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Application of In-situ Hybridization for the Detection and Identification of Avian Malaria Parasites in Paraffin Wax-embedded Tissues from Captive Penguins
... Histological analysis of the exoerythrocytic and erythrocytic stages of avian Plasmodium parasites has been challenging as these require training to visualize histologically. Other authors have used chromogenic in-situ hybridization (CISH) with Plasmodium speci c probes to help label parasites in the tissues [33,35]. Anti-P. ...
Background
Avian malaria is caused by diverse parasite species of the genus Plasmodium, and it affects various bird species. The occurrence of this disease in wild birds is understudied due to the scarce availability of samples. Hence the pathogenicity in these hosts is not completely known. In addition, feral birds may act as reservoirs bridging the transmission cycle from wild migratory birds to domestic and zoo-kept bird species. We report here the occurrence of P. relictum in a feral pigeon.
Case presentation
The bird died unexpectedly, and necropsy revealed an anaemic carcass, with pale organs and hydropericardium. Histopathologic analysis revealed inflammatory infiltrates in lung and liver, and monocytes and Kupffer cells contained hemozoin pigment indicative of phagocytosis of Plasmodium infected erythrocytes. A high erythrocytic infection rate of 18% was evident in tissues and blood vessels in various organs. Furthermore, the thyroid had masses classified as thyroid carcinomas. Immunohistochemistry with anti-P. falciparum HSP70 antibody revealed positive signals in erythrocytes and intravascular leucocytes. Further analysis using Giemsa-stained blood smears revealed a high parasitaemia with an asynchronous infection showing all erythrocytic stages. Molecular diagnosis by PCR identified P. relictum, lineage GRW11 as the etiological agent. The bird presented died most likely due to an acute infection as evidenced by the high blood parasitaemia, leading to major erythrocyte destruction. Further analyses of feral pigeons (n = 22) did not reveal any additional cases with Plasmodium infections.
Conclusion
While our study suggests that Plasmodium infections are rare in pigeons, host conditions like immunosuppression may have influenced the infection outcome in this fatal case due to the tumour.
... In contrast to the results reported from USUV-positive blackbirds in the Netherlands 4 , no exo-erythrocytic stages of haemoprotozoa indicative of avian malaria were observed histologically in the two UK blackbirds positive for Plasmodium DNA. Since histological examination has limited sensitivity, in situ hybridisation could be used to further appraise the clinical significance of this co-infection in the future 16 . ...
Following the first detection in the United Kingdom of Usutu virus (USUV) in wild birds in 2020, we undertook a multidisciplinary investigation that combined screening host and vector populations with interrogation of national citizen science monitoring datasets to assess the potential for population impacts on avian hosts. Pathological findings from six USUV-positive wild passerines were non-specific, highlighting the need for molecular and immunohistochemical examinations to confirm infection. Mosquito surveillance at the index site identified USUV RNA in Culex pipiens s.l. following the outbreak. Although the Eurasian blackbird (Turdus merula) is most frequently impacted by USUV in Europe, national syndromic surveillance failed to detect any increase in occurrence of clinical signs consistent with USUV infection in this species. Furthermore, there was no increase in recoveries of dead blackbirds marked by the national ringing scheme. However, there was regional clustering of blackbird disease incident reports centred near the index site in 2020 and a contemporaneous marked reduction in the frequency with which blackbirds were recorded in gardens in this area, consistent with a hypothesis of disease-mediated population decline. Combining results from multidisciplinary schemes, as we have done, in real-time offers a model for the detection and impact assessment of future disease emergence events.
... Although it is possible that the high homology of both rRNA sequences causes cross-reactivity, we chose this locus because rRNA is supposedly the only target present in sufficient abundance to warrant effective ISH staining of protozoal cells. 12 Nevertheless, verifying the specificity of both probes in situ by staining brain tissue infected with N. caninum has shown no cross-hybridization. In those sections, mainly tissue cysts of N. caninum were detected, which typically have decelerated replication. ...
Histologic examination of aborted material is an essential component in the diagnosis of ovine toxoplasmosis. However, the detection of Toxoplasma gondii in histologic sections, and its differentiation from the closely related protozoan Neospora caninum, is challenging. We developed a chromogenic in situ hybridization (ISH) assay for the identification of T. gondii in paraffin-embedded tissue samples. We examined retrospectively the archived placental tissue of 200 sheep abortion submissions for the presence of T. gondii by immunohistochemistry (IHC), ISH, and real-time PCR (rtPCR). All placental samples that tested positive for T. gondii by rtPCR (9 of 200) were also positive by IHC, with inconclusive IHC staining in an additional 7 rtPCR-negative cases. Further testing for N. caninum of all 200 placentas by rtPCR revealed 7 Neospora-positive cases. T. gondii ISH was positive in 4 of 9 IHC-positive samples and 1 of the 7 N. caninum rtPCR-positive samples. Real-time PCR was used as the reference standard for specificity and sensitivity calculations regarding placenta samples. Specificity of ISH and IHC was 99% and 96-100%, respectively. The sensitivity of ISH (44%) was quite low compared to IHC (100%). The exclusive use of ISH for the detection of T. gondii, and thus for the diagnosis of ovine toxoplasmosis, was not acceptable. However, combined with rtPCR, both ISH and IHC can be useful detection methods to improve histologic evaluation by visualizing the parasite within tissue sections.
... Controlled experimental infections are crucial to get information about exoerythrocytic stages of these parasites. Recently, a molecular chromogenic in situ hybridization method was developed for analysis of exoerythrocytic stages of avian malaria parasites (Dinhopl et al. 2011) and was successfully applied for experimentally infected birds with P. homocircumflexum (Ilgūnas et al. 2016). Experimental infections and analysis of internal organs using in situ hybridization should be used in future studies in all geographical regions. ...
Broadening the field of classical parasitology research by integrating it with ecoimmunology has allowed us a better understanding of the effect of haemo-sporidians and to identify the most relevant factors that affect the health of birds. Despite the recent advances in avian malaria studies, the lack of experimentation remains the main obstacle for a proper characterization of the natural history of parasites and the functioning of the immune system of birds. It is worth considering the complement of both classical and new immunological methodologies to establish reference information and to assess the reliability of previous studies. The new molecular methodologies represent an advantage for distinguishing the involved genes in the immune response on birds when facing haemosporidian infections, avoiding the underestimation of the real prevalence of hemoparasites (e.g., coinfec-tions), and setting up a starting point for new researchers interested in specializing in this area of study. Here, we present important and recent approaches on experimental parasitology demonstrating the negative effects of avian haemosporidians on their avian hosts. Also, we summarize the main advances in this field in avian malaria studies in the neotropics, as well as pinpointing knowledge gaps and future research opportunities. Additionally, we will recapitulate the main contributions and tools used by ecoimmunologists to study immune defences against avian malaria
parasites.
Haemoproteus (Haemoproteus) multipigmentatus, a haemosporidian parasite thought to be specific to
columbiform birds, was detected in passeriform birds on Santiago Island in the Galapagos archipelago.
We surveyed birds along an altitudinal gradient on the islands of Santa Cruz, Isabela and Santiago between
June 2013 and July 2015. Molecular screening of 2254 individuals from 22 species of endemic and
introduced birds revealed clusters of passerine birds positive for H. multipigmentatus on Santiago Island
that coincide with captures of Galapagos doves at sampled sites. Of 507 individuals from 10 species of
endemic passerines sampled on Santiago, 58 individuals from 6 species were found positive (11%
prevalence). However, no gametocytes were found in the blood smears of positive passerines, suggesting
that these species are not competent hosts for the parasite. All 31 doves captured were positive and
gametocytes were found upon microscopic examination of all thin blood smears (averaging 357 gametocytes
per 10,000 erythrocytes). These findings indicate parasite spillover from doves to passerines,
but that passerines are possibly not competent hosts for further parasite transmission. The endemic
Galapagos dove acts as a reservoir host for the introduced H. multipigmentatus, however the effect of this
parasite on passerines has not been studied.We report on these findings because parasites can have large
effects on individual host populations and on the ecology of a community, but may go undetected.
Background
Avian malaria parasites (Plasmodium spp.) and related haemosporidians (Haemosporida) are responsible for diseases which can be severe and even lethal in avian hosts. These parasites cause not only blood pathology, but also damage various organs due to extensive exo-erythrocytic development all over the body, which is not the case during Plasmodium infections in mammals. However, exo-erythrocytic development (tissue merogony or schizogony) remains the most poorly investigated part of life cycle in all groups of wildlife haemosporidian parasites. In spite of remarkable progress in studies of genetic diversity, ecology and evolutionary biology of avian haemosporidians during the past 20 years, there is not much progress in understanding patterns of exo-erythrocytic development in these parasites. The purpose of this review is to overview the main information on exo-erythrocytic development of avian Plasmodium species and related haemosporidian parasites as a baseline for assisting academic and veterinary medicine researchers in morphological identification of these parasites using tissue stages, and to define future research priorities in this field of avian malariology. Methods
The data were considered from peer-reviewed articles and histological material that was accessed in zoological collections in museums of Australia, Europe and the USA. Articles describing tissue stages of avian haemosporidians were included from 1908 to the present. Histological preparations of various organs infected with the exo-erythrocytic stages of different haemosporidian parasites were examined. ResultsIn all, 229 published articles were included in this review. Exo-erythrocytic stages of avian Plasmodium, Fallisia, Haemoproteus, Leucocytozoon, and Akiba species were analysed, compared and illustrated. Morphological characters of tissue stages that can be used for diagnostic purposes were specified. Conclusion
Recent molecular studies combined with histological research show that avian haemosporidians are more virulent than formerly believed. The exo-erythrocytic stages can cause severe disease, especially in non-adapted avian hosts, suggesting the existence of a group of underestimated malignant infections. The development of a given haemosporidian strain can be markedly different in different avian hosts, resulting in significantly different virulence. A methodology combining the traditional histology techniques with molecular diagnostic tools is essential to speed research in this field of avian malariology.
Background
Species of avian malaria parasites (Plasmodium) are widespread, but their virulence has been insufficiently investigated, particularly in wild birds. During avian malaria, several cycles of tissue merogony occur, and many Plasmodium spp. produce secondary exoerythrocytic meronts (phanerozoites), which are induced by merozoites developing in erythrocytic meronts. Phanerozoites markedly damage organs, but remain insufficiently investigated in the majority of described Plasmodium spp. Avian malaria parasite Plasmodium (Giovannolaia) homocircumflexum (lineage pCOLL4) is virulent and produces phanerozoites in domestic canaries Serinus canaria, but its pathogenicity in wild birds remains unknown. The aim of this study was to investigate the pathology caused by this infection in species of common European birds.
Methods
One individual of Eurasian siskin Carduelis spinus, common crossbill Loxia curvirostra and common starling Sturnus vulgaris were exposed to P. homocircumflexum infection by intramuscular sub-inoculation of infected blood. The birds were maintained in captivity and parasitaemia was monitored until their death due to malaria. Brain, heart, lungs, liver, spleen, kidney, and a piece of breast muscle were examined using histology and chromogenic in situ hybridization (ISH) methods.
Results
All exposed birds developed malaria infection, survived the peak of parasitaemia, but suddenly died between 30 and 38 days post exposure when parasitaemia markedly decreased. Numerous phanerozoites were visible in histological sections of all organs and were particularly easily visualized after ISH processing. Blockage of brain capillaries with phanerozoites may have led to cerebral ischaemia, causing cerebral paralysis and is most likely the main reason of sudden death of all infected individuals. Inflammatory response was not visible around the brain, heart and muscle phanerozoites, and it was mild in parenchymal organs. The endothelial damage likely causes dysfunction and failure of parenchymal organs.
Conclusion
Plasmodium homocircumflexum caused death of experimental passerine birds due to marked damage of organs by phanerozoites. Patterns of phanerozoites development and pathology were similar in all exposed birds. Mortality was reported when parasitaemia decreased or even turned into chronic stage, indicating that the light parasitaemia is not always indication of improved health during avian malaria. Application of traditional histological and ISH methods in parallel simplifies investigation of exoerythrocytic development and is recommended in avian malaria research.
Malaria parasite Plasmodium (Novyella) delichoni n. sp. (Haemosporida, Plasmodiidae) was found in a widespread Eurasian songbird, the common house martin Delichon urbicum (Hirundinidae). It is described based on the morphology of its blood stages and segments of the mitochondrial cytochrome b and apicoplast genes, which can be used for molecular identification of this species. Erythrocytic meronts and gametocytes are strictly nucleophilic, and mature gametocytes possess pigment granules of markedly variable size, including large ones (1 μm in length). Due to these features, P. delichoni can be readily distinguished from all described species of avian malaria parasites belonging to subgenus Novyella. Additionally, mature erythrocytic merozoites contain a dense clump of chromatin, a rare character in avian malaria parasites. Erythrocytic merogony is asynchronous. Illustrations of blood stages of the new species are given, and phylogenetic analysis identifies DNA lineages closely related to this parasite. Domestic canary Serinus canaria and Eurasian siskin Carduelis spinus were infected after subinoculation of infected blood obtained from the house martin. Parasitemia was long lasting in both these hosts, but it was high (up to 70 %) in Eurasian siskins and low (up to 1 %) in canaries. Mortality was not observed, and histological examination and chromogenic in situ hybridisation did not reveal secondary exoerythrocytic meronts (phanerozoites) in the exposed birds. It is likely that persistence of this infection occurs due to long-lasting parasitemia in avian hosts. Sporogony was abortive in mosquitoes Culex pipiens pipiens form molestus, Culex quinquefasciatus and Aedes aegypti at gametogenesis or ookinete stages. The new species is absent from juvenile birds at breeding sites in Europe, indicating that transmission occurs at African wintering grounds.
Brener B. & Almosny N.R.P. 2014. Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plas-modium spp.). Pesquisa Veterinária Brasileira 34(12):1234-1240. Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods. Considering the negative impact of avian malaria on penguins, the aim of this study was to detect the presence of Plasmodium spp. in Magellanic penguins using Polymerase Chain Reaction (PCR) and by verifying clinical, hematological, and biochemical alterations in blood samples as well as to verify the likely prognosis in response to infection. Blood samples were obtained from 75 penguins to determine packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), uric acid, total protein, albumin, globulin and aspartate aminotransferase (AST) activity levels. Whole blood samples were used for PCR assays. Plasmodium spp. was detected in 32.0% of the specimens using PCR and in 29.3% using microscopic analyses. Anorexia, diarrhea and neurological disorders were more frequent in penguins with malaria and a significant weight difference between infected and non-infected penguins was detected. PCV and MCV rates showed no significant difference. RBC and WBC counts were lower in animals with avian malaria and leukopenia was present in some penguins. Basophil and lymphocyte counts were lower in infected penguins along with high monocyte counts. There was no significant difference in AST activities between infected and non-infected animals. There was a significant increase in uric acid values, however a decrease in al-bumin values was observed in infected penguins. Based on this study, we concluded that Plasmodium spp. occurs in Magellanic penguins of rehabilitation centers in Southeastern
Native European passerine birds are frequently clinically inapparent carriers of haemosporidian parasites of the genus Plasmodium. Clinical disease and death are only exceptionally reported. In the present study, tissue samples of 233 wild passerine birds found dead in Eastern Austria were examined by in situ hybridization (ISH) and partial cytochrome B gene sequence analysis for the presence, abundance and taxonomic assignment of Plasmodium spp. In 34 cases (14.6 %), ISH yielded a positive result with large numbers of developmental stages in different cell types of the spleen, liver, brain and lung. The abundance of the tissue stages, which was comparable to fatal cases of avian malaria in penguins, suggested a major contribution to the cause of death. Genetic analysis revealed infections with representatives of three different valid species of Plasmodium, Plasmodium elongatum, Plasmodium lutzi and Plasmodium vaughani. Genetically identical parasite lineages had been found in a previous study in penguins kept in the Vienna zoo, providing evidence for the role of wild birds as reservoir hosts. Further, this study provides evidence that several species of Plasmodium are able to abundantly proliferate in endemic wild birds ultimately resulting in mortalities.
Infections with protozoal parasites of the order Trichomonadida are often observed in veterinary medicine. Based on the trichomonad species involved these infections are either asymptomatic or can lead to sometimes serious disease. To further study protozoal agents of the order Trichomonadida the establishment of a method to detect trichomonads directly in the tissue, allowing parasite-lesion correlation, is necessary. Here we describe the design and evaluation of an oligonucleotide probe for chromogenic in situ hybridization, theoretically allowing detection of all hitherto known members of the order Trichomonadida. The probe was designed on a region of the 18S ribosomal RNA gene homologue for all representatives of the order Trichomonadida available in the GenBank. Functionality of the probe was proven using protozoal cultures containing different trichomonads (Monocercomonas colubrorum, Hypotrichomonas acosta, Pentatrichomonas hominis, Trichomitus batrachorum, Trichomonas gallinae, Tetratrichomonas gallinarum, Tritrichomonas foetus, and Tritrichomonas augusta). Furthermore, three different tissue sections containing either T. gallinae, T. foetus or Histomonas meleagridis were tested positive. Additionally, to rule out cross-reactivity of the probe a large number of different pathogenic protozoal agents, fungi, bacteria and viruses were tested and gave negative results. The probe presented here can be considered an important tool for diagnosis of all to date described relevant protozoal parasites of the order Trichomonadida in tissue samples.
Avian Plasmodium and Haemoproteus parasites are easily detected by DNA analyses of infected samples but only correctly assigned to each genus by sequencing and use of a phylogenetic approach. Here, we present a restriction site to differentiate between both parasite genera avoiding the use of those analyses. Alignments of 820 sequences currently listed in GenBank encoding a particular cytochrome B region of avian Plasmodium and Haemoproteus show a shared restriction site for both genera using the endonuclease Hpy CH4III. An additional restriction site is present in Plasmodium sequences that would initially allow differentiation of both genera by differential migration of digested products on gels. Overall 9 out of 326 sequences containing both potential restriction sites do not fit to the general rule. We used this differentiation of parasite genera based on Hpy CH4III restriction sites to evaluate the efficacy of 2 sets of general primers in detecting mixed infections. To do so, we used samples from hosts infected by parasites of both genera. The use of general primers was only able to detect 25% or less of the mixed infections. Therefore, parasite DNA amplification using general primers to determine the species composition of haemosporidian infections in individual hosts is not recommended. Specific primers for each species and study area should be designed until a new method can efficiently discriminate both parasites.
Ten captive-reared African black-footed penguins (Spheniscus demersus) from a large outdoor colony were monitored for avian malaria, using several diagnostic tests. One treatment regimen was evaluated. Thin smear blood evaluation enabled detection of seven parasitemias involving Plasmodium relictum and Plasmodium elongatum in the penguins. Leukocytosis (relative lymphocytosis) was characteristic of infected birds. Parasitemia was detected as early as 21 days prior to onset of clinical signs (depression, anorexia, regurgitation, pale mucous membranes, and respiratory distress). The single bird that died had clinical signs only a few hours prior to its death. Treatment consisted of 0.03 mg of primaquine phosphate base/kg body weight, administered orally once daily for 3 days. Oral chloroquine phosphate therapy, given simultaneously, was administered in an initial loading dose of 10 mg of chloroquine phosphate base/kg body weight, followed by doses of 5 mg/kg at 6, 18 and 24 hours after the initial chloroquine dose. This treatment regimen prevented mortality and cleared parasites from the blood. Recurrences of malaria occurred in two birds that had received this treatment.
Six of seven Hawaii Amakihi (Hemignathus virens) with chronic malarial infections had no increases in peripheral parasitemia, declines in food consumption, or loss of body weight when rechallenged with the homologous isolate of Plasmodium relictum 61 to 62 days after initial infection. Five uninfected control amakihi exposed at the same time to infective mosquito bites developed acute infections with high parasitemias. Reductions in food consumption and loss of body weight occurred in all control birds and three of these individuals eventually died. When surviving birds were rechallenged >2 yr later with either the same parasite isolate or an isolate of P. relictum collected on the island of Kauai, all individuals were immune to superinfection. Chronically infected birds developed antibodies to a common suite of malarial antigens ranging in size from 22 to 170 kDa that were detectable as early as 8 days post infection on immunoblots of SDS-polyacrylamide gels. Antibodies to this suite of malarial antigens persisted as long as 1,248 days after initial infection and were consistently detectable at times when parasites were not easily found by microscopy on Giemsa-stained blood smears. The immunoblotting method that is described here appears to be an effective technique for identifying birds with chronic, low-intensity malarial infections when circulating parasites are not easily detectable by microscopy. Hawaiian honeycreepers that are capable of recovering from acute infections develop concomitant immunity to superinfection, making them functionally immune in areas where malaria transmission has become endemic.
Here, 4 polymerase chain reaction (PCR) assays are compared to test for the presence of avian malaria, including both the Plasmodium and Haemoproteus genera, in 29 different species of African rainforest birds. Two of these PCR assays use primer sets that amplify fragments of the cytochrome b (cyt b) gene of Plasmodium; the other 2 target the 18S ribosomal subunit gene. These PCR assays were performed using genomic DNA extracted from blood and subsequently compared with the results obtained by microscopic examination of blood smears taken from the same individuals. The 2 primer sets amplifying the cyt b gene were found to perform more reliably than those that target the 18S rRNA gene and yielded a substantial number of positive samples that were undetected by blood smear analysis. Of all the individuals screened by PCR, 40% tested positive for avian malaria, whereas 27% tested positive by blood smear analysis. Although sequence variation in the parasites may prohibit the specific alignment of primers and the subsequent PCR amplification of some individuals, PCR, once optimized, is faster, cheaper, and more reliable than blood smear analysis for large-scale screening.
Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).
To investigate the cause of a high seroprevalence of antibodies to Plasmodium spp known to cause avian malaria, but extremely low levels of observed infection, in yellow-eyed penguins, Megadyptes antipodes.
A polymerase chain reaction (PCR) test specific for malarial parasites was applied to DNA extracted from blood samples collected from 143 yellow-eyed penguins from an area where seroprevalence for malarial antibodies was known to be high but no parasites were observed in blood smears.
None of the samples tested positive for malarial parasite DNA using the PCR test. Assuming a sensitivity of 90% for this test, this means that prevalence of infection was 95% likely to be <2.3% in this population during this sampling period.
Serological studies of a population of adult yellow-eyed penguins indicated a high level of exposure to avian malaria parasites, but a correspondingly high level of infection was not observed and no evidence of malarial parasite DNA was found in the current study. Discrepancies between these findings and historical records of Plasmodium spp found in blood smears and post mortem may be explained either by inaccuracy of the serological test used, or by infection occurring in juveniles which is subsequently cleared in surviving adults.
The estimated prevalence of a malaria parasite, Plasmodium mexicanum, of western fence lizards, Sceloporus occidentalis, was compared using two techniques: microscopic examination of blood smears, and nested PCR amplification of the 18S small subunit rRNA gene. Two sites in northern California, USA were investigated, one with known long-term high prevalence of the parasite (30% by blood smear scanning), and one with low prevalence (6%). The nested PCR readily detected very low-level infections (< 1 parasite per 10 000 erythrocytes); such infections are often subpatent by normal microscopic examination. False negatives (scored as not infected after scanning the blood smear, but found infected via PCR) were rare at both sites (4% at the high-prevalence site, 6% at the low-prevalence site). However, a greater proportion of infections was detected only by PCR at the low-prevalence site (50% vs. 9%). If 50% of the infections sustain very weak parasitaemia where lizards are rarely infected, this would accord with hypotheses that predict that parasites should reduce infection growth when transmission is uncommon. The study demonstrates that PCR is a powerful tool to detect very low-level malarial infections in vertebrate hosts, including those with nucleated erythrocytes.
Antibody responses to naturally acquired Plasmodium relictum and P. elongatum infections, blood parasitemia, and disease signs were investigated in 23 naive juvenile African black-footed penguins (Spheniscus demersus). Anti-Plasmodium spp. immunoglobulins were detected by enzyme-linked immunosorbent assay (ELISA) using P. falciparum antigens. All birds rapidly developed antibody to P. relictum and P. elongatum. Five penguins showed detectable parasitemia and signs of the disease. Parasitemia was not related to the timing of the maximal antibody response or to the antibody titer. Two of the five parasitemic birds died and gross examination revealed splenomegaly, hepatomegaly, and congested, edematous lungs. Although the other 17 birds were clearly exposed to the disease, none showed signs of infection. No subsequent episode of parasitemia was observed in individual penguins. A comparison of the fate of 1993 penguins with those from other years showed a great variability in the proportion of birds exhibiting signs of malaria.
Five black-footed penguins (Spheniscus demersus) admitted to the Southern African Foundation for the Conservation of Coastal Birds, in Cape Town, South Africa, died from malaria infection. Evidence for malaria as the cause of death included antemortem clinical signs, parasitemia, splenomegaly, pulmonary edema, and the presence of histologically visible schizonts in the reticuloendothelial system. A portion of the malarial small subunit ribosomal ribonucleic acid gene was detected by polymerase chain reaction from postmortem blood samples from all the birds. A species-specific variable region of this gene was compared with the same region on genes from other known avian malarial organisms, establishing that Plasmodium juxtanucleare was involved.
Here we describe the complete genome sequences of two strains of Usutu virus (USUV), a mosquito-borne member of the genus Flavivirus in the Japanese encephalitis virus (JEV) serogroup. USUV was detected in Austria in 2001 causing a high mortality rate in blackbirds; the reference strain (SAAR-1776) was isolated in 1958 from mosquitoes in South Africa and has never been associated with avian mortality. The Austrian and South African isolates exhibited 97% nucleotide and 99% amino acid identity. Phylogenetic trees were constructed displaying the genetic relationships of USUV with other members of the genus Flavivirus. When comparing USUV with other JEV serogroup viruses, the closest lineage was Murray Valley encephalitis virus (nt: 73%, aa: 82%) followed by JEV (nt: 71%, aa: 81%) and West Nile virus (nt: 68%, aa: 75%). Comparison of the genomes showed that the conserved structural elements and putative enzyme motifs were homologous in the two USUV strains and the JEV serogroup. The factors that determine the severe clinical symptoms caused by the Austrian USUV strain in Eurasian blackbirds are discussed. We also offer a possible explanation for the origins and dispersal of USUV, JEV, and MVEV out of Africa.
An in-situ hybridization (ISH) procedure was developed for the detection of Cryptosporidium sp. in paraffin wax-embedded tissues with a digoxigenin-labelled probe targeting the 18S rRNA. This technique was used in addition to traditional methods, such as haematoxylin and eosin staining, periodic acid-Schiff reaction, transmission electron microscopy and the polymerase chain reaction, to examine the bursa of Fabricius (BF), conjunctiva and other tissues from 20 domestic geese aged 16-36 days for the presence of cryptosporidia. Positive signals were found to a moderate or marked extent in both conjunctival samples (89%) and BF samples (88%) but not in other tissues. Sequencing of the PCR amplification product revealed identity with Cryptosporidium baileyi. The infected geese showed no clinical signs and only scanty histological lesions. These results confirm reports showing that young waterfowl are especially vulnerable to cryptosporidium infection and indicate that the BF and conjunctiva are the preferred sites for the presence of the protozoon. ISH proved a good method for detecting and identifying even small numbers of cryptosporidia in tissue sections.
For use in an in-situ hybridization method, three probes (HM, TR and BL) were designed to hybridize, respectively, with (1) Histomonas meleagridis, (2) Tetratrichomonas gallinarum, and (3) a broad range of micro-organisms, including Blastocystis spp. Mono-eukaryotic cultures were used to test the specificity of the three oligonucleotides and to optimize the hybridization procedure before applying the probes to archived samples of various tissues and to a culture of Trichomonas gallinae. Specific detection of H. meleagridis was possible with the HM probe, but the other two probes were less specific. The TR probe detected members of the Trichomonadidae (Tetr. gallinarum and Tr. gallinae). Positive signals from a great variety of microorganisms, including fungi and protozoa from different animal species, were obtained with the BL probe. However, neither H. meleagridis nor the two members of the Trichomonadidae mentioned above were detected with this probe, allowing the exclusion of these parasites. Use of the three probes makes possible the accurate detection of H. meleagridis and its distinction from other micro-organisms in tissue samples.
This report describes the development of a diagnostic method for protozoal infections of the gastrointestinal tract of captive snakes, based on chromogenic in-situ hybridization with probes designed for the detection of 18S rRNA genes from Cryptosporidium spp., Entamoeba spp., Entamoeba invadens and Monocercomonas spp. The specificity of the probes was confirmed with the help of parasitic cultures and gene sequence analysis. The probes gave clear positive signals. Of 182 snakes examined, seven were positive with the Cryptosporidium probe, 13 with the Entamoeba probe (of which nine were also positive with the E. invadens probe), and 34 with the Monocercomonas probe.
A review and update of avian malaria in the African penguin (Spheniscus demersus)
- M R Cranfield
- M Shaw
- F Beall
- M Skjoldager
- M Ialeggio
Cranfield, M.R., Shaw, M., Beall, F., Skjoldager, M. & Ialeggio, M.
(1990). A review and update of avian malaria in the African penguin
(Spheniscus demersus). Proceedings of the American Association of
Zoo Veterinarians, 21, 243Á248.
Infektionen mit Plasmodium spec. bei Papageitauchern (Fratercula arctica) Kleintierpraxis
- G Loupal
- E Kutzer
Loupal, G. & Kutzer, E. (1995). Infektionen mit Plasmodium spec. bei
Papageitauchern (Fratercula arctica). Kleintierpraxis, 41, 901Á906 (in
German).