehrlichia spp. found may be due to the absence of Amblyomma ticks infesting animals, which should be further investigated.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs.

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n=10) and G2 (uninfected group, n=8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r=0.62, P=0.05), MAT(s) x MAT (ah) (r=0.97, P<0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r= 0.14, P=0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis.

Neospora caninum in birds: A review.

Neospora caninum is an obligate intracellular protozoan parasite that infects domestic and wild animals. Canids are considered to be definitive hosts since they may shed oocysts into the environment through their feces. The disease is recognized as one of the major causes of bovine abortion worldwide, leading to important economic losses in the dairy and beef cattle industries. Previous studies have reported N. caninum infection in different species of birds; infection in birds has been associated with increased seroprevalence and reproductive problems in dairy cattle. Although the role of birds in the epidemiological cycle of neosporosis is unknown, birds are exposed to infection because they feed on the ground and could thus contribute to parasite dissemination. This review is focused on the current state of knowledge of neosporosis in birds.

Cryptosporidium occurrence in ruminants from the North Pioneer mesoregion of Paraná, Brazil.

The aim of this study was to investigate the occurrence of Cryptosporidium in cattle and sheep from the North Pioneer mesoregion of the state of Paraná. For this, 317 stool samples were collected from cattle and sheep on 16 properties in six municipalities in the North Pioneer mesoregion of Paraná. For detection of Cryptosporidium species, molecular analysis was performed using nested-PCR techniques targeting the 18S rRNA gene. Of the 37 beef cows and 115 calves analyzed, four (10.8%) and 14 (12.2%), respectively, were positive for Cryptosporidium. Of the 12 cows and 52 calves, one (8.3%) and 14 (26.9%), respectively, were positive for Cryptosporidium; and of the 42 ewes and 59 lambs, six (14.3%) and 12 (20.3%), respectively were positive for Cryptosporidium. Cattle (15.3%) and sheep (17.8%) were both susceptible to infection. All the properties of the municipalities of Assaí, Ibaiti and, Leópolis presented infected animals. The study showed that Cryptosporidium occurs in most municipalities assessed, that dairy calves had a higher risk (Odds Ratio=2,66, p-value=0,018) for infection than beef calves, and that sheep are just as susceptible to infection as are cattle, and that further Cryptosporidium studies are developed.

First report of Anaplasma marginale infection in goats, Brazil.

Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne bacterium that causes significant economic losses for cattle industries and is increasingly being detected in other animal species. Rhipicephalus microplus is the main vector of this bacterium and may be found parasitizing small ruminants. In northeastern Brazil, multispecies grazing is a common family subsistence practice on smallholder farms possibly facilitating interspecies transmission of pathogens. Considering that A. marginale infection has been previously molecularly described in sheep, this study has aimed to estimate the prevalence of A. marginale and factors associated with the infection in goats from northeastern Brazil. A total of 403 goat blood samples were included in the study. An epidemiological questionnaire was applied to each farm owner addressing age, gender, presence of ticks and multispecies grazing. All samples were screened for A. marginale- and A. ovis-infection using primers targeting the Anaplasma spp. msp4 gene. The identity of A. marginale in the blood was confirmed by PCR amplification of msp5 followed by sequencing. Anaplasma spp. were differentiated by sequencing of the repeat region of the msp1α gene. For the statistical analysis the Chi-square or the Fisher's exact test was used to verify association of the individual factors (age, gender, presence of ticks, and multispecies grazing) with Anaplasma spp. infection. We report the first molecular detection of A. marginale in goats from northeastern Brazil, based on msp1α, msp4 and msp5 gene sequencing analysis. Sequencing of the detected A. marginale msp1α gene revealed the F repeat. Amblyomma parvum and R. microplus were found feeding on animals.

Theileria sp. in water buffaloes from Maranhão State, northeastern Brazil.

Anaplasma marginale and piroplasm species are widespread among Brazilian cattle herds. Both of these tick-borne pathogens hamper livestock production and cause a significant economic impact. Although buffaloes have demonstrated a high level of adaptability, data on tick-borne pathogens are scarcely reported in Brazil. Thus, the aim of this study was to screen water buffaloes from the state of Maranhão for piroplasm and A. marginale occurrence using PCR assays. All samples were negative for A. marginale. One of the 287 (0.35%) water buffaloes tested was positive for Theileria sp. Sequencing of the 18S rDNA fragment (356 bp) showed that the Theileria sp. identified was closely related to the T. buffeli /orientalis group. Future studies on the clinical signs of infection and the main vector in this country are needed.

Hemotropic mycoplasmas infection in water buffaloes (Bubalus bubalis) from northeastern Brazil.

Two species of hemotropic mycoplasmas (HM) are known to infect large domestic ruminants, Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos'. Although HM has been described in cattle worldwide, data in water buffaloes (Bubalus bubalis) remain scarce. Accordingly, the aim was to determine the occurrence of HM in water buffaloes from northeastern Brazil. A total of 101/290 (34.83%) buffaloes were positive for HM (16 M. wenyonii alone, 6 'Ca. M. haemobos' alone and 79 both). This was the first report of M. wenyonii infection in ruminants from Brazil. Clinical signs of hemoplasmosis in buffaloes remain unknown.

Prevalence of Eimeria spp. in calves from dairy farms in northern Paraná state, Brazil.

Bovine coccidiosis is a disease of major importance in cattle herds across the world. The disorder mainly affects young calves, and E. bovis and E. zuernii are considered the most pathogenic species of the genus, however, E. alabamensis have been described in grazing calves. In this study, the prevalence of Eimeria spp. was evaluated in calves on dairy farms in the northern region of the state of Paraná, Brazil. Four hundred calves on 44 dairy farms were tested for the presence of coccidian oocysts. The positives were re-examined and the oocysts were morphometrically analyzed for species identification. All the farms were contaminated and 205 animals (51.25%) presented Eimeria spp. oocysts. Among these, 146 animals (71.22%) were co-infected by two or more species of coccidia. Ten species of Eimeria were identified: E. bovis (in 30.25% of the positive samples), E. alabamensis (26.75%), E. zuernii (22.00%), E. ellipsoidalis (18.50%), E. auburnensis (13.75%), E. canadensis (8.00%), E. cylindrica (7.25%), E. subspherica (5.00%), E. bukidnonensis (3.00%) and E. brasiliensis (0.75%). This study demonstrates the high prevalence of Eimeria spp. in the northern region of Paraná, Brazil, and detection for the first time in our region the pathogenic species E. alabamensis.

Immune response of BALB/c mouse immunized with recombinant MSPs proteins of Anaplasma marginale binding to immunostimulant complex (ISCOM).

Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60-105kDa), rMSP1b (100kDa), rMSP4 (47kDa), and rMSP5 (29kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.

Co-infection with Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' in three cats from Brazil.

The two most common haemotropic Mycoplasma of cats, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' have been identified using molecular techniques in all continents, except Antarctica. We report the first molecular characterization in South America of a dual infection with M haemofelis and 'Candidatus Mycoplasma haemominutum' in three domestic cats. The 16S ribosomal RNA gene was amplified in three anaemic cats in which haemoplasma organisms were seen attached to the erythrocytes in the peripheral blood smear. Bands of the expected size for M haemofelis and 'Candidatus Mycoplasma haemominutum' were observed in all three cats. The 393 bp segment of one of the amplicons had a similarity value of 100% to M haemofelis, whereas the other amplicon, a 192 bp segment, was 100% similar to 'Candidatus Mycoplasma haemominutum'. After diagnosis, two cats received blood transfusion and they were all treated with doxycycline. All three cats recovered uneventfully.

Molecular screening for hemotropic mycoplasmas in captive Barbary sheep (Ammotragus lervia) in southern Brazil.

AIM: This study is part of an active surveillance program for monitoring animal health status in endangered species, and was conducted to screen captive Barbary sheep (Ammotragus lervia) for hemoplasma infection. MATERIALS AND METHODS: A total of 12 blood samples were collected, DNA extracted and further tested by a pan-hemoplasma polymerase chain reaction protocol. RESULTS: Animals were clinically healthy and not infested by ectoparasites. Although housekeeping gene DNA was successfully amplified, all the Barbary sheep samples tested negative for Mycoplasma sp. CONCLUSION: Notwithstanding the negative results, molecular pathogen surveys on Barbary sheep and other exotic wild mammals may provide insights regarding infection of endangered species caused by captivity stress in association with exposure to new pathogens worldwide.