Avian haemosporidian parasites in captive and free-ranging, wild birds from zoological institutions in Switzerland: Molecular characterization and clinical importance.

Avian haemosporidian parasites are widespread and infect birds from a broad variety of avian families with diverse consequences ranging from subclinical infections to severe and fatal disease. This study aimed to determine the occurrence and diversity of avian haemosporidia including associated clinical signs and pathomorphological lesions in captive and free-ranging, wild birds from two zoos and the near environment in Switzerland. Blood samples from 475 birds, including 230 captive and 245 free-ranging, wild individuals belonging to 42 different avian species from 15 orders were examined for the presence of avian haemosporidian DNA by a one-step multiplex PCR designed to simultaneously detect and discriminate the genera Plasmodium, Haemoproteus and Leucocytozoon by targeting mitochondrial genome sequences. Positive samples were additionally tested using a nested PCR targeting the cytochrome b gene of Plasmodium and Haemoproteus. The obtained amplicons were bidirectionally sequenced. This study revealed haemosporidian DNA in 42 samples, belonging to ten host species. The most commonly detected lineage was Plasmodium relictum SGS1, which was identified in 29 birds (Phoenicopterus roseus: n = 24, Alectoris graeca: n = 1, Lamprotornis superbus: n = 1, Somateria mollissima: n = 1, Spheniscus demersus: n = 1, Tetrao urogallus crassirostris: n = 1), followed by Haemoproteus sp. STRURA03 in six avian hosts (Bubo bubo: n = 5, Bubo scandiacus = 1), Plasmodium relictum GRW11 in four individuals (Phoenicopterus roseus: n = 3, Spheniscus demersus: n = 1) and Plasmodium elongatum GRW06 in one Alectura lathami lathami. A Phalacrocorax carbo was infected with Plasmodium relictum, but the exact lineage could not be determined. One mixed infection with P. relictum and Haemoproteus sp. was detected in a Bubo scandiacus. Only five individuals (Spheniscus demersus: n = 2, Somateria mollissima: n = 1, Bubo scandiacus: n = 1, Alectoris graeca: n = 1) showed clinical and pathomorphological evidence of a haemosporidian infection.

Syngamus trachea in free-ranging white stork (Ciconia ciconia) nestlings in Switzerland.

Syngamosis is a disease caused by the strongylid nematode Syngamus trachea, which infects the respiratory tract of various bird species around the world. The parasite appears to be harmful for a wide variety of avian orders, occasionally leading to a fatal outcome, particularly in young birds. The aim of this study was to examine the parasitic fauna in deceased or euthanized, free-ranging white storks nesting at the Zoo Basel in 2019 and 2020; and to assess the extent to which these parasites contributed to the wild birds' death. In five out of 24 necropsied white storks, an infection with S. trachea was diagnosed based on morphological analysis of adult nematode stages and eggs, in combination with PCR amplification and sequencing of DNA extracted from female worms. The main pathological changes affected the white storks' respiratory tract and a mixed cell tracheitis was diagnosed in the histopathological examination of three of the five infected birds. Some birds displayed additional lesions compatible with syngamosis, namely partially degenerated parasitic structures with concurrent granulomatous inflammation in the lung and multifocal acute hemorrhages in the bronchi and parabronchi. Coprological examinations (fecal flotation technique, fecal sedimentation technique, sodium acetate acetic acid formalin procedure and Ziehl-Neelsen staining) from the intestinal content as well as a PCR for Toxoplasma gondii on brain, lung, heart, liver, and spleen tissue yielded negative results in all examined individuals. In the absence of further major pathological findings, S. trachea was assumed to have significantly contributed to the death of the infected birds.

Causes of Abortions in South American Camelids in Switzerland-Cases and Questionnaire.

Over the last decade, South American camelids (SAC) have gained increasing popularity in Switzerland. They are used for several purposes such as fiber and meat production, as companion or guard animals and for trekking activities. The purpose of this study was to investigate the frequency and reasons for pregnancy loss and perinatal death in SAC herds. Within the scope of this study, early embryonic losses could not be identified, as pregnancy examinations by ultrasonography are not performed routinely. Aborted and stillborn fetuses were collected, necropsied and analyzed for infectious abortifacients. A nationwide survey among breeders was carried out. During a 1.5-year period, only eight cases of aborted or stillborn alpaca and llama (out of a population of 6550 animals) were reported by the breeders, and their causes were subsequently analyzed. In half of the cases, Coxiella burnetii was identified in the fetoplacental material. Abortions and stillbirths were reported to be rare in Swiss herds. As a conclusion, recording of embryonic losses through ultrasound training of veterinarians should be impaired and breeders motivated to have abortions and perinatal mortality examined. Special focus should be laid on C. burnetii due to its zoonotic risk.

A Review of the Parasite Fauna of the Black-Bellied Pangolin, Phataginus tetradactyla LIN. (Manidae), From Central Africa with the Description of Intraproboscis sanghae n. gen., n. sp. (Acanthocephala: Gigantorhynchidae).

A new archiacanthocephalan in the family Gigantorhynchidae, Intraproboscis sanghae n. gen., n. sp. is described from females collected from the African black-bellied pangolin Phataginus tetradactyla Linn. (Manidae) in the Central African Republic. A dichotomous key to the genera of Gigantorhynchidae is provided. The specimens presented are distinct from those of the genus Gigantorhynchus Hamann, 1892 that have only 1 or 2 circles of hooks (crowns) at the apical end of the proboscis and are found in South American mammals, except for Gigantorhynchus pesteri Tadros, 1966 from baboons in Rhodesia (Zimbabwe), Africa (Amin, 2013). They superficially resemble those of the other gigantorhynchid genus Mediorhynchus Van Cleave, 1916, especially in the organization of the truncate-cone proboscis and the position of the receptacle. Species of Mediorhynchus are bird parasites. The new genus, Intraproboscis, now the third genus in Gigantorhynchidae; however, is distinguished from Mediorhynchus by having a simple proboscis receptacle that is completely suspended within the proboscis, the passage of the retractor muscles through the receptacle into the body cavity posteriorly, absence of neck, and presence of a parareceptacle structure (first finding in the Archiacanthocephala) and a uterine vesicle; among other features, including the differential dorsoventral thickness of the body wall. The receptacle in Mediorhynchus is complex, with many accessory muscles and retractor muscles passing into the body cavity dorsally and ventrally. Our specimens reached 180 mm in length and the proboscis had 34-36 rows of 6-7 ventrally lamellated, rooted hooks each anteriorly, and 15-17 spinelike hooks each posteriorly. Micropores extended into the anterior and posterior proboscis and energy dispersive x-ray analysis (EDXA) of anterior hooks showed high levels of calcium and phosphorus but negligible traces of sulfur. Spinelike hooks in the posterior proboscis had lower levels of Ca and P and slightly higher levels of S. Molecular and phylogenetic analyses based on the 18S rDNA gene placed I. sanghae in a clade with the archiacanthocephalans Mediorhynchus, Moniliformis, Macracanthorhynchus, Oncicola, and Oligacanthorhynchus.

Fatal avian malaria in captive Atlantic puffins (Fratercula arctica) in Switzerland.

Avian malaria is a vector-borne disease caused by Plasmodium species, which may affect a broad spectrum of bird families worldwide. In most endemic and migratory birds, Plasmodium infections seem not to cause severe harm; however, non-indigenous species kept in human care such as penguins may experience high morbidity and mortality rates. Fatal avian malaria may also occur in other non-native seabirds such as puffins (Fratercula spp.), but reported cases are scarce. The aim of this study was to analyze seven cases of sudden death in captive Atlantic puffins (Fratercula arctica) at Berne Animal Park in Switzerland between 2010 and 2020, and to determine the involvement of haemosporidian parasites in the fatal outcome. In all cases, lymphoplasmacytic inflammation, necrotic lesions in several organs and presence of protozoan stages within tissues/erythrocytes or accumulation of iron-based pigment were observed histologically. A one-step multiplex PCR designed to simultaneously detect and discriminate haemosporidia belonging to the genera Plasmodium, Haemoproteus and Leucocytozoon, and a nested PCR detecting Plasmodium and Haemoproteus infections were performed on DNA extracted from formalin-fixed and paraffin-embedded (FFPE) or fresh liver and spleen tissues from five and two birds, respectively. Plasmodium spp. DNA was detected in the tissues from six of seven birds by the one-step multiplex PCR and in five of seven individuals by the nested PCR protocol. Direct sequencing of the amplification products allowed the molecular identification of Plasmodium relictum SGS1 as the involved species in three individuals and Plasmodium matutinum LINN1 in two of these fatal cases. In one bird, no haemosporidian DNA could be amplified from FFPE tissues despite of suggestive histopathological findings. These results indicate that avian malaria represents an important cause of death in captive puffins and it should be considered as a differential diagnosis in unclear or fatal cases in this threatened bird species.

An experimental genetically attenuated live vaccine to prevent transmission of Toxoplasma gondii by cats.

Almost any warm-blooded creature can be an intermediate host for Toxoplasma gondii. However, sexual reproduction of T. gondii occurs only in felids, wherein fertilisation of haploid macrogametes by haploid microgametes, results in diploid zygotes, around which a protective wall develops, forming unsporulated oocysts. Unsporulated oocysts are shed in the faeces of cats and meiosis gives rise to haploid sporozoites within the oocysts. These, now infectious, sporulated oocysts contaminate the environment as a source of infection for people and their livestock. RNA-Seq analysis of cat enteric stages of T. gondii uncovered genes expressed uniquely in microgametes and macrogametes. A CRISPR/Cas9 strategy was used to create a T. gondii strain that exhibits defective fertilisation, decreased fecundity and generates oocysts that fail to produce sporozoites. Inoculation of cats with this engineered parasite strain totally prevented oocyst excretion following infection with wild-type T. gondii, demonstrating that this mutant is an attenuated, live, transmission-blocking vaccine.

RNA-Seq analysis during the life cycle of Cryptosporidium parvum reveals significant differential gene expression between proliferating stages in the intestine and infectious sporozoites.

Cryptosporidium parvum is a major cause of diarrhoea in humans and animals. There are no vaccines and few drugs available to control C. parvum. In this study, we used RNA-Seq to compare gene expression in sporozoites and intracellular stages of C. parvum to identify genes likely to be important for successful completion of the parasite's life cycle and, thereby, possible targets for drugs or vaccines. We identified 3774 protein-encoding transcripts in C. parvum. Applying a stringent cut-off of eight fold for determination of differential expression, we identified 173 genes (26 coding for predicted secreted proteins) upregulated in sporozoites. On the other hand, expression of 1259 genes was upregulated in intestinal stages (merozoites/gamonts) with a gene ontology enrichment for 63 biological processes and upregulation of 117 genes in 23 metabolic pathways. There was no clear stage specificity of expression of AP2-domain containing transcription factors, although sporozoites had a relatively small repertoire of these important regulators. Our RNA-Seq analysis revealed a new calcium-dependent protein kinase, bringing the total number of known calcium-dependent protein kinases (CDPKs) in C. parvum to 11. One of these, CDPK1, was expressed in all stages, strengthening the notion that it is a valid drug target. By comparing parasites grown in vivo (which produce bona fide thick-walled oocysts) and in vitro (which are arrested in sexual development prior to oocyst generation) we were able to confirm that genes encoding oocyst wall proteins are expressed in gametocytes and that the proteins are stockpiled rather than generated de novo in zygotes. RNA-Seq analysis of C. parvum revealed genes expressed in a stage-specific manner and others whose expression is required at all stages of development. The functional significance of these can now be addressed through recent advances in transgenics for C. parvum, and may lead to the identification of viable drug and vaccine targets.

Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.

BACKGROUND: The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into. RESULTS: To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites. CONCLUSIONS: The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.

Comparación de técnicas parasitológicas para el examen de heces de perro / Comparison of parasitological techniques for the examination of dog feces

Se evaluaron comparativamente las siguientes técnicas para el examen parasitológico de heces de perro: la técnica de Sheater modificada (flotación por la solución de azúcar), la de sulfato de zinc (flotación por la solución de esta sal) y la de Ritchie modificada, denominada también de formol-éter (sedimentación con solución salina formolada y extracción de grasas por éter). Se detectaron parásitos en 25 de 40 muestras de materia fecl de perros que se examinaron por las tres técnicas. No hubo diferencias entre las técnicas de Sheater y sulfato de zinc en la cantidad de detecciones logradas (37 y 38 respectivamente) pero el valor correspondiente a la de formol-éter (16) fue significativamente menor. Las cantidades de huevos de toxocara canis y trichuris vulpis (213 y 334 respectivamente) detectadas por Sheater en materia fecal en fresco, fueron significativamente mayores que las correspondientes a sulfato de zinc (44 y 13 respectivamente). Utilizando materia fecal con formol con huevos de T. vulpis, los resultados fueron similares (Sheater: 61 vs sulfato de zinc: 0). Las cantidades de quistes de giardia detectadas por formol-éter en materia fecal en fresco y con formol, fueron significativamente mayores que las detectadas por sulfato de zinc (87 y 19 vs 36 y 3, respectivamente. En 180 muestras examinadas la cantidad de detecciones de quistes giardia sp efectuadas por medio de formol-éter fue significativamente mayor por Sheater (15 vs 1, respectivamente). La técnica de Sheater fue más eficiente que la de sulfato de zinc en la detección de huevos de nematodes y la de formol-éter más eficiente para la detección de quistes de giardia sp. De acuerdo a los resultados obtenidos se recomienda que, salvo indicaciones especiales cada muestra que requiera examen parasitológico sea procesada por las técnicas de Sheater de formol-éter