Sequence subtyping of Trichomonas gallinae from Bonelli's eagle (Aquila fasciata) during four years (2014-2017) reveals that MLS type is associated with lesions.

Avian trichomonosis is a parasitic disease that affects wild birds. The objective of this work was to determine the importance of avian trichomonosis in Bonelli's eagles to improve conservation measures in this population. One hundred and eighty-eight birds were studied: 181 chicks, two juveniles, one subadult and four adults. The birds were externally examined and gross lesions at the oropharynx registered. Samples from the oropharyngeal cavity were obtained for Trichomonas spp. detection by culture and PCR, and positive samples were subjected to a multilocus sequence typing approach, including the ITS1/5.8S/ITS2 region (ITS), ribosomal RNA small subunit (18S) and Fe-hydrogenase gene (FeHyd). Global prevalence of T. gallinae infection was 37.8% in total, 45.5% in nestlings. Thirty-three percent of the birds developed lesions that ranged from mild (n = 41) to moderate (n = 14) or severe (n = 7). Multilocus sequence typing analysis showed five different MLS types, ITS-A/18S-VI/FeHyd-A1 and ITS-D/18S-II/Fe-C4 being the most frequent. An association between ITS-A/18S-VI/FeHyd-A1 and moderate or severe lesions was observed, but birds with type ITS-A/18S-VI/FeHyd-A2 also developed lesions. On the contrary, birds with MLS type ITS-D/18S-II/FeHyd-C4 displayed only a low proportion of mild lesions. Chicks raised in nests were at higher risk for T. gallinae infection and development of lesions than chicks raised in captivity. Discordances between samples cultured in TYM and samples subjected to PCR from oropharyngeal swabs were observed, swab-ITS-PCR being more sensitive.RESEARCH HIGHLIGHTS 45.5% of Bonelli's eagles in the nest carried T. gallinae and 39.4% showed lesions.PCR from oral swabs showed higher sensitivity than culture in TYM for detection of T. gallinae.MLS types ITS-A/18S-VI/Fe-A1 (and A2) are a risk factor for the development of lesions.

Oral trichomonosis: Description and severity of lesions in birds in Spain.

Avian trichomonosis is a parasitic disease caused by the flagellated protozoan Trichomonas gallinae. Columbiformes are the reservoir host of the parasite, with high levels of infection, but also other domestic and wild birds from a variety of orders are susceptible to the infection and development of gross lesions. In this paper we describe the type and severity of lesions in wild birds in four avian orders (Accipitriformes, Falconiformes, Strigiformes and Columbiformes). A total of 94 clinical cases diagnosed of trichomonosis were selected for the categorization of their lesions in the upper digestive tract. The affected birds were classified into three different categories (mild, moderate and severe) based on size (in relation to the tracheal opening), depth and location of the lesions. Mild cases are those with small and superficial lesions far from the oropharyngeal opening; moderate cases possess larger and deeper lesions, and severe cases very large and deep lesions that impede swallowing or affect the skull. Mild lesions were found in 10.6 % of cases; moderate lesions were observed in 18.1 % of the birds and severe lesions in 71.3 %. Treatment outcomes in birds with either mild or moderate lesions were favorable, while severe lesions were related to poor body score, leading to death or euthanasia in most cases. A relationship between severe lesions and avian order was found, with a higher percentage of birds with this type in Falconiformes, Columbiformes and Strigiformes. Multifocal lesions were more frequent in Columbiformes and Falconiformes. In Strigiformes, 93.3 % of birds showed lesions in the upper jaw. This study seeks to further understanding of avian trichomonosis and to provide information that will be useful to veterinarians and related professionals for assessment, prognosis and treatment choice for these birds.

Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur.

Genetic characterization of oropharyngeal trichomonad isolates from wild birds indicates that genotype is associated with host species, diet and presence of pathognomonic lesions.

Oropharyngeal trichomonad isolates of wild birds from Spain were studied. A total of 1688 samples (1214 of predator birds and 474 of prey species) from wildlife recovery centres and scientific bird-ringing campaigns were analysed from 2011 to 2013. The overall infection prevalence was 20.3% (11.4% in predator birds and 43.3% in prey species). Pathognomonic lesions were present in 26% of the infected birds (57.3% in predator birds and 4.9% in prey species). The most commonly parasitized species were the goshawk (Accipiter gentilis, 74.5%) and the rock pigeon (Columba livia, 79.4%). Host species in which the parasite has not been previously analysed by polymerase chain reaction and sequencing in Spain are also reported: Columba palumbus, Streptopelia turtur, Pica pica, A. gentilis, Accipiter nisus, Asio otus, Bubo bubo, Buteo buteo, Circus aeruginosus, Circus cyaneus, Falco naumanni, Falco peregrinus, Neophron percnopterus, Otus scops, Pernis apivorus and Strix aluco. Sequence analysis of the ITS1/5.8S/ITS2 region revealed five different genotypes and also some mixed infections. A relationship between genotype and host species was observed, but only two genotypes (ITS-OBT-Tg-1and ITS-OBT-Tg-2) were widely distributed. Genotype ITS-OBT-Tg-1 was most frequently found in predator birds and statistically associated with pathognomonic lesions. Non-strict ornithophagous species were at higher risk to develop disease than ornithophagous ones. Genotypes ITS-OBT-Tcl-1 and ITS-OBT-Tcl-2 are new reports, and ITS-OBT-Tvl-5 is reported for the first time in Spain. They showed higher genetic homology to Trichomonas canistomae and Trichomonas vaginalis than to Trichomonas gallinae, indicating the possibility of new species within this genus.

Encephalomyelitis by Toxoplasma gondii in a captive fossa (Cryptoprocta ferox).

Encephalomyelitis due to Toxoplasma gondii was diagnosed in a fossa (Cryptoprocta ferox). The animal had ataxia, atrophy of hind limb muscles and progressive wasting before dying 12 months after the onset of clinical signs. Toxoplasmosis was suspected antemortem based on clinical signs and the detection of T. gondii DNA by PCR on EDTA-blood from live animal. Necropsy revealed necrotizing gastritis and severe emaciation. The main histological lesions included non-suppurative encephalomyelitis, with dilation of myelin sheaths and swollen axons in the spinal cord, and multifocal gliosis in the brain with intralesional protozoan cysts that stained positive for T. gondii immunohistochemistry. To the authors' knowledge, this is the first report of toxoplasmosis in a fossa, and a new host record.

High prevalence of Blastocystis sp. in pigs reared under intensive growing systems: frequency of ribotypes and associated risk factors.

Three hundred and ninety-five pig fecal samples were analyzed looking for Blastocystis sp. using optical microscopy and PCR. A global prevalence of 46.8% has been observed in this study, although relative values of prevalence differ notably according to the strata examined, ranging from 9.3% in sows to 75% in weaners. Statistic analysis of the data included several risk factors such as different management systems, date of sample collection, fecal consistency, age and sex of the animals. The presence of the parasite was statistically associated to the variables "age" and "date of sample collection", being more prevalent in weaners and grower pigs and warm season. Random fragment-length polymorphism (RFLP-PCR) analysis of positive PCR samples revealed a high homology in the digestion pattern, appearing as two ribotypes. The results were further confirmed by sequencing of ten randomly selected samples, showing that the samples obtained in this study were included in two genotypes: genotype I previously named by Noël et al. [Noël, C., Dufernez, F., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Ho, L.-Ch., Singh, M., Wintjens, R., Sogin, M.L., Capron, M., Pierce, R., Zenner, L., Viscogliosi, E., 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J. Clin. Microbiol. 43, 348-355], in which Blastocystis sp. sequences from humans, pigs and cattle were included, and genotype II, which only included Blastocystis hominis sequences obtained from human and other primates. This is the first report including Blastocystis sequences from swine origin in genotype II.

Inhibition of bovine T lymphocyte responses by extracts of the stomach worm Ostertagia ostertagi.

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the nematode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products were also shown to be secreted by the late L4. The suppressive activity is reversible if the L4 products are removed from culture. There is no immediate effect on proliferating cells and the L4SE must be in culture for 24-48 h before suppression is observable. The L4SE caused slight but not statistically significant decreases in the percentage of T cells and increases in B cell percentages in cultures when compared with cultures stimulated with Con A alone. No changes were seen in percentage of cells positive for markers for CD4, CD8, gammadelta T cells, or monocytes/macrophages as a consequence of the addition of L4SE. In contrast, there was a strong and significant reduction in the expression of the IL-2 receptors in cells cultured in the presence of the worm extract. There was no evidence of either necrosis or apoptosis resulting from the presence of L4 products in culture. The expression of messenger RNA for interleukin-2, -4, -13, tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (gamma-IFN) was decreased when L4SE was included in cultures of Con A-stimulated cells compared to cultures stimulated with Con A only. In contrast, messenger RNA expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) was increased in cells growing in the presence of L4 products. The potential role of these cytokines during ostertagiosis is discussed.

Correlation between in vitro and in vivo infectivity of Leishmania infantum clones.

Eleven clones of a single strain of Leishmania infantum (MCAN/ES/88/ISS441, Doba) were analyzed for biological behavior in vivo and in vitro. Different clones showed differences in growth dependent upon the two culture media employed. All clones displayed only slight differences in H2O2 and NaNO2 sensitivity compared to the original strain, whereas in vitro infectivity for mouse peritoneal macrophages differed significantly among the clones. In vivo infections in hamsters correlated strongly with in vitro infectivity. The phenotypic differences found suggest a polyclonal structure for the Leishmania infantum strain studied.

Serodiagnosis of haemonchosis with a somatic antigen (Hc26) in several breeds of sheep.

Sera from 53 sheep belonging to Castellano, Churro, Manchego, and Merino breeds were analyzed to test the diagnostic value of a 26-kD antigen from adult Haemonchus contortus at prepatency and early and late patency of experimental haemonchosis. Animals that received zero, 1, or 2 infections with the parasite were tested. In addition, sera from 20 experimentally infected and 10 noninfected Texel sheep were used to test the antigen. Sera from 37 infected animals at prepatency as well as at patency in primary and secondary infection were found positive with the 26-kD antigen. However, sera from 10 animals with the lowest worm burdens (second infection) did not recognize the antigen during early patency (day 28 postinfection). IgG1 was the only isotype implicated in antigen recognition because IgG2, IgA, and IgM, in the same sera, showed no reactivity with the peptide. Antigen specificity was confirmed because hyperimmune sera against infective larvae and adult stages of the most common gastrointestinal nematodes found in natural infections in sheep (Trichostrongylus colubriformis and Teladorsagia circumcincta) did not recognize this peptide. The antigen was recognized only by anti-adult H. contortus hyperimmune sera and appeared to be absent in the L3 parasite stage. In addition, the partial N-terminal amino acid sequence of the diagnostic peptide is reported.

Short report: improved diagnosis and follow-up of canine leishmaniasis using amastigote-based indirect immunofluorescence.

A comparison of an indirect immunofluorescence test using promastigotes (IFATp) or cultured amastigotes (IFATa) in the diagnosis and follow-up of canine leishmaniasis caused by Leishmania infantum was carried out. Results obtained with both diagnostic methods were in good agreement although the IFATa titration was more sensitive than the currently used IFATp without losing specificity. The higher sensitivity of the amastigote-based IFAT resulted in an earlier diagnosis in the absence of clinical signs. Both methods showed comparable results for monitoring the clinical evolution of naturally infected and treated (meglumine antimoniate plus allopurinol) dogs.

Partial anaerobiosis induces infectivity of Leishmania infantum promastigotes.

Leishmania infantum stationary-phase promastigotes could acquire infectivity via preincubation in a partially anaerobic medium (95% air/5% CO2) for 16 h before the infection, whereas promastigotes were efficiently destroyed when no CO2 was present. Incubation of L. infantum promastigotes with additional glucose (20 and 50 mM) greatly increased infection parameters in the absence of CO2; this is consistent with a "reverse Pasteur effect." Results showed that culture at 33 degrees C permitted survival and amastigote multiplication (a nearly 10-fold increase in amastigotes as compared with those observed in 37 degrees C cultures). This finding was obtained with the two strains of L. infantum tested (Doba and PB75).

Serum antibody response of Castellana sheep to Haemonchus contortus infection and challenge: relationship to abomasal worm burdens.

Primary and secondary serum antibody responses to Haemonchus contortus were studied in Castellana sheep. Ten-month-old sheep were infected (200 L3/kg live weight (lw)) and challenged (400 L3/kg lw) or uninfected and equally challenged with H. contortus. Primary infections induced a partially protective response upon challenge, characterized by higher serum protein levels, longer prepatent periods, lower fecal egg counts, and significant reduction in the establishment rate of the parasite and abomasal adult and L4 worm burdens. The resistant status of the infected and challenged sheep was not clearly related either to the serum specific antibody levels (IgG: IgG1, IgG2; IgM; IgA) estimated by ELISA or to immunodetection patterns in the Western blots.

Infection-induced protection against Haemonchus contortus in merino and manchego sheep. Relationship to serum antibody response.

Resistance to primary and secondary infections with Haemonchus contortus was studied in 10-month-old Manchego and Merino sheep. No notable interbreed differences were observed after primary infections in the parameters determined (prepatency period, faecal egg output, abomasal worm burden). Previously infected sheep (200 L-3/kg live weight (lw)) from both breeds showed notable protection after challenge (400 L-3/kg lw), evidenced by lower eggs/g faeces (epg) values and worm burdens. A protective response in the Manchego breed was associated with arrested development of fourth stage larvae in the abomasal mucosa, whereas in the Merino breed a more rapid expulsion mechanism seems to be involved. Serum antibody levels (IgG, IgA) were infective dose-dependent and protection from re-infection was not clearly related to the parasite-specific IgG response estimated by enzyme-linked immunosorbent assay (ELISA) and Western blotting.

Characterization of protective immune responses in local lymphoid tissues after drug-attenuated infections with Ostertagia ostertagi in calves.

In the present study, cell-surface markers and cytokine gene expression of lymphocytes from the local lymph nodes were studied 9 days after primary infection with Ostertagia ostertagi in previously naive calves or in calves previously immunized with multiple, chemically attenuated infections. Changes in lymphocyte populations were assessed by flow cytometry utilizing monoclonal antibodies specific for bovine cell-surface markers. Changes observed in the percentages of lymphoid populations after challenge were similar in animals immunized by either three or five drug-attenuated infections. In both immunized groups, the CD4+/CD8+ ratio was significantly higher than in naive animals after challenge infections. In addition, both immunized groups showed significantly lower levels of Ig-bearing cells upon experimental challenge when compared to animals with a primary experimental infection. No differences were observed in the number of gammadelta or interleukin 2 receptor (IL-2R) positive cells. The levels of mRNA for IL-4, IL-10, IL-15, IFN-gamma and TGF-beta1 were examined by competitive RT-PCR. After challenge, the levels of these cytokines were lower in animals immunized by five drug-attenuated infections, and in the case of IL-4 and TGF-beta1, these differences were statistically significant. These results indicate that animals exhibiting protection from reinfection with O. ostertagi do not show a shift to higher percentages of Ig+ cells characteristic of a primary infection. In addition, protected animals appear to show a decreased IL4 and TGF-beta1 response upon challenge when compared to non-immune animals.

Identification and partial purification of a 26 kilodalton antigen of adult Haemonchus contortus.

Adult H. contortus soluble extracts were fractionated by means of gel filtration (S-200) and anion exchange chromatography (DEAE-Sephacel). Fractions from both analyses were checked by ELISA and western blotting (WB) with sera from lambs infected with H. contortus, monospecific heterologous sera (anti-Trichostrongylus colubriformis and anti-Teladorsagia circumcinta) and sera from naturally infected sheep with mixed trichostrongylid infections. High cross reactivity was seen between H. contortus and heterologous sera, particularly with the anti-T. colubriformis serum, when fractions from gel filtration were checked by ELISA. Individual fractions containing the highest positive/negative and positive/heterologous ratios were pooled and analysed by SDS-PAGE. One of the pools (A4) containing 2 regions around 48-55 and 25-27 kDa were strongly recognized by homologous sera in WB. Similar results were obtained with the first peak eluted in the DEAE-Sephacel chromatography with NaCl 0.1 M. The pooled fraction A4 from gel filtration was further fractionated by anion exchange chromatography and the peak obtained with the NaCl gradient contained a ca. 26 kDa antigen apparently specific for the diagnosis of H. contortus infections in lambs.

Cross antigenicity among ovine trichostrongyloidea. Preliminary report.

A preliminary trial on the extent of cross-antigenicity among the sheep strongylids Haemonchus contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta and Nematodirus battus in 2.5- to 4-month-old lambs has been carried out using ELISA and Western blotting (WB). Cross antigenicity was tested using soluble extracts from adult and third stage larvae (L3) of H. contortus as antigenic source probed with sera from lambs with monospecific heterologous infections. There was cross-antigenicity between L3 of H. contortus and Trichostrongylus colubriformis in ELISA and WB. Immunodetection results with adult H. contortus antigen showed a closer relationship to Teladorsagia circumcincta. Certain heterologous sera reacted with H. contortus antigens more strongly than the homologous one, but sera from the H. contortus-infected animals had reactivity around the 25 kDa region from adult antigens which could have potential diagnostic use.

Lamb serum recognition of infective larvae and adult Haemonchus contortus antigens.

Sixteen- to eighteen-week-old lambs were infected with 2500 3rd stage larvae (L-3) of Haemonchus contortus or kept as uninfected controls. Two months later all animals were challenged with 5000 L-3 of this parasite. Soluble antigens of H. contortus L-3 and adult worms were analyzed by SDS-PAGE and Western blotting during experimental infection and challenge. Antigens from both sources, particularly of low molecular weight under reducing conditions, were recognised by the pooled sera of infected lambs. No single L-3 antigen was recognised by all infected lambs, whereas two peptides having around 25 and 26 kDa from adults were recognised by infected animals during the patency and could be of potential use in the diagnosis of lamb haemonchosis.

Serum IgG response of Manchego lambs to infections with Haemonchus contortus and preliminary characterization of adult antigens.

Manchego lambs (16-18 weeks old) were infected with 2500 infective larvae (L3) of Haemonchus contortus and challenged 2 months later with 5000 L3. The serum IgG anti-Haemonchus response was estimated by enzyme-linked immunosorbent assay (ELISA) using soluble proteins from adults and L3. Previously infected Manchego lambs failed to mount a protective immune response against challenge, at least as assessed by faecal egg counts and pre-patency periods. Primary infection did not provoke any rise in specific anti-parasite serum antibodies, whereas a weak but significant rise was observed in challenged 6.5-month-old lambs which was very similar in both infected and non-infected lambs. The serum IgG anti-parasite response was higher against larval antigens than adult soluble proteins. Preliminary characterization of adult and larval soluble proteins by electrophoresis under reducing and denaturing conditions and Western blotting showed high cross-reactivity of both extracts. Immunoblots of adult H. contortus probed with infected and challenged lambs' sera did not yield conclusive results, although some low molecular weight peptides were recognized.